Aptamer-based analyte monitoring system

Abstract

Described herein are variations of analyte monitoring systems, including analyte monitoring devices. For example, the analyte monitoring device may include a skin-piercing microneedle array for use in measuring an analyte, such as in a continuous manner. The microneedle array may include at least one microneedle including a working electrode containing an analyte-sensing aptamer that selectively and reversibly binds to the analyte. The working electrode may, for example, include at least one thiol-based passivation molecule that provides protection of the working electrode from contaminants and interferents. For example, a sensor may be configured to generate a signal indicative of the concentration of an analyte in a fluid, the sensor including a working electrode including an electrode material, a biorecognition layer at least partially disposed on the electrode material and including a biorecognition element that selectively and reversibly binds to the analyte, and an at least partially dried hydrogel disposed on the biorecognition layer.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 478,482, filed January 4, 2023, and U.S. Provisional Patent Application No. 63 / 506,035, filed June 2, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] (Statement Regarding Federally Sponsored Research) This invention was made with U.S. Government support under Contract No. FA8650-18-2-5402 pursuant to a grant awarded by the Air Force Research Laboratory. The U.S. Government has certain rights in this invention.

[0003] (Reference to the Electronic Sequence Listing) The contents of the electronic sequence listing submitted electronically herewith (file name: BLNQ_003_03WO_SeqList_ST26.xml, size: 34,700 bytes, and creation date: January 2, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0004] Nucleic acid-based electrochemical sensors are a versatile technology that enables affinity-based detection of a wide variety of molecular targets, regardless of their intrinsic electrochemical activity or enzymatic reactivity. Additionally, their modular interface and ease of fabrication allow for rapid prototyping and sensor development. However, this technology has limitations in terms of long-term stability that complicate its translation into clinically valuable platforms such as continuous molecular monitors. For example, the biorecognition surface of a sensor for biomolecules typically has a relatively complex molecular structure in which one or more macromolecules are arranged in an ordered manner. These macromolecules act as biorecognition elements that can selectively bind or otherwise interact with the desired analyte. Therefore, the biorecognition surface is relatively delicate and prone to degradation and damage caused by, for example, mechanical perturbation, desiccation, and radiation exposure. Therefore, methods and materials are needed that can improve sensor stability while maintaining sensor sensitivity. Summary of the Invention [Means for solving the problem]

[0005] Described herein are devices, systems, and methods relating to sensors configured to generate a signal indicative of the concentration of an analyte in a fluid.

[0006] According to one embodiment, the present disclosure further relates to a working electrode including an electrode material, a biorecognition element disposed on the electrode material and configured to selectively and reversibly bind to an analyte in a fluid, a first thiol-based passivation molecule disposed on the electrode material, and a second, different thiol-based passivation molecule disposed on the electrode material. In some embodiments, the first thiol-based passivation molecule is 6-mercapto-1-hexanol, and the second, different thiol-based passivation molecule is 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate. In some embodiments, the distal end of the first thiol-based passivation molecule, the second, different thiol-based passivation molecule, or both, includes one or more of a hydrophilic moiety, a hydrophobic moiety, a charged moiety, and a zwitterionic moiety. In some embodiments, the distal end of the first thiol-based passivation molecule, the second different thiol-based passivation molecule, or both, comprises a zwitterionic moiety, wherein the zwitterionic moiety is a zwitterionic phosphorylcholine head group. In some embodiments, the first thiol-based passivation molecule, the second different thiol-based passivation molecule, or both, is a zwitterionic peptide. In some embodiments, the biorecognition element is an aptamer. In some embodiments, the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array.

[0007] According to one embodiment, the present disclosure further relates to a method of fabricating a working electrode for an analyte sensor configured to generate a signal indicative of the concentration of an analyte in a fluid, the method including: providing a working electrode including an electrode material and a biorecognition element deposited on the electrode material, where the biorecognition element is configured to selectively and reversibly bind to an analyte; applying a first thiol-based passivation molecule to the electrode material for a first predetermined period of time; and applying a second, different thiol-based passivation molecule to the electrode material for a second predetermined period of time. In some embodiments, the first thiol-based passivation molecule is 6-mercapto-1-hexanol (MCH) and the second, different thiol-based passivation molecule is 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate (PC). In some embodiments, applying the first thiol-based passivating molecule and the second thiol-based passivating molecule comprises contacting the electrode material with a solution comprising the first thiol-based passivating molecule and a second, different thiol-based passivating molecule in a ratio of MCH to PC of about 1 mM to about 30 mM MCH to about 2.5 mM to about 30 mM PC.

[0008] According to an embodiment, the present disclosure further relates to a wearable device comprising an electrochemical aptamer-based sensor comprising a first thiol-based passivation molecule and a second, different thiol-based passivation molecule on an electrode material, wherein the first thiol-based passivation molecule comprises 6-mercapto-1-hexanol (MCH) and the second, different thiol-based passivation molecule comprises 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate (PC).

[0009] According to one embodiment, the present disclosure further relates to a sensor configured to generate a signal indicative of an analyte concentration in a fluid, the sensor including a working electrode including an electrode material, a biorecognition layer at least partially disposed on the electrode material and including a biorecognition element that selectively and reversibly binds to the analyte, and an at least partially dried hydrogel disposed on the biorecognition layer. In some embodiments, the hydrogel contains about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% water compared to the hydrogel's fully hydrated state. In some embodiments, the hydrogel is completely dried. In some embodiments, the sensor further includes a microelectrode array, and the working electrode is part of the microelectrode array. In some embodiments, the biorecognition element is an aptamer.

[0010] According to one embodiment, the present disclosure further relates to a method of manufacturing a sensor configured to generate a signal indicative of the concentration of an analyte in a fluid, the method comprising: (a) providing a working electrode including an electrode material and a biorecognition layer at least partially disposed on the electrode material, the biorecognition layer including a biorecognition element that selectively and reversibly binds to an analyte; (b) applying a hydrogel onto the biorecognition layer; and (c) drying the hydrogel to an at least partially dried state. In some embodiments, the hydrogel contains about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% water compared to the hydrogel's fully hydrated state. In some embodiments, the hydrogel is completely dried. In some embodiments, the sensor further includes a microelectrode array, and the working electrode is part of the microelectrode array. In some embodiments, the biorecognition element is an aptamer.

[0011] According to one embodiment, the present disclosure further relates to a sensor including a working electrode comprising an electrode material, a biorecognition layer at least partially disposed on the electrode material and including a biorecognition element that selectively and reversibly binds to an analyte, and a hydrogel disposed on the biorecognition layer, wherein the sensor is sterilized by exposure to radiation, and the sterilized sensor is configured to generate a signal indicative of the concentration of the analyte in a fluid.

[0012] In some embodiments, the radiation is ultraviolet radiation, gamma radiation, X-ray radiation, or electron beam radiation. In some embodiments, the radiation is electron beam radiation. In some embodiments, the biorecognition layer is degraded by about 2% or less, about 5% or less, about 10% or less, about 15% or less, or about 20% or less upon exposure to the radiation. In some embodiments, the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array. In some embodiments, the biorecognition element is an aptamer.

[0013] According to one embodiment, the present disclosure further relates to a method of sterilizing a sensor configured to generate a signal indicative of the concentration of an analyte in a fluid, the method comprising: (a) providing a working electrode including an electrode material and a biorecognition layer including a biorecognition element that selectively and reversibly binds to the analyte, wherein the biorecognition layer is at least partially disposed on the electrode material; (b) applying a hydrogel onto the biorecognition layer; and (c) sterilizing the working electrode by exposure to radiation. In some embodiments, the radiation is ultraviolet radiation, gamma radiation, x-ray radiation, or electron beam radiation. In some embodiments, the radiation is electron beam radiation. In some embodiments, the biorecognition layer is degraded by about 2% or less, about 5% or less, about 10% or less, about 15% or less, or about 20% or less upon exposure to radiation. In some embodiments, the biorecognition element is an aptamer.

[0014] According to one embodiment, the present disclosure further relates to a sensor configured to generate a signal indicative of the concentration of an analyte in a fluid, the sensor comprising: a working electrode comprising an electrode material; a biorecognition layer at least partially disposed on the electrode material and including a biorecognition element that selectively and reversibly binds to the analyte; and a hydrogel disposed on the biorecognition layer, wherein the biorecognition layer degrades at a rate of about 3% or less per day when stored at an ambient temperature of about 15° C. to about 30° C. and an ambient humidity of about 10% to about 80% relative humidity. In some embodiments, the hydrogel comprises about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% water compared to the hydrogel's fully hydrated state. In some embodiments, the hydrogel is completely dried. In some embodiments, the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array. In some embodiments, the biorecognition element is an aptamer.

[0015] According to one embodiment, the present disclosure further relates to a working electrode configured to generate a signal indicative of the concentration of an analyte in a fluid, the working electrode including: an electrode material; a biorecognition element disposed on the electrode material that selectively binds to the analyte, the biorecognition element being functionalized with a redox-active molecule and configured to undergo a conformational change upon binding to the analyte to move the redox-active molecule toward or away from the electrode material; and a passivation element disposed on the electrode material, the passivation element comprising a zwitterionic peptide. In some embodiments, the C-terminus of the C-terminal cysteine ​​is a modified C-terminus having a free carboxyl group or a neutral charge. In some embodiments, the N-terminus of the N-terminal cysteine ​​is a modified N-terminus having a free amine group or a neutral charge. In some embodiments, the zwitterionic peptide consists of the peptide sequence X-(KX)x-PyC or CPy-(KX)xK, where x is 1 to 5, X is a glutamic acid residue or an aspartic acid residue, K is a lysine residue, P is a proline residue, and C is a cysteine ​​residue. In some embodiments, the biorecognition element is an aptamer.

[0016] According to an embodiment, the present disclosure further relates to a working electrode configured to generate a signal indicative of the concentration of an analyte in a fluid, the working electrode comprising: an electrode material; a biorecognition element disposed on the electrode material that selectively binds to the analyte; and a passivation element comprising a zwitterionic peptide disposed on the electrode material, the zwitterionic peptide comprising an amino acid sequence of X-(KX)x-PyC or CPy-(KX)xK, where x is 0, 1, or 2; X is a glutamic acid residue or an aspartic acid residue; K is a lysine residue; P is a proline residue; and C is a cysteine ​​residue.

[0017] In some embodiments, the zwitterionic peptide has an amino acid sequence of X-(KX)x-PyC, including a C-terminal cysteine, where the C-terminus of the C-terminal cysteine ​​is a modified C-terminus with a free carboxyl group or a neutral charge. In some embodiments, the zwitterionic peptide has an amino acid sequence of CPy-(KX)xK, including an N-terminal cysteine, where the N-terminus of the N-terminal cysteine ​​residue is a modified N-terminus with a free amine group or a neutral charge. In some embodiments, the zwitterionic peptide consists of the amino acid sequence of any one of SEQ ID NOs: 2-33. In some embodiments, the biorecognition element is an aptamer.

[0018] According to one embodiment, the present disclosure further relates to a zwitterionic peptide comprising the amino acid sequence X-(KX)x-PyC or CPy-(KX)xK, where x is 0, 1, or 2, X is a glutamic acid residue or an aspartic acid residue, K is a lysine residue, P is a proline residue, and C is a cysteine ​​residue. In some embodiments, the amino acid sequence of the zwitterionic peptide is X-(KX)x-PyC, comprising a C-terminal cysteine, the C-terminus of which is a modified C-terminus with a free carboxyl group or a neutral charge. In some embodiments, the amino acid sequence of the zwitterionic peptide is CPy-(KX)xK, comprising an N-terminal cysteine, the N-terminus of which is a modified N-terminus with a free amine group or a neutral charge. In some embodiments, the zwitterionic peptide consists of the amino acid sequence of any one of SEQ ID NOs: 2-33. In some embodiments, the biorecognition element is an aptamer.

[0019] According to one embodiment, the present disclosure further relates to a method for fabricating a working electrode, the method comprising: depositing gold on a substrate to create a gold surface; depositing a biorecognition element on the gold surface, the biorecognition element being functionalized with a redox-active molecule and configured to undergo a conformational change upon binding to an analyte to move the redox-active molecule toward or away from the gold surface; and depositing a passivation element comprising a zwitterionic peptide on the gold surface.

[0020] Both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. Additional features and / or variations may be provided in addition to those described herein. For example, implementations described herein may be directed to various combinations and subcombinations of the disclosed features. [Brief explanation of the drawings]

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain aspects of the subject matter disclosed herein and, together with the description, serve to explain some of the principles associated with the disclosed implementations.

[0022] [Figure 1] 1 shows an exemplary schematic diagram of an analyte monitoring system having a microneedle array.

[0023] [Figure 2A] 1 shows an exemplary schematic diagram of an analyte monitoring device.

[0024] [Figure 2B] 1 shows an exemplary schematic of microneedle insertion depth in an analyte monitoring device.

[0025] [Figure 3A] 1A-1C show top perspective, side, bottom, and exploded views, respectively, of an analyte monitoring device. [Figure 3B] 1A-1C show top perspective, side, bottom, and exploded views, respectively, of an analyte monitoring device. [Figure 3C] 1A-1C show top perspective, side, bottom, and exploded views, respectively, of an analyte monitoring device. [Figure 3D] 1A-1C show top perspective, side, bottom, and exploded views, respectively, of an analyte monitoring device.

[0026] [Figure 4A] 1A-1C show exploded perspective, exploded side, bottom perspective, side, and top perspective views, respectively, of a sensor assembly in an analyte monitoring device. [Figure 4B] 1A-1C show exploded perspective, exploded side, bottom perspective, side, and top perspective views, respectively, of a sensor assembly in an analyte monitoring device. [Figure 4C] 1A-1C show exploded perspective, exploded side, bottom perspective, side, and top perspective views, respectively, of a sensor assembly in an analyte monitoring device. [Figure 4D] 1A-1C show exploded perspective, exploded side, bottom perspective, side, and top perspective views, respectively, of a sensor assembly in an analyte monitoring device. [Figure 4E] 1A-1C show exploded perspective, exploded side, bottom perspective, side, and top perspective views, respectively, of a sensor assembly in an analyte monitoring device.

[0027] [Figure 4F] 1A-1C show exploded perspective, exploded side, and side views, respectively, of a sensor assembly in an analyte monitoring device. [Figure 4G] 1A-1C show exploded perspective, exploded side, and side views, respectively, of a sensor assembly in an analyte monitoring device. [Figure 4H] 1A-1C show exploded perspective, exploded side, and side views, respectively, of a sensor assembly in an analyte monitoring device.

[0028] [Figure 5A] 1 shows an exemplary schematic diagram of a microneedle array. [Figure 5B] 5B shows an exemplary schematic diagram of a microneedle of the microneedle array shown in FIG. 5A.

[0029] [Figure 6] FIG. 1 shows an exemplary schematic diagram of a microneedle array used to sense multiple analytes, at least one of the analytes being cortisol.

[0030] [Figure 7A] FIG. 1 shows a cross-sectional side view of a pillar-shaped microneedle with a tapered distal end. [Figure 7B] 7B and 7C are images showing a perspective view and a detailed view, respectively, of one embodiment of the microneedle shown in FIG. 7A. [Figure 7C] 7B and 7C are images showing a perspective view and a detailed view, respectively, of one embodiment of the microneedle shown in FIG. 7A.

[0031] [Figure 8] FIG. 1 shows an exemplary schematic diagram of a pillar-shaped microneedle with a tapered distal end.

[0032] [Figure 9] FIG. 1 shows a cross-sectional side view of a pillar-shaped microneedle with a tapered distal end.

[0033] [Figure 10A] 1A and 1B show exemplary schematic diagrams of a microneedle array and a microneedle, respectively. [Figure 10B] 1A and 1B show exemplary schematic diagrams of a microneedle array and a microneedle, respectively. [Figure 10C] 1 shows a detailed partial view of an exemplary variation of a microneedle. [Figure 10D] 1 shows a detailed partial view of an exemplary variation of a microneedle. [Figure 10E] 1 shows a detailed partial view of an exemplary variation of a microneedle. [Figure 10F] 1 shows a detailed partial view of an exemplary variation of a microneedle.

[0034] [Figure 11A] 1 illustrates exemplary variations of microneedles. [Figure 11B] 1 illustrates exemplary variations of microneedles.

[0035] [Figure 12A] 1 shows an exemplary schematic diagram of a microneedle array configuration. [Figure 12B] 1 shows an exemplary schematic diagram of a microneedle array configuration. [Figure 12C] 1 shows an exemplary schematic diagram of a microneedle array configuration. [Figure 12D] 1 shows an exemplary schematic diagram of a microneedle array configuration.

[0036] [Figure 13A]1A and 1B show perspective and orthogonal views, respectively, of an exemplary variation of a die containing a microneedle array. [Figure 13B] 1A and 1B show perspective and orthogonal views, respectively, of an exemplary variation of a die containing a microneedle array.

[0037] [Figure 14A] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14B] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14C] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14D] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14E] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14F] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14G] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14H] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14I] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations. [Figure 14J] 1A-1D show exemplary schematic diagrams of different variations of microneedle array configurations.

[0038] [Figure 15] 1 shows an exemplary schematic diagram of a planar microelectrode array.

[0039] [Figure 16A] 1A and 1B show exemplary schematic diagrams of the layered structure of the working, counter, and reference electrodes, respectively. [Figure 16B] 1A and 1B show exemplary schematic diagrams of the layered structure of the working electrode, counter electrode, and reference electrode, respectively. [Figure 16C] 1A and 1B show exemplary schematic diagrams of the layered structure of the working electrode, counter electrode, and reference electrode, respectively.

[0040] [Figure 16D] 1A and 1B show exemplary schematic diagrams of the layered structure of the working electrode, counter electrode, and reference electrode, respectively. [Figure 16E] 1A and 1B show exemplary schematic diagrams of the layered structure of the working electrode, counter electrode, and reference electrode, respectively. [Figure 16F] 1A and 1B show exemplary schematic diagrams of the layered structure of the working electrode, counter electrode, and reference electrode, respectively.

[0041] [Figure 16G] 1A and 1B show exemplary schematic diagrams of the layered structure of the working electrode, counter electrode, and reference electrode, respectively. [Figure 16H] 1A and 1B show exemplary schematic diagrams of the layered structure of the working electrode, counter electrode, and reference electrode, respectively. [Figure 16I] 1A and 1B show exemplary schematic diagrams of the layered structure of the working electrode, counter electrode, and reference electrode, respectively.

[0042] [Figure 16J] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer.

[0043] [Figure 16K] 16A shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer as shown in FIG. 16J and a biocompatible layer is applied over the biorecognition layer.

[0044] [Figure 16L] 16A-16J show exemplary schematic diagrams of layered structures of working electrodes in which the biorecognition element is an analyte-binding aptamer as shown in FIG. 16J, and the biorecognition layer includes a multicomponent passivation element.

[0045] [Figure 16M]16A-16J show exemplary schematic diagrams of layered structures of working electrodes in which the biorecognition element is an analyte-binding aptamer as shown in FIG. 16J and the biorecognition layer includes a multicomponent passivation element.

[0046] [Figure 17A] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17B] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17C] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17D] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17E] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17F] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17G] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17H] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17I] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17J] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer. [Figure 17K] 1 shows an exemplary schematic diagram of the layered structure of a working electrode in which the biorecognition element is an analyte-binding aptamer.

[0047] [Figure 18A] FIG. 1 is a flow diagram of an embodiment of a method for applying a passivation element to a working electrode as part of a biorecognition layer. [Figure 18B]FIG. 1 is a flow diagram of an embodiment of a method for applying a passivation element to a working electrode as part of a biorecognition layer.

[0048] [Figure 19A] 1 shows an exemplary schematic diagram of a housing of an analyte monitoring device including a user interface with an indicator light element. [Figure 19B] 1 shows an exemplary schematic diagram of a housing of an analyte monitoring device including a user interface with an indicator light element.

[0049] [Figure 20A] 1 shows an exemplary schematic diagram of an illumination mode in an analyte monitoring device for displaying analyte measurement data. [Figure 20B] 1 shows an exemplary schematic diagram of an illumination mode in an analyte monitoring device for displaying analyte measurement data. [Figure 20C] 1 shows an exemplary schematic diagram of an illumination mode in an analyte monitoring device for displaying analyte measurement data.

[0050] [Figure 21A] 21A and 21B show exemplary schematics of an exemplary working electrode functionalized with a cortisol-binding aptamer before (FIG. 21A) and after (FIG. 21B) application of a hydrogel biocompatible layer. [Figure 21B] 21A and 21B show exemplary schematics of an exemplary working electrode functionalized with a cortisol-binding aptamer before (FIG. 21A) and after (FIG. 21B) application of a hydrogel biocompatible layer.

[0051] [Figure 21C] FIG. 21C shows an exemplary schematic diagram of an exemplary microelectrode array including the working electrode shown in FIG. 21B.

[0052] [Figure 22A] 10 shows plots illustrating cortisol detection using a working electrode covered with a hydrogel containing a cortisol-binding aptamer before and after drying overnight.

[0053] [Figure 22B] 10 shows plots showing cortisol detection using a bare (not hydrogel-covered) working electrode containing the cortisol-binding aptamer before and after drying overnight.

[0054] [Figure 23A] 1 shows plots showing cortisol detection using a working electrode covered with a hydrogel containing a cortisol-binding aptamer before and after 2.5 days of drying, where the electrode is encapsulated with a hydrogel biocompatible layer.

[0055] [Figure 23B] 10 shows plots showing cortisol detection using a bare (not hydrogel-covered) working electrode containing the cortisol-binding aptamer before and after 2.5 days of drying.

[0056] [Figure 24A] 10 shows plots illustrating cortisol detection using a working electrode covered with a hydrogel containing a cortisol-binding aptamer before and after drying for 2.5 days.

[0057] [Figure 24B] 10 shows plots showing cortisol detection using a bare (not hydrogel-covered) working electrode containing the cortisol-binding aptamer before and after 2.5 days of drying.

[0058] [Figure 24C] 10 shows plots showing initial cortisol detection using a bare working electrode containing the cortisol-binding aptamer or a working electrode covered with hydrogel as soon as fabricated.

[0059] [Figure 24D] 10 shows plots showing cortisol detection using a bare working electrode or a working electrode covered with a hydrogel containing a cortisol-binding aptamer after drying for 2.5 days.

[0060] [Figure 25A]10 shows plots illustrating cortisol detection using a working electrode covered with a hydrogel containing a cortisol-binding aptamer before and after e-beam sterilization.

[0061] [Figure 25B] 1 shows plots showing cortisol detection using a bare (not hydrogel-covered) working electrode containing the cortisol-binding aptamer before and after e-beam sterilization.

[0062] [Figure 25C] 10 shows plots showing cortisol detection using a bare working electrode or a hydrogel-covered working electrode containing a cortisol-binding aptamer after e-beam sterilization.

[0063] [Figure 26] 1 shows exemplary zwitterionic peptides for use in the sensors described herein.

[0064] [Figure 27] 27 shows plots illustrating cortisol detection in a control sensor using 6-mercaptohexanol as the passivating element and an experimental cortisol sensor using the zwitterionic peptide shown in FIG. 26 as the passivating element.

[0065] [Figure 28] 1 shows plots of E-AB sensor stability over time using various multi-component passivation elements. [Figure 29] 1 shows plots of E-AB sensor stability over time using various multi-component passivation elements. [Figure 30] 1 shows plots of E-AB sensor stability over time using various multi-component passivation elements.

[0066] [Figure 31] 1 shows a plot of peak current measured for E-AB sensors at different frequencies, each having a different multi-component passivation element. [Figure 33] 1 shows a plot of peak current measured for E-AB sensors at different frequencies, each having a different multi-component passivation element.

[0067] [Figure 32] 1 shows a plot of measured gain for E-AB sensors at different frequencies, each having a different multi-component passivation element. [Figure 34] 1 shows a plot of measured gain for E-AB sensors at different frequencies, each having a different multi-component passivation element.

[0068] [Figure 35] 1 shows a plot of noise levels for E-AB sensors at different frequencies, each having a different multi-component passivation element.

[0069] [Figure 36] 1 shows a scanning electron micrograph of an exemplary biocompatible layer deposited on a microneedle array. DETAILED DESCRIPTION OF THE INVENTION

[0070] Non-limiting examples of various aspects and variations of the present invention are described herein and illustrated in the accompanying drawings.

[0071] As used herein, the term "a" or "an" can refer to one or more of that entity, i.e., it can refer to a plurality of referents. Similarly, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein. In addition, reference to an "element" by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be only one element.

[0072] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method employed to determine the value or the variation that exists between samples measured. Unless otherwise stated or clear from the context, the term "about" means within 10% above or below the reported numerical value (except where such numerical value would be greater than 100% or less than 0% of the possible values). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or each of the values ​​recited in the series of ranges, unless otherwise indicated. As used herein, the terms "about" and "approximately" are used interchangeably.

[0073] Aptamers are single-stranded oligonucleotides or peptides that fold into a predetermined structure to selectively bind to a specific analyte (which may be referred to as a target), which may be, for example, a protein, peptide, hormone, nucleic acid, or small molecule. Aptamers with affinity for a desired target can be conventionally selected from large oligonucleotide libraries by a process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Through an iterative process, non-binding aptamers are discarded, and aptamers that bind to the proposed target are amplified by polymerase chain reaction (PCR). The iterative process may include counterselection (using interferents and structurally similar molecules) to discard aptamers with insufficient selectivity for the analyte. Furthermore, conformational changes in aptamers resulting from target binding and dissociation can be used to induce electrical, electrochemical, or chemical changes that can be utilized to visualize target binding / dissociation via assays or sensors. If necessary, selected aptamers can be further modified (e.g., by truncation and mutation) to improve the conformational changes of the aptamer and thereby improve the sensor signal. These properties make aptamers attractive "bio-recognition" elements for use in detecting one or more desired analytes.

[0074] Since the development of continuous glucose monitors, electrochemical biosensors have attracted widespread attention due to their potential value in biomedical applications. While glucose monitors achieve selectivity through the surface-immobilized enzyme glucose oxidase, another class of electrochemical biosensors uses nucleic acids instead of enzymes. These nucleic acid-based electrochemical sensors (NBEs) rely on the affinity of surface-bound oligonucleotides for specific target molecules. Upon target binding, a signal is generated via a change in the electron transfer rate of either a covalently bound or solvated redox reporter. For example, in NBEs, such as electrochemical aptamer-based sensors, the surface of the working electrode can be functionalized with an aptamer (e.g., an analyte-binding aptamer) configured to selectively and reversibly bind to a given analyte. The aptamer can then be modified by the addition of a redox-active molecule. The aptamer can be configured such that upon binding to the analyte, the analyte-binding aptamer undergoes a conformational change that either moves the redox-active molecule closer to or further away from the electrode. The movement of redox-active molecules can be detected as an analyte concentration-dependent electrochemical signal. Because NBEs rely on affinity instead of target reactivity, they can be developed for many molecules of interest: from complementary nucleic acids to small molecule drugs, protein biomarkers, and even whole viruses or cells. However, NBEs have limitations that prevent their translation into clinically valuable platforms such as wearable analyte monitors.

[0075] One such limitation of NBEs is their lack of compatibility with long-term storage. NBEs rapidly degrade when stored dry. Drying out of NBEs can cause damage to the sensing structure itself and / or the chemicals linking the sensing chemicals to the electrodes. While hydrating the sensing surface could improve the ability to preserve the sensing chemicals, this is not feasible for a variety of reasons, including, for example, packaging and shipping.

[0076] Thus, described herein is a biocompatible layer comprising a hydrogel that can be added to an NBE platform and provide a means of preserving the biorecognition element (i.e., sensing chemical) even when dried. The hydrogel biocompatible layer can allow the NBE platform to be shipped, packaged, and / or stored safely and stably. When compared to a control sensor that does not include a hydrogel biocompatible layer, a sensor comprising the hydrogel biocompatible layer described herein can experience zero loss of sensor signal when dried overnight. Furthermore, the hydrogel biocompatible layer can also protect the biorecognition element from e-beam sterilization.

[0077] Another such limitation of NBEs is their lack of stability over time. For example, while NBEs are relatively stable in blood and serum in vitro, they exhibit significant drift when deployed directly in vivo. This problem likely arises, at least in part, due to degradation of the target-recognizing sensing chemicals and nonspecific adsorption of cells and other components to the sensor surface. Nonspecific adsorption, sometimes referred to as fouling, can increase undesirable background current. When sensors are exposed to blood or serum, proteins from these fluids deposit on the surface in a monolayer. These proteins, forming a fouling layer, simultaneously restrict the conformational dynamics of nucleic acids bound to the electrode and reduce the efficiency of electron transfer between the electrode and the redox reporter. Fouling-induced disruption of conformational dynamics reduces the total signal gain possible from target binding, while impaired electron transfer reduces the total signal output regardless of target concentration. Collectively, these effects limit the signaling precision and signaling lifetime of NBEs.

[0078] To address these concerns, mathematical correction and / or sensor surface modification have been used. Surface modification, as an example, involves approaches to passivate the sensor surface. For sensors containing gold electrodes, this involves utilizing thiol chemistry on gold self-assembly to form a monolayer containing short-chain alkyl thiols for electrode surface passivation. With particular reference to NBE sensors and further discussed above, a typical E-AB sensor architecture contains sensing chemistry on a portion of the electrode surface, while the remainder of the electrode surface is also covered with thiol-linked small molecule blocking groups. In this way, the small molecule blocking groups function as passivation elements on the sensor surface. To date, only a single type of small molecule thiol has been explored. However, such approaches are often insufficient because the passivation element must simultaneously achieve multiple, sometimes competing, goals.

[0079] For example, the passivating element must have sufficient intermolecular interactions with itself (meaning molecules of the same type) to form a densely packed layer on the gold surface, thereby providing good passivation. Good passivation is key to good resolution of MB peaks (resulting in more accurate measurements) and long-term stability. At the same time, the passivating element must not be so well passivated that it blocks electron transfer from the redox probe. The longer each passivating molecule is (e.g., 6 carbons vs. 10 carbons vs. 16 carbons), the stronger its intermolecular attraction and the more densely packed it is, resulting in better passivation and long-term stability, but the electron transport from the redox probe becomes less efficient. This is because electrons must pass through the passivating element to reach the electrode surface, and as the thickness of the passivating element increases, the possibility of electron transport from the redox reporter decreases. Therefore, the measured current decreases across the homologous series. Thus, thick layers have very little (or no) signal from the MBs and a low signal-to-noise ratio (SNR). Conversely, thin layers have a strong signal from the MBs but are poorly passivated and therefore less stable.

[0080] In another example, the passivation element must be compatible with the aptamer and redox probe so as not to interfere with or prevent binding to the target analyte or prevent conformational switching of the aptamer. For example, the use of a passivation element terminated with a hydrophobic moiety can reduce signal transmission because the aptamer and / or redox probe may interact with the hydrophobic passivation element, becoming "attached" to its surface or partially embedded within it. Either interaction prevents the conformational switching mechanism (similar to the irreversible denaturation observed in proteins). A functional "blocking" layer can be achieved by using a passivation element terminated with a hydrophilic moiety (i.e., a polar or charged moiety), which may be a polar hydroxyl group, a charged sulfonate, a carboxylate, a multivalent group, or a zwitterionic group (i.e., equal positive and negative charges). However, the hydrophilic head group can allow greater ingress of aqueous electrolyte into the passivation element. Therefore, if the passivation element is a relatively thin (e.g., six-carbon) thiol layer, it may be prone to degradation.

[0081] Given the above, the challenge of balancing thickness, packing density, and hydrophilicity of a passivation element is easily understood. An ideal passivation element would provide additional benefits, such as preventing biofouling and providing covalent attachment points (e.g., vinyl, epoxide, acrylate, methacrylate, benzophenone, azide) for subsequent biocompatible protective layers, thereby improving the overall mechanical integrity of the analyte sensor. However, there are limits to what a single small molecule thiol can achieve. In fact, it is unlikely that a single molecule can meet each of the above requirements. Furthermore, there are limits to the types of thiol molecules that are commercially available or can be easily synthesized by chemists. While there may be many theoretical thiols that could function as blocking groups, there are technical and practical barriers to discovering and utilizing them. Therefore, described herein is a multi-component passivation element that simultaneously provides improvements in thickness, packing density, and hydrophilicity. Generally, a multi-component passivation element can include a thiol mixture containing two or more thiols forming the multi-component passivation element, each thiol providing its own advantages while minimizing the disadvantages of the other thiols. In this manner, the E-AB sensors described herein can exhibit improved fidelity, improved long-term stability, improved biocompatibility, reduced biofouling, and / or improved structural integrity.

[0082] Specifically, the multi-component passivation elements described herein improve stability by suppressing the oxygen reduction reaction at the electrode surface (thereby reducing sensor degradation), suppressing electrode capacitance, reducing variability in sensor response to target molecules (e.g., improving manufacturing batch yield and / or reducing sensor degradation), and / or reducing variability in detection limits for target molecules. Additionally, the multi-component passivation elements reduce sensor degradation by increasing baseline current and shifting redox probe peak current values ​​when measured against a baseline.

[0083] In addition to, or instead of, the multi-component passivation elements described herein, the passivation elements described herein may include zwitterions. The zwitterions may, in some cases, be peptides, in which case they may be referred to as "zwitterionic peptides." As described herein, using zwitterionic peptides as components in the passivation elements in the sensors described herein may provide several advantages, including, but not limited to: (1) improved hydrophilicity of the electrode surface; (2) better blocking of nonspecific adsorption of proteins on the electrode surface in vivo; (3) reduced need for biologically incompatible reagents in the manufacturing process; (4) odorlessness, thereby eliminating the need for fume hoods during the manufacturing process; and (5) reduced toxicity of electrodes functionalized therewith when applied to or inserted within a subject.

[0084] In some embodiments, the multi-component passivation elements described herein can include both small molecule thiols and zwitterions, such as a mixture of small molecule thiols and zwitterions. The small molecule thiols can include one or more different small molecule thiols described herein. Similarly, the zwitterions can include one or more different zwitterions described herein. Thus, the multi-component passivation elements can include one or more small molecule thiols and one or more zwitterions in any combination, as described in more detail herein.

[0085] The following are analyte monitoring systems that may utilize a hydrogel biocompatible layer and a multi-component passivation element. In some variations, the hydrogel additive and passivation element may be utilized in conjunction with an analyte monitoring system, which may include a microneedle array. The following description is intended to be exemplary, and aspects related to aptamer-based approaches for measuring and monitoring analytes consistent with the present subject matter are not limited to the exemplary analyte monitoring devices and exemplary microneedle arrays described herein. Analyte Monitoring Systems

[0086] As generally described herein, an analyte monitoring system can include an analyte monitoring device worn by a user and including one or more sensors for monitoring an analyte in the user. The sensor can include, for example, one or more electrodes configured to perform electrochemical detection of the analyte. The analyte monitoring device can communicate sensor data to an external computing device for storage, display, and / or analysis of the sensor data.

[0087] For example, as shown in FIG. 1 , analyte monitoring system 100 may include an analyte monitoring device 110 worn by a user, which may be a continuous analyte monitoring device. Analyte monitoring device 110 may include, for example, a microneedle array including at least one electrochemical sensor for detecting and / or measuring an analyte in the user's bodily fluid. In some variations, the analyte monitoring device may be applied to the user using a suitable applicator 160 or may be applied manually. Analyte monitoring device 110 may include one or more processors for performing analysis of the sensor data and / or a communications module (e.g., a wireless communications module) configured to communicate the sensor data to mobile computing device 102 (e.g., a smartphone) or another suitable computing device. In some variations, mobile computing device 102 may include one or more processors for executing a mobile application to process the sensor data (e.g., display the data, analyze the data for trends, etc.) and / or provide suitable alerts or other notifications related to the sensor data and / or its analysis. It should be understood that, in some variations, the mobile computing device 102 may perform the sensor data analysis locally, while other computing devices may alternatively or additionally analyze the sensor data and / or communicate information related to such analysis to the mobile computing device 102 (or other suitable user interface) for display to a user. Further, in some variations, the mobile computing device 102 may be configured to communicate the sensor data and / or analysis of the sensor data to one or more storage devices 106 (e.g., a server) via the network 104 for archiving data and / or other suitable information related to a user of the analyte monitoring device.

[0088] The analyte monitoring devices described herein have features that improve several advantageous properties of continuous analyte monitoring devices, such as reducing sensor degradation and / or damage over time and improving stability, helping to minimize changes in sensor response throughout storage and operation of the analyte monitoring device.

[0089] Various aspects of exemplary variations of the analyte monitoring system and methods of use are described in further detail below. Analyte Monitoring Devices

[0090] As shown in FIG. 2A , in some variations, the analyte monitoring device 110 may generally include a housing 112 and a microneedle array 140 extending outwardly from the housing. The housing 112 may be a wearable housing configured to be attached to a user's skin, such that the microneedle array 140 extends at least partially into the user's skin. For example, the housing 112 may include an adhesive such that the analyte monitoring device 110 is a simple and easy skin-adhesive patch for application to a user. The microneedle array 140 may include one or more electrochemical sensors (e.g., electrodes) configured to pierce the user's skin and measure analytes accessible after the microneedle array 140 pierces the user's skin. In some variations, the analyte monitoring device 110 may be integrated or self-contained as a single unit, and the unit may be disposable (e.g., used for a period of time and replaced with another instance of the analyte monitoring device 110).

[0091] The electronics system 120 is disposed at least partially within the housing 112 and can include various electronic components, such as a sensor circuit 124 configured to perform signal processing (e.g., biasing and readout of an electrochemical sensor, converting analog signals from the electrochemical sensor to a digital signal, etc.). The electronics system 120 can also include at least one microcontroller 122 for controlling the analyte monitoring device 110, at least one communications module 126, at least one power source 130, and / or various other suitable passive circuitry 127. The microcontroller 122 can be configured, for example, to interpret digital signals output from the sensor circuit 124 (e.g., by executing routines programmed in firmware), perform various suitable algorithms or mathematical transformations (e.g., calibration, etc.), and / or route processed data to or from the communications module 126. In some variations, the communications module 126 can include a suitable wireless transceiver (e.g., a Bluetooth transceiver, etc.) for communicating data with the external computing device 102 via one or more antennas 128. For example, the communications module 126 may be configured to provide unidirectional and / or bidirectional communication of data with an external computing device 102 paired with the analyte monitoring device 110. The power source 130 may provide power to the analyte monitoring device 110, such as for the electronics system. The power source 130 may include a battery or other suitable power source and, in some variations, may be rechargeable and / or replaceable. The passive circuitry 127 may include various non-powered electrical circuits (e.g., resistors, capacitors, inductors, etc.) that provide interconnections between other electronic components, etc. The passive circuitry 127 may be configured to perform, for example, noise reduction, biasing, and / or other purposes. In some variations, the electronic components within the electronics system 120 may be disposed on one or more printed circuit boards (PCBs), which may be, for example, rigid, semi-rigid, or flexible. Further details of the electronics system 120 are described further below.

[0092] In some variations, the analyte monitoring device 110 may further include one or more additional sensors 150 to provide additional information that may be relevant to user monitoring. For example, the analyte monitoring device 110 may further include at least one temperature sensor (e.g., a thermistor) configured to measure skin temperature, thereby enabling temperature compensation of sensor measurements obtained by the microneedle array electrochemical sensor.

[0093] In some variations, the microneedle array 140 of the analyte monitoring device 110 may be configured to pierce a user's skin. As shown in FIG. 2B, when the device 110 is worn by a user, the microneedle array 140 may extend into the user's skin such that electrodes on distal regions of the microneedles reside in the dermis. Specifically, in some variations, the microneedles may be designed to penetrate the skin and access upper skin regions of the skin (e.g., the papillary dermis and upper reticular dermis) to allow the electrodes to access the interstitial fluid surrounding the cells of these layers. For example, in some variations, the microneedles may generally have a height ranging from at least 350 μm to about 515 μm. In some variations, one or more microneedles may extend from the housing such that the distal ends of the electrodes on the microneedles are positioned less than about 5 mm from the skin-contacting surface of the housing, less than about 4 mm from the housing, less than about 3 mm from the housing, less than about 2 mm from the housing, or less than about 1 mm from the housing.

[0094] In contrast to conventional continuous monitoring devices that typically include sensors implanted about 8 mm to about 10 mm below the skin surface in the subcutaneous tissue or adipose layer of the skin, the analyte monitoring device 110 has a shallower microneedle insertion depth of about 0.25 mm (so that the electrodes are implanted in the upper cutaneous region of the skin), which provides many advantages, including access to the skin interstitial fluid, which contains many potential biomolecules that can serve as analytes for detection.

[0095] Furthermore, when the microneedle array is in the upper skin region, the lower dermis below the microneedle array contains very high levels of vascularization and perfusion to support dermal metabolism, which allows for thermoregulation (via vasoconstriction and / or vasodilation) and provides a barrier function that helps stabilize the sensing environment around the microneedles. Yet another advantage of a shallower insertion depth is that the upper dermal layers lack pain receptors, thereby reducing the sensation of pain when the microneedle array pierces the user's skin, providing a more comfortable and less invasive user experience.

[0096] Furthermore, the shallower microneedle insertion depth of the analyte monitoring device 110 results in a reduced time delay in analyte detection compared to conventional continuous analyte monitoring devices. Such shallower insertion depth positions the sensor surface in close proximity (e.g., within a few hundred micrometers or less) to the dense, well-perfused capillary bed of the reticular dermis, resulting in negligible diffusion lag from the capillaries to the sensor surface. The diffusion time is t=x 2The diffusion delay is related to the diffusion distance according to ∂t / (2D), where t is the diffusion time, x is the diffusion distance, and D is the mass diffusivity of the analyte of interest. Thus, positioning the analyte sensing element twice as far from the analyte source in the capillary increases the diffusion delay time by four times. Thus, conventional analyte sensors (located in the poorly vascularized adipose tissue below the dermis) experience significantly longer diffusion distances from the vasculature in the dermis, resulting in substantial diffusion latencies (e.g., typically 5-20 minutes). In contrast, the shallower microneedle insertion depth of the analyte monitoring device 110 benefits from low diffusion latencies from the capillaries to the sensor, thereby reducing the time delay in analyte detection and providing more accurate results in real time or near real time. For example, in some embodiments, the diffusion latency can be less than 10 minutes, less than 5 minutes, or less than 3 minutes.

[0097] Furthermore, when the microneedle array is in the upper skin region, the lower dermis below the microneedle array contains very high levels of vascularization and perfusion to support dermal metabolism, which allows for thermoregulation (via vasoconstriction and / or vasodilation) and provides a barrier function that helps stabilize the sensing environment around the microneedles. Yet another advantage of a shallower insertion depth is that the upper dermal layers lack pain receptors, thereby reducing the sensation of pain when the microneedle array pierces the user's skin, providing a more comfortable and less invasive user experience.

[0098] Thus, the analyte monitoring devices and methods described herein enable improved continuous monitoring of a user's analytes. For example, as described above, the analyte monitoring devices can be simple and easy to apply, thereby improving ease of use and user compliance. Furthermore, analyte measurement in skin interstitial fluid can provide highly accurate analyte detection. Furthermore, compared to conventional continuous analyte monitoring devices, insertion of the microneedle array and its sensors can be less invasive and less painful for the user. Additional advantages of other aspects of the analyte monitoring devices and methods are further described below. housing

[0099] As described above, the analyte monitoring device may include a housing. The housing may at least partially enclose or surround other components (e.g., electronic components) of the analyte monitoring device, such as for protection of such components. For example, the housing may be configured to help prevent dust and moisture from entering the analyte monitoring device. In some variations, an adhesive layer may attach the housing to a surface (e.g., skin) of a user while allowing the microneedle array to extend outward from the housing into the user's skin. Furthermore, in some variations, the housing may generally include rounded edges or corners and / or may be low-profile so as to be atraumatic and reduce interference with clothing worn by the user, etc.

[0100] Figures 3A-3D illustrate an embodiment of analyte monitoring device 110. Figures 3A-3D show a top perspective view, a side view, a bottom view, and an exploded view of analyte monitoring device 110, respectively.

[0101] The analyte monitoring device 110 may include a housing that at least partially surrounds or encloses other components (e.g., electronic components) of the analyte monitoring device 110, such as to protect such components. For example, the housing may be configured to help prevent dust and moisture from entering the analyte monitoring device 110. In some variations, an adhesive layer may attach the housing to a surface (e.g., skin) of a user while allowing the microneedle array 140 to extend outward from the housing into the user's skin. Furthermore, in some variations, the housing may generally include rounded edges or corners and / or may be low-profile to reduce interference with clothing worn by the user, etc.

[0102] 3A-3D, an exemplary variation of analyte monitoring device 110 may include a housing cover 320 and a base plate 330 configured to at least partially enclose the internal components of analyte monitoring device 110. For example, housing cover 320 and base plate 330 may provide an enclosure for a sensor assembly 350 including a microneedle array 140 and electronic components. When assembled, microneedle array 140 extends outward from a portion of base plate 330 in a skin-facing direction (e.g., the underside) of analyte monitoring device 110.

[0103] The housing cover 320 and the base plate 330 may include one or more rigid or semi-rigid protective shell components that can be coupled to one another via, for example, suitable fasteners (e.g., mechanical fasteners), mechanical interlocking or mating features, and / or engineered fits. The housing cover 320 and the base plate 330 may include rounded edges and corners and / or other atraumatic features. When coupled to one another, the housing cover 320 and the base plate 330 may form an interior volume that houses internal components, such as the sensor assembly 350. For example, the internal components disposed within the interior volume may be arranged in a compact, low-profile stack as the sensor assembly 350.

[0104] Analyte monitoring device 110 may include one or more adhesive layers for attaching analyte monitoring device 110 (e.g., housing cover 320 and base plate 330 coupled together) to a surface (e.g., skin) of a user. As shown in FIG. 3D , the one or more adhesive layers may include inner adhesive layer 342 and outer adhesive layer 344. Inner adhesive layer 342 may adhere to base plate 330, and outer adhesive layer 344 may adhere to inner adhesive layer 342 and provide adhesive on its outward-facing side for (e.g., temporary) adhesion to the user's skin. Inner adhesive layer 342 and outer adhesive layer 344 together act as a double-sided adhesive for adhering analyte monitoring device 110 to the user's skin. Outer adhesive layer 344 may be protected by a release liner that the user removes to expose the adhesive prior to application to the skin. In some variations, a single adhesive layer is provided. In some variations, the outer adhesive layer 344, the inner adhesive layer 342, and / or the single adhesive layer may have an outer periphery that extends farther than the outer periphery or perimeter of the housing cover 320 and the base plate 330. This may increase the surface area for attachment and increase the stability of the retention or attachment to the user's skin. The inner adhesive layer 342, the outer adhesive layer 344, and / or the single adhesive layer each have an opening that allows the outwardly extending microneedle array 140 to pass through, as described further below. The openings in the inner adhesive layer 342 and the outer adhesive layer 344 may generally be aligned with one another, but in some variations, they may be different sizes, with one opening being smaller than the other. In some variations, the openings are substantially the same size.

[0105] Base plate 330 has a first surface (e.g., an outer exposed surface) opposite a second surface and functions as a support and / or connecting structure as well as a protective cover for sensor assembly 350. Base plate 330 is sized and shaped to attach to housing cover 320. Base plate 330 may be shaped to fit securely within housing cover 320 such that outer edges of base plate 330 align with corresponding edges of the opening in housing 320. This alignment may be such that there is no gap between the outer edges of base plate 330 and the corresponding edges of the opening in housing cover 320.

[0106] A connecting member 332 may be formed in a central or near-central region of the first surface of the base plate 330. The connecting member 332 is a protrusion (e.g., a protruding hub) having a sidewall extending from the first surface of the base plate 330 and a first surface substantially parallel to the first surface of the base plate 330. The sidewall extends from an edge of the first surface of the connecting member 332 to the first surface of the base plate 330. The remainder of the first surface of the base plate 330 surrounding the connecting member 332 may be flat or substantially flat. One or more connector features 336 extend outward from the sidewall of the connecting member 332 to releasably engage with corresponding connectors of a microneedle enclosure, as described further below. The first surface and sidewall of the connecting member 332 partially define a cavity. The cavity may be further defined by a portion of the base plate 330 adjacent to (e.g., below) the connecting member 332. The cavity has an opening on the second surface of the base plate 330 and is accessible. An aperture 334 is formed through the first surface of the connecting member 332. The aperture 334 can be sized and shaped such that the microneedle array 140 fits securely within and extends through the aperture 334. For example, the sidewalls of the microneedle array 140 can align with the corresponding sidewalls of the aperture 334. In some variations, the aperture 334 can be sized and shaped to correspond to the area surrounding the microneedle array 140. The openings in the inner adhesive layer 342 and the outer adhesive layer 344 (or a single adhesive layer) are sized such that the connecting member 332 extends through the opening without interfering with the adhesive layers. For example, the diameter of the opening in the inner adhesive layer 342 and the diameter of the opening in the outer adhesive layer 344 are larger than those of the connecting member 332. In some variations, the opening in the inner adhesive layer 342 and / or the opening in the outer adhesive layer 344 (or the opening in the single adhesive layer) is adjacent to a sidewall of the connecting member 332 with clearance to accommodate one or more connector features 336. In some variations, one or more slits or notches may be formed in the inner adhesive layer 342, the outer adhesive layer 344, and / or the single adhesive layer, extending from the opening to aid in placement of the respective adhesive layer.

[0107] Although the housing cover 320 and base plate 330 shown in FIGS. 3A-3D are substantially circular, with the housing cover 320 having a dome shape, in other variations, the housing cover 320 and base plate 330 may have any suitable shape. For example, in other variations, the housing cover 320 and base plate 330 may be generally prismatic, elliptical, triangular, rectangular, pentagonal, hexagonal, or other suitable shapes. The outer adhesive layer 344 (or single adhesive layer) may extend outward from the housing cover 320 and base plate 330 and extend beyond the outer periphery of the housing cover 320. The outer adhesive layer 344 (or single adhesive layer) may be circular, as shown in FIGS. 3A-3D, or may have an elliptical, triangular, rectangular, pentagonal, hexagonal, or other suitable shape, and need not be the same shape as the housing cover 320 and / or base plate 330.

[0108] 4A-4E show an embodiment of a sensor assembly 350 of an analyte monitoring device 110 in an exploded perspective view, an exploded side view, a bottom perspective view, a side view, and a top perspective view, respectively.

[0109] The sensor assembly 350 includes microneedle array components and electronics for implementing the analyte detection and processing aspects of the microneedle array-based continuous analyte monitoring device 110 for analyte detection and measurement. In some variations, the sensor assembly 350 is a compact, low-profile laminate that is at least partially contained within an interior volume defined by the housing cover 320 and the base plate 330.

[0110] In some variations, the sensor assembly 350 includes a microneedle array assembly 360 and an electronics assembly 370 that connect to each other to implement the microneedle array analyte detection and processing aspects described further herein. In some variations, the electronics assembly 370 includes a main printed circuit board (PCB) 450 to which the electronic components are connected, and the microneedle array assembly 360 includes a secondary printed circuit board (PCB) 420 to which the microneedle array 140 is connected.

[0111] In some variations, the microneedle array assembly 360 includes, in addition to the secondary PCB 420 and the microneedle array 140, an epoxy skirt 410 and a secondary PCB connector 430. The microneedle array 140 is coupled to the top side (e.g., the outward-facing side) of the secondary PCB 420 so that the individual microneedles of the microneedle array 140 are exposed, as described with reference to Figures 3A-3D. The secondary PCB connector 430 is coupled to the back side, opposite the top side, of the secondary PCB 420. The secondary PCB connector 430 may be an electromechanical connector and may be communicatively coupled to the primary PCB 450 via a primary PCB connector 470 on the top side (e.g., the outward-facing side) of the primary PCB 450 to enable signal communication between the secondary PCB 420 and the primary PCB 450. For example, signals from the microneedle array 140 can be communicated to the primary PCB 450 via the secondary PCB 420, the secondary PCB connector 430, and the primary PCB connector 470.

[0112] The secondary PCB 420 may, in part, determine the distance that the microneedle array 140 protrudes from the housing's backplate 330. Thus, the height of the secondary PCB 420 may be selected to help ensure that the microneedle array 140 is properly inserted into a user's skin. During microneedle insertion, the first surface (e.g., the outward-facing surface) of the connecting member 332 of the backplate 330 may act as a stop for microneedle insertion. If the secondary PCB 420 has a low height and its top surface is flush or nearly flush with the first surface of the connecting member 332, the connecting member 332 may prevent the microneedle array 140 from being fully inserted into the skin.

[0113] In some variations, other components (e.g., electronic components such as sensors or other components) may also be connected to secondary PCB 420. For example, secondary PCB 420 may be sized and shaped to accommodate electronic components on the top or back side of secondary PCB 420.

[0114] 3C and 3D , the epoxy skirt 410 may be deposited along the edge (e.g., periphery) of the microneedle array 140 to provide a tight fit of the microneedle array 140 within the apertures 334 formed in the connecting members 332 of the base plate 330 and / or to soften sharp edges along the microneedle array 140. For example, the epoxy skirt 410 may occupy the portion of the apertures 334 not filled by the microneedle array 140 and / or the portion of the cavity defined in the base plate 330 not filled by the secondary PCB 420. The epoxy skirt 410 may also provide a transition from the edge of the microneedle array 140 to the edge of the secondary PCB 420. In some variations, the epoxy skirt 410 may be replaced or supplemented by a gasket (e.g., a rubber gasket) or the like.

[0115] Electronics assembly 370 with primary PCB 450 includes battery 460 coupled to the back side of primary PCB 450, opposite the top side to which primary PCB connector 470 is coupled. In some variations, battery 460 may be coupled to the top side of primary PCB 450 and / or in other arrangements.

[0116] Figures 4F-4H show an embodiment of an alternative variation of sensor assembly 350 of analyte monitoring device 110. Perspective exploded, side exploded, and side views of sensor assembly 350 are provided in Figures 4F-4H, respectively.

[0117] As shown, the sensor assembly 350 incorporates an additional PCB component, the intermediate PCB 425. In some variations, the intermediate PCB 425 is part of the microneedle array assembly 360 and is positioned between and connected to the secondary PCB 420 and the microneedle array 140. The intermediate PCB 425 may be added to increase the height of the microneedle array assembly 360, thereby extending the microneedle array 140 a greater distance from the base plate 330, which may aid in insertion of the microneedle array 140 into a user's skin. The microneedle array 140 is coupled to the top side (e.g., the outward-facing side) of the intermediate PCB 425 such that the individual microneedles of the microneedle array 140 are exposed, as described with reference to FIGS. 3A-3D . The secondary PCB 420 is bonded to the back side, opposite the top side, of the intermediate PCB 425, and the secondary PCB connector 430 is bonded to the back side, opposite the top side, of the secondary PCB 420. An epoxy skirt 410 (which may be replaced or supplemented by an equivalent gasket) provides a transition from the edge of the microneedle array 140 to the edge of the intermediate PCB 425.

[0118] The intermediate PCB 425 with the secondary PCB 420 determines, in part, the distance the microneedle array 140 protrudes through the aperture 334 of the backplate 330. The incorporation of the intermediate PCB 425 provides additional height that helps ensure that the microneedle array 140 is properly inserted into the user's skin. In some variations, the upper side (e.g., the outward-facing side) of the intermediate PCB 425 extends out through the aperture 334, such that the first surface (e.g., the exposed top surface) of the connecting member 332 surrounding the aperture 334 does not prevent the microneedle array from being fully inserted into the skin. In some variations, the upper side (e.g., the outward-facing side) of the intermediate PCB 425 does not extend out of the aperture 334, but the increased height (by incorporating the intermediate PCB 425) ensures that the microneedle array 140 protrudes a sufficient distance from the housing's backplate 330.

[0119] In some variations, a microneedle enclosure may be provided for releasable attachment to the analyte monitoring device 110. The microneedle enclosure may provide a protective environment or enclosure in which the microneedle array 140 may be safely contained, thereby ensuring the integrity of the microneedle array 140 during certain stages of manufacturing and shipping of the analyte monitoring device 110 prior to application of the analyte monitoring device 110. The microneedle enclosure is releasable or removable from the analyte monitoring device 110 so that the microneedle array 140 is exposed and ready for insertion into a user's skin, as described further herein.

[0120] In some variations, the microneedle enclosure provides an enclosed, sealed environment in which the microneedle array 140 can be contained, thereby providing an environment in which the microneedle array 140 can be sterilized. For example, the microneedle enclosure with the microneedle array 140 can be subjected to a sterilization process in which the sterilization penetrates the microneedle enclosure such that the microneedle array 140 is also sterilized. Because the microneedle array 140 is contained within the enclosed environment, the microneedle array 140 remains sterile until it is removed from the enclosed environment. User Interface

[0121] In some variations, the analyte monitoring system may provide user status, analyte monitoring device status, and / or other suitable information directly via a user interface on the analyte monitoring device (e.g., a display, indicator lights, etc., as described below). Thus, in contrast to analyte monitoring systems that may solely communicate information to a separate peripheral device (e.g., a cell phone, etc.), which may then communicate that information to the user, in some variations, such information may be provided directly by the analyte monitoring device. Advantageously, in some variations, such a user interface on the analyte monitoring device may reduce the need for a user to constantly maintain a separate peripheral device to monitor user status and / or analyte monitoring device status (which may be impractical due to cost, inconvenience, etc.). Additionally, the user interface of the analyte monitoring device may reduce risks associated with loss of communication between the analyte monitoring device and a separate peripheral device, such as the user having an inaccurate understanding of the current analyte level (e.g., leading the user to assume that the analyte level is high when it is actually low, which could lead the user to self-administer an incorrect dose of medication or to withhold a therapeutic intervention when medically necessary, for example).

[0122] Furthermore, the ability to communicate information to the user via the analyte monitoring device itself, independent of a separate peripheral device, can reduce or eliminate the need to maintain compatibility between the analyte monitoring device and the separate peripheral device when the separate peripheral device is upgraded (e.g., replaced with a new device model or other hardware, run a new version of an operating system or other software, etc.).

[0123] Thus, in some variations, the housing may include a user interface, such as a visual, audible, and / or tactile interface, to provide information regarding a user condition based on the analyte measurements and / or the status of the analyte monitoring device and / or other suitable information.

[0124] Examples of user states based on analyte measurements that may be communicated via the user interface include information indicative of the analyte measurement in the user, such as the analyte concentration in a bodily fluid such as skin interstitial fluid or bloodstream, the analyte measurement being below a predetermined analyte measurement threshold or range, within a predetermined analyte measurement range, or above a predetermined analyte measurement threshold or range, an increase or decrease in the analyte measurement over time, a rate of change in the analyte measurement, an analyte variability indicating the standard deviation of the analyte measurement over a period of time, information regarding trends in the analyte measurement, and / or other suitable alerts related to the analyte measurement.

[0125] Examples of analyte monitoring device status that may be communicated via the user interface include device operating mode (e.g., related to device warm-up status, analyte monitoring status, battery power status such as low battery, etc.), device error status (e.g., operational error, pressure-induced sensing decay, failure, failure mode, etc.), device power status, device life status (e.g., expected end of sensor life), status of connection between the device and a mobile computing device, etc.

[0126] In some variations, the user interface may default to enabled or “on” state to communicate such information at least whenever the analyte monitoring device is performing analyte measurements or whenever the analyte monitoring device is powered on, thereby helping to ensure that information is continuously available to the user. For example, user interface elements may communicate via displays or indicator lights (e.g., as described below) to flag for user attention or recommend corrective action, as well as when user and / or device status is normal. Thus, in some variations, the user need not perform an action to initiate a scan to learn the current analyte measurement level; such information may always be readily available to the user. However, in some variations, the user may perform an action to temporarily disable the user interface (e.g., similar to a “snooze” button), such as for a predetermined period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.), at which time the user interface will automatically be re-enabled, or until a second action is performed to re-enable the user interface.

[0127] In some variations, the user interface of the housing can include a display configured to visually communicate information. The display can include, for example, a display screen (e.g., an LCD screen, an OLED display, an electrophoretic display, an electrochromic display, etc.) configured to display alphanumeric text (e.g., numbers, letters, etc.), symbols, and / or suitable graphics for conveying information to a user. For example, the display screen can include numerical information, text information, and / or graphics (e.g., sloping lines, arrows, etc.) of information such as user status and / or the status of the analyte monitoring device. For example, the display screen can include text or graphic representations of analyte measurement levels, trends, and / or recommendations. For example, the display screen can include text and / or graphic representations related to recommendations for physical activity, meditation, rest, food, dietary supplements, and / or medical consultations.

[0128] As another example, the display on the housing may include one or more indicator lights (e.g., including LEDs, OLEDs, lasers, electroluminescent materials, or other suitable light sources, waveguides, etc.) that can be controlled in one or more predetermined illumination modes to communicate different statuses and / or other suitable information. The indicator lights may be controlled to illuminate multiple colors (e.g., red, orange, yellow, green, blue, and / or purple, etc.) or only one color. For example, the indicator lights may include multicolor LEDs. As another example, the indicator lights may include transparent or translucent material (e.g., acrylic) disposed over one or more different colored light sources (e.g., LEDs) such that the different colored light sources can be selectively activated to illuminate the indicator light in selected colors. Activation of the light sources may occur simultaneously or sequentially. The indicator lights may have any suitable form (e.g., raised from the housing body, flush, recessed, etc.) and / or shape (e.g., circular or other polygonal, ring, elongated strip, etc.). In some variations, the indicator light may have the size and / or shape of a pinhole that emits the same intensity of light as a larger light source, but requires significantly less power, helping to conserve on-board power of the analyte monitoring device.

[0129] The indicator lights on the display may be illuminated in one or more different ways to communicate different types of information. For example, the indicator lights may be selectively illuminated or extinguished to convey information (e.g., lighting "on" indicates one state and lighting "off" indicates another state). Additionally or alternatively, the indicator lights may be illuminated at a color or intensity selected to convey information (e.g., lighting of a first color or intensity indicates a first state and lighting of a second color or intensity indicates a second state). Additionally or alternatively, the indicator lights may be illuminated in a time pattern selected to convey information (e.g., lighting of a first time pattern indicates a first state and lighting of a second time pattern indicates a second state). For example, the indicator light may be selectively illuminated in one of a number of predetermined time patterns that vary in illumination frequency (e.g., repeating illumination at a fast or slow frequency), regularity (e.g., cyclically repeating illumination vs. intermittent illumination), duration of illumination "on" time, duration of illumination "off" time, rate of change of illumination intensity, duty cycle (e.g., ratio of illumination "on" time to illumination "off" time), etc., and each predetermined time pattern may indicate a respective state.

[0130] Additionally or alternatively, in some variations, the display may include multiple indicator lights that may be collectively illuminated in one or more predetermined lighting modes or sequences according to one or more predetermined spatial and / or temporal patterns. For example, in some variations, some or all of the indicator lights arranged on the display may be illuminated synchronously or sequentially to indicate a particular state. Thus, a selected subset of the indicator lights (e.g., the spatial arrangement of the illuminated indicator lights) and / or the manner in which they are illuminated (e.g., the illumination sequence, the illumination rate, etc.) may indicate a particular state. Additionally or alternatively, multiple indicator lights may be illuminated simultaneously or sequentially to increase the diversity of the color palette. For example, in some variations, red, green, and blue LEDs may be illuminated in rapid succession to create the impression of white light to the user.

[0131] It should be further appreciated that one or more of the above lighting modes can be combined in any suitable manner (e.g., combinations of various colors, intensities, brightness, luminosity, contrast, timing, position, etc.) to convey information. Additionally or alternatively, an ambient light sensor can be incorporated into the device body to enable dynamically adjusted light levels of the indicator lights to compensate for environmental light conditions and conserve power. The ambient light sensor, in some variations, can be used in conjunction with a motion sensor (e.g., as described in more detail below) to further determine an appropriate period for the analyte monitoring device to enter a power-saving mode or reduced-power state. For example, detection of darkness and a lack of motion by the analyte monitoring device can indicate that the wearer of the analyte monitoring device is asleep, which can cause the analyte monitoring device to transition to a power-saving mode or reduced-power state.

[0132] 19A shows an exemplary variation of an analyte monitoring device 1900 including a user interface 1920 having a plurality of indicator lights (1922, 1924a-1924c). The indicator lights 1922 may be selectively illuminated to indicate, for example, a device status (e.g., an operating mode, an error state, a power state, a life state, etc.). While the indicator lights 1922 are in the form of a symbol (e.g., a logo), it should be understood that in other variations, the indicator lights 1922 may have any suitable shape (e.g., text, other geometric shapes, etc.). The indicator lights 1924a, 1924b, 1924c may be selectively illuminated to indicate a user status (e.g., information representative of an analyte measurement). It should be understood that while the indicator lights 1924a, 1924b, 1924c are linear elements extending across the user interface (e.g., a cord across a circular display), in other variations the indicator lights 1924a, 1924b, 1924c have other suitable shapes (e.g., wavy lines, circles, etc.). In some variations, a one-dimensional array of indicator lights of any suitable shape can be arranged on the housing (e.g., arranged in rows, columns, arcs, etc.). Alternatively, the housing can include a multi-dimensional array of indicator lights of any suitable shape.

[0133] Further, in some variations, the indicator lights may include icons (e.g., symbols) that can indicate analyte information (e.g., an up arrow indicating an upward trend in the analyte measurement level, a down arrow indicating a downward trend in the analyte measurement level), the status of the analyte monitoring device (e.g., an exclamation point indicating a device error condition), and / or other suitable information. Additionally or alternatively, iconography within the indicator lights may be used to communicate recommendations to the user, such as action recommendations. Iconography may have the advantage, for example, of communicating recommendations to the user in a more universal or language-independent manner (e.g., not requiring language translation to tailor the device to different geographic regions or user preferences, etc.). In one example, an increase in analyte levels may be correlated with increased stress in the user. For example, as shown in FIG. 19B , in some variations, the user interface of the analyte monitoring device 1900′ may include a running person icon 1926 indicating a recommendation that the user engage in physical activity, a tree icon 3128 indicating a recommendation that the user go outside, and / or a thinking head icon 1932 indicating a recommendation that the user meditate.

[0134] 19A and 19B, each of the indicator lights 1924a, 1924b, 1924c may be illuminated exclusively to indicate a different analyte measurement (e.g., within target range, below target range, significantly below target range, above target range, significantly above target range, etc.). Additionally, the indicator lights 1924a, 1924b, 1924c may be positioned adjacent to one another such that they may be selectively illuminated in a progressive sequence to convey trend information of the analyte measurements (e.g., a progressive sequence of illumination in a first direction corresponding to an increase in the analyte measurement, a progressive sequence of illumination in a second direction corresponding to a decrease in the analyte measurement, the pace of illumination progression in the first or second direction corresponding to the rate of increase or decrease in the analyte measurement, etc.). 19A and 19B show one device status light 1922 and three user status lights 1924a, 1924b, 1924c, it should be understood that in other variations, the analyte monitoring device may include any suitable number of lights, such as one, two, three, four, five, or more device status lights and one, two, three, four, five, or more user status lights. Further details regarding exemplary operation of user interface 1920 to communicate device status and / or user status are provided below (see, e.g., FIGS. 20A-20C). Microneedle Array

[0135] As shown in the schematic diagram of FIG. 5A, in some variations, a microneedle array 510 for use in analyte sensing may include one or more microneedles 510 protruding from a substrate surface 502. The substrate surface 502 may be, for example, a substantially planar semiconductor (e.g., silicon) substrate, and the one or more microneedles 510 may protrude perpendicularly from the plane. Generally, as shown in FIG. 5B, the microneedle 510 may include a body portion 512 (e.g., a shaft) and a tapered distal portion 514 configured to pierce a user's skin. In some variations, the tapered distal portion 514 may terminate in an insulated distal tip 516. The microneedle 510 may further include an electrode 520 on the surface of the tapered distal portion. In some variations, electrode-based measurements may be performed at the interface between an electrode placed within the body and interstitial fluid (e.g., on the outer surface of the entire microneedle). In some variations, the microneedle 510 may have a solid core (e.g., a solid body portion), while in some variations, the microneedle 510 may include one or more lumens that may be used, for example, for drug delivery or sampling of skin interstitial fluid. Other microneedle variations, as described below, may similarly include either a solid core or one or more lumens.

[0136] The microneedle array 500 may be formed at least in part from a semiconductor (e.g., silicon) substrate and include various layers of material applied and shaped using various suitable microelectromechanical systems (MEMS) fabrication techniques (e.g., deposition and etching techniques), as described further below. The microneedle array can be reflow soldered to a circuit board, similar to a typical integrated circuit. Furthermore, in some variations, the microneedle array 500 may include a three-electrode configuration including a working (sensing) electrode having an electrochemical sensing coating (including a biorecognition element such as an aptamer or enzyme) that enables detection of an analyte, a reference electrode, and a counter electrode. In other words, the microneedle array 500 may include at least one microneedle 510 including a working electrode, at least one microneedle 510 including a reference electrode, and at least one microneedle 510 including a counter electrode. Further details of these types of electrodes are described in more detail below.

[0137] In some variations, the microneedle array 500 may include multiple microneedles that are insulated such that the electrodes on each microneedle in the multiple microneedles are individually addressable and electrically isolated from all other electrodes on the microneedle array. The resulting individual addressability of the microneedle array 500 may allow greater control over the function of each electrode, since each electrode can be probed separately. For example, the microneedle array 500 may be used to provide multiple independent measurements of a given analyte, thereby improving the sensing reliability and accuracy of the device. Furthermore, in some variations, the electrodes of multiple microneedles may be electrically connected to generate enhanced signal levels. As another example, the same microneedle array 500 may additionally or alternatively be interrogated to simultaneously measure multiple analytes to provide a more comprehensive assessment of a physiological condition. For example, as shown in the schematic diagram of FIG. 6, the microneedle array may include a portion of microneedles for detecting a first analyte A, a second portion of microneedles for detecting a second analyte B, and a third portion of microneedles for detecting a third analyte C. It should be understood that the microneedle array may be configured to detect any suitable number of analytes (e.g., 1, 2, 3, 4, 5 or more, etc.), provided that at least one of the analytes detected is an analyte.

[0138] In some variations of microneedles (e.g., microneedles having a working electrode), the electrode 520 may be positioned proximal to the insulated distal apex 516 of the microneedle. In other words, in some variations, the electrode 520 does not cover the apex of the microneedle. Rather, the electrode 520 may be offset from the apex or tip of the microneedle. The electrode 520 being proximal to or offset from the insulated distal apex 516 of the microneedle advantageously provides more accurate sensor measurements. For example, this arrangement prevents electric field concentration at the microneedle apex 516 during manufacturing, thereby avoiding uneven deposition of sensing chemistry on the electrode surface 520, which could result in erroneous sensing. The electrode 520 may be configured to have an annular shape and may include a distal edge 521 a and a proximal edge 521 b.

[0139] As another example, positioning the electrode 520 offset from the microneedle apex further improves sensing accuracy by reducing undesirable signal artifacts and / or erroneous sensor readings caused by stresses during microneedle insertion. The distal apex of the microneedle is the first area to penetrate the skin and, therefore, experiences the greatest stresses caused by mechanical shear phenomena associated with tearing or cutting the skin. If the electrode 520 were positioned at the apex or tip of the microneedle, this mechanical stress could peel off the electrochemical sensing coating on the electrode surface when the microneedle is inserted and / or cause a small but interfering amount of tissue to be transported onto the active sensing portion of the electrode. Therefore, positioning the electrode 520 sufficiently offset from the microneedle apex can improve sensing accuracy. For example, in some variations, the distal edge 520 of the electrode 521a may be positioned at least about 10 μm (e.g., about 20 μm to about 30 μm) from the distal apex or tip of the microneedle, as measured along the longitudinal axis of the microneedle.

[0140] The body portion 512 of the microneedle 510 may further include an electrically conductive pathway extending between the electrode 520 and a back electrode or other electrical contact (e.g., disposed on the back surface of the microneedle array substrate). The back electrode may be soldered to a circuit board and may be in electrical communication with the electrode 520 via the conductive pathway. For example, during use, the in vivo sensed current measured at the working electrode (inside the dermis) is interrogated by the back electrical contact, and the electrical connection between the back electrical contact and the working electrode is facilitated by the conductive pathway. In some variations, this conductive pathway may be facilitated by a metal via running through the interior of the microneedle body portion (e.g., shaft) between the proximal and distal ends of the microneedle. Alternatively, in some variations, the conductive pathway may be provided by the entire body portion formed from a conductive material (e.g., doped silicon). In some of these variations, the entire substrate on which the microneedle array 500 is constructed may be electrically conductive, and each microneedle 510 of the microneedle array 500 may be electrically isolated from adjacent microneedles 510 as described below. For example, in some variations, each microneedle 510 in the microneedle array 500 may be electrically isolated from adjacent microneedles 510 using an insulating barrier comprising an electrically insulating material (e.g., a dielectric material such as silicon dioxide) surrounding the conductive pathway extending between the electrode 520 and the backside electrical contact. For example, the body portion 512 may include an insulating material that forms a sheath around the conductive pathway, thereby preventing electrical communication between the conductive pathway and the substrate. Other exemplary variations in structures that allow for electrical isolation between microneedles are described in more detail below.

[0141] Such electrical isolation between microneedles within a microneedle array allows the sensors to be individually addressable. This individual addressability advantageously allows for independent, parallelized measurements between sensors, as well as dynamic reconfiguration of sensor assignments (e.g., for different analytes). In some variations, the electrodes of the microneedle array can be configured to provide redundant analyte measurements, which is an advantage over conventional analyte monitoring devices. For example, redundancy can improve performance by improving accuracy (e.g., averaging multiple analyte measurements from different microneedles to reduce the impact of extremely high or low sensor signals on analyte level determinations) and / or can improve device reliability by reducing the likelihood of overall failure.

[0142] In some variations, as described in further detail below with each different variation of microneedle, the microneedle array may be formed at least in part by suitable semiconductor and / or MEMS fabrication techniques and / or mechanical cutting or dicing. Such processes may be advantageous, for example, to enable large-scale, cost-effective manufacture of microneedle arrays. Microneedle structure

[0143] Further exemplary variations of microneedle structures incorporating one or more of the microneedle features described above for microneedle arrays in analyte monitoring devices are described herein.

[0144] In some variations, the microneedle may have a generally cylindrical body portion and a tapered distal portion carrying an electrode. For example, FIGS. 7A-7C show exemplary variations of a microneedle 700 extending from a substrate 702. FIG. 7A is a schematic side cross-sectional view of the microneedle 700, FIG. 7B is a perspective view of the microneedle 700, and FIG. 7C is a detailed perspective view of the distal portion of the microneedle 700. As shown in FIGS. 7B and 7C, the microneedle 700 may include a cylindrical body portion 712, a tapered distal portion 714 terminating in an insulated distal apex 716, and a ring electrode 720. The ring electrode 720 includes a conductive material (e.g., Pt, Ir, Au, Ti, Cr, Ni, combinations thereof, etc.) disposed on the tapered distal portion 714, e.g., on a segment thereof, and includes a distal edge 721 a and a proximal edge 721 b. 7A, the ring electrode 720 may be proximal to (or offset from or spaced apart from) the distal apex 716. The ring electrode 720 may be electrically insulated from the distal apex 716 by a distal insulating surface 715a comprising an insulating material, for example, SiO. For example, the distal edge 721a of the ring electrode 720 may be adjacent to the proximal edge of the distal insulating surface 715a of the insulated distal apex 716. In some variations, the distal edge 721 a of the ring electrode 720 may be proximal (e.g., immediately proximal, adjacent, abutting) the proximal edge of the distal apices 716 (the proximal edge of the distal insulating surface 715 a), while in other variations, the distal edge 721 a of the ring electrode 720 may be distal (e.g., immediately distal, adjacent) to the proximal edge of the insulated distal apices 716 (the proximal edge of the distal insulating surface 715 a), but remain proximal to the apices themselves. Thus, in some variations, the ring electrode 720 may overlie a portion of the distal insulating surface 715 a, but remain proximal to (and may be offset from) the insulated distal apices themselves.

[0145] 7A, the proximal edge 721b of the ring electrode 720 may be distal to, or in some variations offset or spaced from, the cylindrical body portion 712. In some variations, the proximal edge 721b of the ring electrode 720 may also be electrically insulated from the cylindrical body portion 712 by a second distal insulating surface 715b comprising an insulating material (e.g., SiO2) at the proximal end or region of the tapered distal portion 714. For example, the proximal edge 721b of the ring electrode 720 may be proximate the distal edge of the second distal insulating surface 715b. In some variations, the proximal edge 721 b of the ring electrode 720 may be proximal (e.g., immediately proximal, adjacent, abutting) the distal edge of the second distal insulating surface 715 b, while in other variations, the proximal edge 721 b of the ring electrode 720 may be distal (e.g., immediately distal, adjacent) to the distal edge of the second distal insulating surface 715 b but remain proximal to the cylindrical body portion 712. Thus, in some variations, the ring electrode 720 may overlie a portion of the second distal insulating surface 715 b but remain proximal to (and offset from) the cylindrical body portion 712. As shown in FIG. 7A and in some other variations, the ring electrode 720 may be on only a portion of the surface of the tapered distal portion 714 and may or may not extend all the way to the cylindrical body portion 712.

[0146] The electrode 720 may be in electrical communication with a conductive core 740 (e.g., a conductive path) that passes along the body portion 712 to a backside electrical contact 730 (e.g., made from a Ni / Au alloy) or another electrical pad in or on the substrate 702. For example, the body portion 712 may include a conductive core material (e.g., heavily doped silicon). As shown in FIG. 7A, in some variations, an insulating moat 713 comprising an insulating material (e.g., SiO) may be disposed around (e.g., around the periphery of) the body portion 712 and extend at least partially through the substrate 702. The insulating moat 713 may thus serve to prevent electrical contact between, for example, the conductive core 740 and the surrounding substrate 702. The insulating moat 713 may further extend onto the surface of the body portion 712. The top and / or bottom surfaces of the substrate 702 may also include a substrate insulating layer 704 (e.g., SiO). Thus, the insulation provided by the insulating moat 713 and / or substrate insulator 704 may at least partially contribute to the electrical isolation of the microneedles 700, enabling individual addressability of the microneedles 700 within a microneedle array. Furthermore, in some variations, the insulating moat 713 extending onto the surface of the body portion 712 may function to increase the mechanical strength of the microneedle 700 structure.

[0147] The microneedle 700 can be formed, at least in part, by a suitable MEMS fabrication technique, such as plasma etching, also known as dry etching. For example, in some variations, the insulating moat 713 around the body portion 712 of the microneedle may be fabricated by first forming a trench in a silicon substrate by deep reactive ion etching (DRIE) from the backside of the substrate, and then filling the trench with a SiO2 / polycrystalline silicon (poly-Si) / SiO2 sandwich by low-pressure chemical vapor deposition (LPCVD) or other suitable process. In other words, the insulating moat 713 passivates the surface of the body portion 712 of the microneedle and can continue as a recessed feature in the substrate 702 near the proximal portion of the microneedle. By comprising a primarily silicon compound, the insulating moat 713 can provide good filling and adhesion to adjacent silicon walls (e.g., the conductive core 740, the substrate 702, etc.). The sandwich structure of insulating moat 713 can further help provide an excellent coefficient of thermal expansion (CTE) match with the adjacent silicon, thereby advantageously reducing failure, cracking, and / or other thermally induced weaknesses of insulating structure 713.

[0148] The tapered distal portion can be formed by isotropic dry etching from the front side of the substrate, and the body portion 712 of the microneedle 700 can be formed by DRIE. A front metal electrode 720 can be deposited and patterned on the distal portion by specialized lithography (e.g., electron beam evaporation) that allows metal deposition on the desired annular region of the electrode 720 without coating the distal tip 716. Additionally, a Ni / Au backside electrical contact 730 can be deposited by a suitable MEMS fabrication technique (e.g., sputtering).

[0149] The microneedle 700 may have any suitable dimensions. By way of example, in some variations, the microneedle 700 may have a height of about 300 μm to about 500 μm. In some variations, the tapered distal portion 714 may have a tip angle of about 60 degrees to about 80 degrees and a top diameter of about 1 μm to about 15 μm. In some variations, the surface area of ​​the ring electrode 720 may be about 9000 μm. 2 ~about 11000μm 2 , or approximately 10,000 μm 2 FIG. 8 illustrates various dimensions of an exemplary variation of a cylindrical microneedle having a tapered distal portion and a ring electrode, similar to the microneedle 700 described above. The cylindrical microneedle of FIG. 8, like the microneedle 700 described above, comprises a cylindrical body portion, a tapered distal portion terminating in an insulated distal apex, a contact trench formed in the tapered distal portion, and a ring electrode (labeled "Pt" in FIG. 8) disposed on the tapered distal portion and covering the contact trench. The ring electrode can comprise a conductive material (e.g., Pt, Ir, Au, Ti, Cr, Ni, combinations thereof, etc.). In some variations, the contact trench may have a width of about 1 μm (micrometer), about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, or about 20 μm as shown in Figure 8. The ring electrode may include a distal edge and a proximal edge, and in some variations, the distance between the distal and proximal edges of the ring electrode may be about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, or about 60 μm as shown in Figure 8. In some variations, as shown in FIG. 8 by the dimension callouts 60 μm and 20 μm, the annular electrode may overlie a portion of the insulating surface of the contact trench, the tapered distal portion (labeled "oxide" in FIG. 8).

[0150] 9 shows another exemplary variation of a microneedle 900 having a generally cylindrical body portion. The microneedle 900 may be similar to the microneedle 700 described above, except as described below. For example, like the microneedle 700, the microneedle 900 may include a cylindrical body portion 912 and a tapered distal portion 914 terminating in an insulated distal apex 916. The microneedle 900 may further include an annular electrode 920 comprising a conductive material and disposed on the tapered distal portion 914 at a location proximal to (or offset or spaced from) the distal apex 916. Other elements of the microneedle 900 are numbered similarly to corresponding elements of the microneedle 700.

[0151] However, compared to microneedle 700, microneedle 900 may have a sharper tip at distal apex 916 and a modified insulating moat 913. For example, distal apex 916 may have a sharper tip angle, such as about 25 degrees to about 45 degrees, and an apex radius of less than about 100 nm, which provides a sharper microneedle profile that can penetrate skin more easily, at a lower speed, with less energy, and / or with less trauma. Furthermore, in contrast to insulating moat 713 (which extends through substrate 702 along the height of microneedle body portion 712, as shown in FIG. 7A), modified insulating moat 913 may extend only through substrate 902, such that the sandwich structure filling the trench (e.g., created by DRIE as described above) forms only a buried feature within the substrate. Although the sidewalls of the microneedle 900 are shown in FIG. 9 as extending approximately perpendicular to the substrate surface, it should be understood that because the modified insulating moat 913 need not extend the entire height of the microneedle body portion 712, in some variations the sidewalls of the microneedle 900 may be sloped at a non-orthogonal angle to the substrate (e.g., the sidewalls may have a slight positive taper of between about 1 degree and about 10 degrees, or between about 5 degrees and about 10 degrees).

[0152] In some variations, the remainder of the microneedle surface 900 (other than the ring electrode 920) may include insulating material extending from the substrate insulator 904. For example, a layer of insulating material (e.g., SiO) may extend from the front surface of the substrate 902 to provide body portion insulation 918 and may further extend over the proximal edge 921b of the electrode 920, as shown in FIG. 9 . Another region of insulating material may similarly cover the distal edge 921a of the electrode 920, insulating the distal tip 916. Such regions of insulating material and / or modified insulating moat 913 may help prevent electrical contact between the conductive core 940 and the surrounding substrate 902. Thus, similar to the microneedle 700, the microneedle 900 can maintain electrical isolation for addressability within a microneedle array. In some variations, the process of forming microneedles 900 may result in higher yields and / or offer lower manufacturing costs compared to the process of forming microneedles 700.

[0153] The microneedle 900 can have any suitable dimensions. By way of example, the microneedle 900 can include a height of about 400 μm to about 600 μm, or about 500 μm, in some variations. In some variations, the tapered distal portion 914 can have a tip angle of about 25 degrees to about 45 degrees, with a tip radius of less than about 100 nm. Additionally, the microneedle can have a shaft diameter of about 160 μm to about 200 μm.

[0154] 10A-10F show another exemplary variation of a microneedle 1000 having a generally cylindrical body portion extending from a substrate 1002 having an upper surface 1004. The microneedle 1000 may be similar to the microneedle 800 described above, except as described below. For example, as shown in FIG. 10B, similar to the microneedle 800, the microneedle 1000 may include a cylindrical body portion 1012 and a tapered distal portion disposed on a cylinder 1013 and terminating in an insulated distal apex 1016. The cylinder 1013 may be insulated and may have a smaller diameter than the cylindrical body portion 1012. The microneedle 1000 may further include a ring electrode 1020 comprising a conductive material and disposed on the tapered distal portion proximal to (or offset or spaced from) the distal apex 1016. Other elements of the microneedle 1000 shown in FIGS. 10A-10F are numbered similarly to corresponding elements of the microneedle 800.

[0155] 10B, 10C, and 10F, the tapered distal portion 1014, and more particularly, the electrode 1020 on the tapered distal portion 1014 of the microneedle 1000, may include a tip contact trench 1022. This contact trench may be configured to establish an ohmic contact between the electrode 1020 and the underlying conductive core 1040 of the microneedle. In some variations, the shape of the tip contact trench 1022 may include an annular recess formed in the surface of the tapered distal portion 1014. In some variations, the shape of the tip contact trench 1022 may include an annular recess formed in the surface of the conductive core 1040 (e.g., in contact with a conductive path within the body portion of the microneedle or otherwise within the body portion). In some variations, the tip contact trench 1022 may be formed in the insulating material on the tapered distal portion 1014 and may have a depth approximately equal to the thickness of the insulating material (e.g., distal insulating surface 1015a and / or second distal insulating surface 1015b). In some examples, the depth of the contact trench may be greater than the thickness of the insulating material such that the contact trench extends beyond the surface of the conductive core 1040 (e.g., into the conductive core 1040). The electrode 1020 may overlie the tip contact trench 1022 such that an ohmic contact is established between the electrode 1020 and the conductive core 1040. In some variations, the electrode 1020 may extend beyond the tip contact trench 1022 such that when the material of the electrode 1020 is deposited on the conductive core 1040, the electrode 1020 with the tip contact trench 1022 can have a stepped profile in side view. Thus, the tip contact trench 1022 may advantageously help to ensure contact between the electrode 1020 and the underlying conductive core 1040. Any of the other microneedle variations described herein may have similar tip contact trenches to help ensure contact between the electrode (which may be, for example, a working electrode, a reference electrode, a counter electrode, etc.) and the conductive pathway within the microneedle.

[0156] 11A and 11B show additional various dimensions of exemplary variations of a cylindrical microneedle having a tapered distal portion and a ring electrode, similar to the microneedle 1000 described above. For example, the microneedle variations shown in FIGS. 11A and 11B can have a tapered distal portion with a taper angle of generally about 80 degrees (or about 78 degrees to about 82 degrees, or about 75 degrees to about 85 degrees) and a cone diameter of about 140 μm (or about 133 μm to about 147 μm, or about 130 μm to about 150 μm). The cone of the tapered distal portion may be positioned on the cylinder such that the combined height of the cone and cylinder is about 110 μm (or about 99 μm to about 116 μm, or about 95 μm to about 120 μm). The ring electrode on the tapered distal portion can have an outer or base diameter of about 106 μm (or about 95 μm to about 117 μm, or about 90 μm to about 120 μm) and an inner diameter of about 33.2 μm (or about 30 μm to about 36 μm, or about 25 μm to about 40 μm). The length of the ring electrode measured along the slope of the tapered distal portion can be about 57 μm (or between about 55 μm and about 65 μm), and the total surface area of ​​the electrode can be about 12700 μm. 2 (or approximately 12,500 μm 2 ~Approx. 12900μm 2 Between or about 12000 μm 2 ~about 13000μm 2 As shown in FIG. 11B, the electrode can further have a tip contact trench extending around the conical central region of the tapered distal portion, the contact having a width of about 11 μm (or about 5 μm to about 50 μm, about 10 μm to about 12 μm, or about 8 μm to about 14 μm) as measured along the slope of the tapered distal portion, and a trench depth of about 1.5 μm (or about 0.1 μm to about 5 μm, or about 0.5 μm to about 1.5 μm, or about 1.4 μm to about 1.6 μm, or about 1 μm to about 2 μm). The microneedle has an insulated distal tip with a diameter of about 5.5 μm (or about 5.3 μm to about 5.8 μm, or about 5 μm to about 6 μm).

[0157] Details of exemplary variations of microneedle array configurations are described in further detail below.

[0158] As described above, each microneedle in a microneedle array may include an electrode. In some variations, multiple different types of electrodes may be included within the microneedles of the microneedle array. For example, in some variations, the microneedle array may function as an electrochemical cell capable of electrolytic operation using three types of electrodes. In other words, the microneedle array may include at least one working electrode, at least one counter electrode, and at least one reference electrode. Thus, the microneedle array may include three different electrode types, but one or more of each electrode type may form a complete system (e.g., the system may include multiple separate working electrodes). Furthermore, multiple separate microneedles may be electrically joined to form an effective electrode type (e.g., a single working electrode may be formed from two or more connected microneedles having working electrode sites). Each of these electrode types may include a metallization layer and one or more coatings or layers on the metallization layer that help facilitate the function of that electrode.

[0159] Generally, the working electrode is the electrode where the oxidation and / or reduction reaction of interest occurs for detection of the analyte of interest. The counter electrode functions to source (source) or sink (store) the electrons necessary to sustain the electrochemical reaction at the working electrode via electrical current. The reference electrode functions to provide a reference potential for the system; that is, the potential to which the working electrode is biased is referenced to the reference electrode. A fixed, time-varying, or at least controlled potential relationship is established between the working and reference electrodes, and, within practical limits, current is not sourced from or sunk to the reference electrode. Furthermore, to implement such a three-electrode system, the analyte monitoring device can include a suitable potentiostat or electrochemical analog front end for maintaining a fixed potential relationship between the working and reference electrode contingencies in the electrochemical system (via an electronic feedback mechanism), while dynamically varying the counter electrode to the potential required to sustain the redox reaction of interest. Microneedle array configuration

[0160] A plurality of microneedles (e.g., any of the microneedle variations described herein, each of which may have a working electrode, counter electrode, or reference electrode as described above) can be arranged in a microneedle array. Considerations for how to configure the microneedles include factors such as the desired insertion force for penetrating the skin with the microneedle array, optimization of electrode signal levels and other performance aspects, and manufacturing cost and complexity.

[0161] For example, a microneedle array may include multiple microneedles spaced at a predetermined pitch (the distance between the center of one microneedle and the center of its nearest neighboring microneedle). In some variations, the microneedles may be spaced at a pitch sufficient to distribute the force applied to a user's skin to penetrate the microneedle array (e.g., avoiding a "needle-rat" effect). As the pitch increases, the force required to insert the microneedle array tends to decrease and the depth of penetration tends to increase. However, it has been found that the pitch only begins to affect insertion force at low values ​​(e.g., less than about 150 μm). Thus, in some variations, the microneedles of the microneedle array may have a pitch of at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, or at least 750 μm. For example, the pitch may be about 200 μm to about 800 μm, about 300 μm to about 700 μm, or about 400 μm to about 600 μm. In some variations, the microneedles may be arranged in a periodic grid, and the pitch may be uniform in all directions and across the entire area of ​​the microneedle array. Alternatively, the pitch may be different when measured along different axes (e.g., X, Y directions), and / or some areas of the microneedle array may include a smaller pitch and other areas may include a larger pitch.

[0162] Additionally, for more consistent penetration, the microneedles may be spaced equidistant from one another (e.g., the same pitch in all directions). To that end, in some variations, the microneedles within the microneedle array may be arranged in a hexagonal configuration, as shown in Figures 12A-12C, 13A-13B, and 14A-14J. Alternatively, the microneedles within the microneedle array may be arranged in a rectangular array (e.g., a square array) or in another suitable symmetrical manner.

[0163] Another consideration for determining the configuration of a microneedle array is the overall signal level provided by the microneedles. Generally, the signal level at each microneedle is consistent with the total number of microneedle elements in the array. However, the signal level can be enhanced by electrically interconnecting multiple microneedles within the array. For example, an array with a large number of electrically connected microneedles is expected to generate a greater signal strength (and therefore, improved accuracy) than an array with fewer microneedles. However, a larger number of microneedles on a die increases die cost (for a given pitch) and also requires greater force and / or speed for insertion into the skin. In contrast, a smaller number of microneedles on a die reduces die cost and allows for insertion into the skin with reduced applied force and / or speed. Furthermore, in some variations, a smaller number of microneedles on a die may reduce the overall footprint area of ​​the die, potentially reducing undesirable local edema and / or erythema. Thus, in some variations, a balance of these factors can be achieved with a microneedle array comprising 37 microneedles as shown in Figures 13A-13B, or a microneedle array comprising 7 microneedles as shown in Figures 12A-12C. However, in other variations, the number of microneedles in the array may be fewer (e.g., from about 5 to about 35, from about 5 to about 30, from about 5 to about 25, from about 5 to about 20, from about 5 to about 15, from about 5 to about 100, from about 10 to about 30, from about 15 to about 25, etc.) or the number of microneedles in the array may be greater (e.g., greater than 37, greater than 40, greater than 45, etc.).

[0164] Furthermore, as described in more detail below, in some variations, only a subset of the microneedles in the microneedle array may be active during operation of the analyte monitoring device. For example, some of the microneedles in the microneedle array may be inactive (e.g., no signal is read from the electrodes of the inactive microneedles). In some variations, some of the microneedles in the microneedle array may be activated at a particular time during operation and remain active for the remainder of the operational life of the device. Furthermore, in some variations, some of the microneedles in the microneedle array may additionally or alternatively be deactivated at a particular time during operation and remain inactive for the remainder of the operational life of the device.

[0165] Considering the characteristics of the die for a microneedle array, the die size is a function of the number of microneedles in the microneedle array and the pitch of the microneedles. Manufacturing costs should also be considered, as a smaller die size contributes to lower costs because it increases the number of dies that can be formed from a single wafer of a given area. Furthermore, a smaller die size is also less susceptible to brittle fracture due to the relative fragility of the substrate.

[0166] Furthermore, in some variations, microneedles at the periphery of the microneedle array (e.g., near the edge or border of the die, near the edge or border of the housing, near the edge or border of an adhesive layer on the housing, along the outer edge of the microneedle array, etc.) may be found to have better performance (e.g., sensitivity) due to better penetration compared to microneedles at the center of the microneedle array or die. Thus, in some variations, the working electrode may be located mostly or entirely on microneedles located at the periphery of the microneedle array to obtain more accurate and / or precise analyte measurements.

[0167] 13A and 13B show exemplary schematic diagrams of 37 microneedles arranged in an exemplary variation of a microneedle array 1300. The 37 microneedles may be arranged in a hexagonal array with, for example, a needle-to-needle center-to-center pitch of about 750 μm (or about 700 μm to about 800 μm, or about 725 μm to about 775 μm) between the center of each microneedle and the center of its immediately neighboring microneedle in any direction. FIG. 13A shows an exemplary schematic diagram of an exemplary variation of a die containing the microneedle arrangement. Exemplary dimensions of the die (e.g., about 4.4 mm by about 5.0 mm) and the microneedle array 1300 are shown in FIG. 13B.

[0168] 12A and 12B show perspective views of an exemplary schematic of seven microneedles 1210 arranged in an exemplary variation of a microneedle array 1200. The seven microneedles 1210 are arranged in a hexagonal array on a substrate 1202. As shown in FIG. 12A, electrodes 1220 are disposed on distal portions of the microneedles 1210 extending from a first surface of the substrate 1202. As shown in FIG. 12B, proximal portions of the microneedles 1210 are conductively connected to respective backside electrical contacts 1230 on a second surface of the substrate 1202 opposite the first surface of the substrate 1202. FIGS. 12C and 12D show plan and side views of an exemplary schematic of a microneedle array similar to the microneedle array 1200. As shown in Figures 12C and 12D, the seven microneedles are arranged in a hexagonal array with a center-to-center needle pitch of about 750 µm between the center of each microneedle and the center of its immediately adjacent microneedle in any direction. In other variations, the center-to-center needle pitch may be, for example, about 700 µm to about 800 µm, or about 725 µm to about 775 µm. The microneedles may have an approximate outer axial diameter of about 170 µm (or about 150 µm to about 190 µm, or about 125 µm to about 200 µm) and a height of about 500 µm (or about 475 µm to about 525 µm, or about 450 µm to about 550 µm).

[0169] Furthermore, the microneedle arrays described herein can have a high degree of configurability with respect to where the working, counter, and reference electrodes are located within the microneedle array, and this configurability can be facilitated by the electronics system.

[0170] In some variations, the microneedle array can include electrodes distributed symmetrically or asymmetrically within the microneedle array into two or more groups, each group featuring the same or different numbers of electrode components depending on signal sensitivity and / or redundancy requirements. For example, electrodes of the same type (e.g., working electrodes) may be distributed bilaterally or radially symmetrically within the microneedle array. For example, FIG. 14A shows a variation of a microneedle array 1400A including two symmetric groups of seven working electrodes (WE), with the two working electrode groups labeled "1" and "2." In this variation, the two working electrode groups are distributed bilaterally symmetrically within the microneedle array. The working electrodes are generally positioned between a central region of three reference electrodes (RE) and an outer peripheral region of 20 counter electrodes (CE). In some variations, each of the two working electrode groups can include seven working electrodes electrically connected between them (e.g., to enhance the sensor signal). Alternatively, only a portion of one or both of the working electrode populations may include multiple electrodes electrically connected therebetween. As yet another alternative, the working electrode populations may include working electrodes that are freestanding and not electrically connected to other working electrodes. Furthermore, in some variations, the working electrodes may be distributed across the microneedle array in an asymmetric or random configuration.

[0171] As another example, FIG. 14B shows a variation of a microneedle array 1400B that includes four symmetric groups of three working electrodes (WE), with the four working electrode groups labeled "1," "2," "3," and "4." In this variation, the four working electrode groups are radially symmetrically distributed in the microneedle array. Each working electrode group is adjacent to and symmetrically positioned with one of the two reference electrode (RE) components in the microneedle array. The microneedle array also includes counter electrodes (CE) positioned around the periphery of the microneedle array, except for two electrodes at the vertices of a hexagon that may be inactive or used for other features or modes of operation.

[0172] In some variations, only a portion of the microneedle array may include active electrodes. For example, Figure 14C shows a variation of a microneedle array 1400C having 37 microneedles and a reduced number of active electrodes, including four working electrodes (labeled "1," "2," "3," and "4") in a bilateral symmetrical arrangement, 22 counter electrodes, and three reference electrodes. The remaining eight electrodes of the microneedle array are inactive. In the microneedle array shown in Figure 19C, each working electrode is surrounded by a group of counter electrodes. The two groups of such clusters of working and counter electrodes are separated by a row of three reference electrodes.

[0173] As another example, FIG. 14D shows a variation of microneedle array 1400D having 37 microneedles and a reduced number of active electrodes, including four working electrodes (labeled "1," "2," "3," and "4") in a bilateral symmetrical arrangement, 20 counter electrodes, and three reference electrodes; the remaining 10 electrodes of the microneedle array are inactive.

[0174] As another example, FIG. 14E shows a variation of a microneedle array 1400E having 37 microneedles and a reduced number of active electrodes, including four working electrodes (labeled "1," "2," "3," and "4"), 18 counter electrodes, and two reference electrodes. The remaining 13 electrodes of the microneedle array are inactive. The inactive electrodes are located along a partial periphery of the overall microneedle array, thereby reducing the effective size and shape of the active microneedle array to a smaller hexagonal array. Within the active microneedle array, the four working electrodes are generally arranged in a radially symmetrical fashion, with each working electrode surrounded by a group of counter electrodes.

[0175] FIG. 14F shows another exemplary variation of a microneedle array 1400F having 37 microneedles and a reduced number of active electrodes, including four working electrodes (labeled "1," "2," "3," and "4"), two counter electrodes, and one reference electrode. The remaining 30 electrodes of the microneedle array are inactive. The inactive electrodes are arranged in two layers around the periphery of the overall microneedle array, thereby reducing the effective size and shape of the active microneedle array to a smaller hexagonal array centered on the reference electrode. Within the active microneedle array, the four working electrodes are in a bilateral symmetrical arrangement, and the counter electrodes are equidistant from the central reference electrode.

[0176] Figure 14G shows another exemplary variation of a microneedle array 1400G having 37 microneedles and a reduced number of active electrodes. The active electrodes of the microneedle array 1400G are arranged similarly to the microneedle array 1400F shown in Figure 14F, except that the microneedle array 1400G includes one counter electrode and two reference electrodes, with a smaller, hexagonal array of active microneedles centered around the counter electrode. Within the active microneedle arrangement, the four working electrodes are in a bilateral symmetrical arrangement, and the reference electrodes are equidistant from the central counter electrode.

[0177] Figure 14H shows another exemplary variation of a microneedle array 1400H having seven microneedles. The microneedle arrangement includes two microneedles designated as independent working electrodes (1 and 2), a counter electrode condition consisting of four microneedles, and a single reference electrode. There is symmetry in the arrangement of the working and counter electrodes, which are equidistant from the central reference electrode. Furthermore, the working electrode is placed as far away from the center of the microneedle array (e.g., at the periphery of the die or array) as possible to take advantage of locations where the working electrode is expected to have higher sensitivity and overall performance.

[0178] Figure 14I shows another exemplary variation of a microneedle array 1400I having seven microneedles. The microneedle arrangement includes four microneedles assigned as two independent groups (1 and 2) of two working electrodes each, a counter electrode consisting of two microneedles, and a single reference electrode. There is a symmetry in the arrangement of the working and counter electrodes, which are equidistant from the central reference electrode. Furthermore, the working electrodes are placed as far away from the center of the microneedle array (e.g., the periphery of the die or array) as possible to take advantage of locations where the working electrodes are expected to have higher sensitivity and overall performance.

[0179] Figure 14J shows another exemplary variation of a microneedle array 1400J having seven microneedles. The microneedle arrangement includes four microneedles designated as independent working electrodes (1, 2, 3, and 4), a counter electrode condition consisting of two microneedles, and a single reference electrode. There is a symmetry in the arrangement of the working and counter electrodes, which are equidistant from the central reference electrode. Furthermore, the working electrode is placed as far away from the center of the microneedle array (e.g., the periphery of the die or array) as possible to take advantage of locations where the working electrode is expected to have higher sensitivity and overall performance.

[0180] While Figures 14A-14J show exemplary variations of microneedle array configurations, it should be understood that these figures are not limiting and other microneedle configurations (including different numbers and / or distributions of working, counter, and reference electrodes, as well as different numbers and / or distributions of active and inactive electrodes, etc.) may be suitable for other variations of microneedle arrays.

[0181] Further details of exemplary variations of microneedle array configurations are described in more detail below. Electrode-structure

[0182] As described above, each microneedle in the microneedle array may include an electrode (e.g., electrode 520 of microneedle 510 as shown in FIG. 5B , electrode 720 of microneedle 710 as shown in FIGS. 7A and 7C , electrode 920 of microneedle 910 as shown in FIG. 9 , electrode 1020 of microneedle 1010 as shown in FIGS. 10B and 10C , and electrode 1220 of microneedle 1210 as shown in FIG. 12A ). In some variations, multiple different types of electrodes may be included within the microneedles of the microneedle array. For example, in some variations, the microneedle array may function as an electrochemical cell capable of electrolytic operation using three types of electrodes. In other words, the microneedle array may include at least one working electrode coupled to a first microneedle, at least one counter electrode coupled to a second microneedle, and at least one reference electrode coupled to a third microneedle. Thus, a microneedle array may include three different electrode types, but one or more of each electrode type may form a complete system (e.g., a system may include multiple separate working electrodes). Additionally, multiple separate microneedles may be electrically joined to form an effective electrode type (e.g., a single working electrode may be formed from two or more connected microneedles having working electrode sites). Each of these electrode types may include a metallization layer and one or more coatings or layers on the metallization layer that help facilitate the function of that electrode.

[0183] While the electrodes (including the working electrode) are primarily described herein above as being coupled to microneedles, it should be understood that the electrodes described herein, including the working electrode, counter electrode, and reference electrode, may be coupled to or otherwise incorporated into other structures, such as flat surfaces, probe tips, the exterior or interior surfaces of tubular structures, etc. In some variations, one or more of the electrodes described herein may be planar electrodes, and in some cases, may be planar microelectrodes (e.g., having a width or diameter of less than 500 microns). FIG. 15 shows, by way of example, a microelectrode array 1500 including a plurality of planar microelectrodes 1510.

[0184] Generally, the working electrode is an electrode that selectively detects an analyte of interest through a redox reaction using electrochemical methods. The counter electrode functions to source (source) or sink (store) the electrons necessary to sustain the electrochemical reaction at the working electrode via electrical current. The reference electrode functions to provide a reference potential for the system; that is, the potential to which the working electrode is biased is referenced to the reference electrode. A fixed, time-varying, or at least controlled potential relationship is established between the working and reference electrodes, and, within practical limits, current is not sourced from or sunk to the reference electrode. Furthermore, to implement such a three-electrode system, the analyte monitoring device can include a suitable potentiostat or electrochemical analog front end for maintaining (via an electronic feedback mechanism) a fixed or controlled potential relationship between the working and reference electrode contingencies in the electrochemical system, while dynamically varying the counter electrode to the potential required to sustain the redox reaction of interest.

[0185] Turning to multiple thiol-based passivation approaches for the E-AB sensors of the present disclosure consistent with implementation aspects of the present subject matter, aspects of the working, counter, and reference electrodes are provided. working electrode

[0186] As described above, the working electrode is the electrode where analyte detection occurs. In some variations, analyte sensing may be performed at the interface between the working electrode placed in the body and interstitial fluid (e.g., on the outer surface of the entire microneedle). In some variations, the working electrode may include an electrode material and a biorecognition layer on which a biorecognition element (e.g., an aptamer) is immobilized to facilitate selective analyte quantification. In some variations, the biorecognition layer may also function as an interference-blocking layer, helping to prevent endogenous and / or exogenous species from directly oxidizing (or reducing) at the electrode. In some variations, the biorecognition layer may include a passivation element deposited on the electrode material. The passivation element prevents interference with the electrochemical reaction at the electrode surface and minimizes capacitance. In some variations, the passivation element may include a passivation molecule that can be directly or indirectly chemisorbed onto the electrode material surface. In some variations, the passivation molecule may be a molecule that includes a thiol as a functional group (which may be referred to herein as a "thiol-based passivation molecule"), such as a small molecule ("thiol-based small molecule") or a peptide. In some variations, the passivation element may be a multi-component passivation element and may include two or more types of thiol-based passivation molecules, which may be, for example, two or more types of thiol-based small molecules.

[0187] In some variations, the electrode surface is functionalized with redox-active molecules via aptamer immobilization, and analyte binding to these surface sites follows the Michaelis-Menten model. Upon binding to the analyte, the analyte-binding aptamer undergoes a conformational change that either moves the redox-active molecule closer to or further away from the electrode. The redox-active molecule is maintained in its oxidized state, and sweeps to more negative potentials reduce the redox-active molecule within the range of electron transfer. The final relationship is nonlinear, and comparing the signal gain to the logarithm of the analyte concentration is quasi-linear over a limited range. The relationship of the redox current detected at the working electrode is expressed as follows: i=-nFA·dΓO / dt(3) where n is the stoichiometric number of electrons that moderate the redox reaction, F is Faraday's constant, A is the electrode surface area, and dΓO / dt is the change in surface concentration of the oxidized form of the redox active molecule over time.

[0188] Furthermore, because the detection current is a direct function of the electrode surface area A, increasing the surface area of ​​the electrode can increase the sensitivity of the sensor (e.g., amperes per mole of analyte). For example, multiple single working electrodes can be grouped into an array of two or more elements to increase the total effective sensing surface area. Additionally or alternatively, for redundancy, multiple working electrodes can be operated as a parallelized sensor to obtain multiple independent measurements of the concentration of the analyte of interest. The working electrode can operate as an anode (so that the analyte is oxidized at its surface) or as a cathode (so that the analyte is reduced at its surface). i. Biometric Recognition Element

[0189] In some variations, the biorecognition element in the biorecognition layer can be a molecule that selectively binds to a given analyte. In some variations, the biorecognition element in the biorecognition layer can be a molecule that selectively and reversibly binds to a given analyte. In some examples, the biorecognition element can be an oligonucleotide. In some variations, the oligonucleotide can be DNA or RNA. The oligonucleotide can be functionalized at the 3' or 5' end. One end can provide a chemical moiety for surface immobilization ("immobilization moiety"), such as an amine, aldehyde, carboxylic acid, thiol, disulfide, azide, n-hydroxysuccinimide (NHS), maleimide, vinyl, silane, chlorosilane, methoxysilane, ethoxysilane, or acetylene group. The immobilization moiety can be separated from the oligonucleotide sequence by a linker selected for its ability to create distance between the oligonucleotide sequence and the surface to which it is immobilized. The linker may also be selected for compatibility with other chemical layers on the electrode surface, such as hydrocarbon linkers with lengths equal to or similar to those of the hydrocarbon chains used in the self-assembled monolayer coating the remainder of the electrode surface. The opposite end of the oligonucleotide may be functionalized with one or more redox-active molecules that function as probes, such as methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, hydroquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, π-extended tetrathiafulvalene (exTTF), or carboxy-X-rhodamine. These redox-active molecules may also be attached to the oligonucleotide via custom linkers. The backbone of the oligonucleotide may be modified to enhance stability under physiological conditions. For example, RNA arrays incorporating L-ribose or DNA arrays incorporating L-deoxyribose, as opposed to the natural dextrorotatory sugar, can be used to protect the oligonucleotide from degradation by enzymes in the body.In some variations, backbone modifications can include replacing ribose in RNA or deoxyribose in DNA with 2'-O-methylribose, which also has the effect of protecting against enzymatic cleavage under physiological conditions.

[0190] In some variations, the oligonucleotide may comprise a region having a nucleotide sequence complementary to a given nucleic acid analyte, e.g., a viral or bacterial gene or regulatory region, hi some variations, the oligonucleotide may be an aptamer.

[0191] In some examples, the biorecognition element can be a peptide, which can be an antibody or a portion thereof, such as a nanobody (also known as a VHH antibody) that comprises an antigen-binding fragment of a heavy chain-only antibody that selectively binds to a given analyte.

[0192] In some examples, the molecule may be an aptamer ("analyte-binding aptamer"). Aptamers are peptides or single-stranded oligonucleotides that fold into a predetermined structure that selectively binds to a specific analyte (which may be referred to as a target), which may be, for example, a protein, peptide, hormone, nucleic acid, or small molecule. The recognition and binding of an aptamer to its target involves three-dimensional shape-dependent interactions as well as hydrophobic interactions, base stacking, and intercalation, and is typically reversible by dissociation. Aptamers with affinity for a desired target are conventionally selected from large oligonucleotide libraries by a process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Non-binding aptamers are discarded through an iterative process, and aptamers that bind to the proposed target are amplified by polymerase chain reaction (PCR). The iterative process may include counterselection (using interfering substances and structurally similar molecules) to discard aptamers with insufficient selectivity for the analyte. Multiple rounds of SELEX can be performed with increasing stringency to further enrich the oligonucleotide pool until one or more oligonucleotides with the desired degree of affinity and selectivity for the desired target are selected for use.

[0193] In some variations, the analyte-binding aptamer is a cortisol-binding aptamer defined by the following DNA sequence: 5'-GGACGACGCCAGAAGTTTACGAGGATATGGTAACATAGTCGT-3' (SEQ ID NO: 1), where G, A, C, and T represent typical DNA nucleotides including guanine, adenine, cytosine, and thymine, respectively.

[0194] In some variations, analyte-binding aptamers may be selected not for maximum affinity for the analyte, but for an intermediate affinity such that a portion of the population of selected aptamers having bound analyte molecules is sensitive to the physiological concentration range of the analyte in skin interstitial fluid, which may be about 1 pmol / L to about 10 mmol / L or about 0.001 μmol / L to about 1 μmol / L. In some variations, the selection criteria for analyte-binding aptamers may include analyte-binding aptamers having about 10% to about 75% "on" gain from the minimum to the maximum analyte concentration and / or about 10% to about 40% "off" gain from the minimum to the maximum analyte concentration. An "on-gain" signal may refer to a set of square-wave voltammetry parameters (frequency, peak value, step height) selected to maximize the current signal obtained in the presence of the target analyte. An "off-gain" signal may refer to a set of square-wave voltammetry parameters selected to minimize the current signal obtained in the presence of the target analyte. The sensitivity of aptamers to analytes in skin interstitial fluid advantageously makes it possible to avoid interference or signal degradation over time from biofouling or irreversible changes to aptamer structure due to folding or damage.

[0195] The analyte-sensing aptamer can be functionalized with a redox-active molecule, such as methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, hydroquinone, gallocyanine, indophenol, neutral red, dabcyl, exTTF, or carboxy-X-rhodamine. When the aptamer is an oligonucleotide, the redox-active molecule can be functionalized at the 3' or 5' end of the aptamer. The specific and reversible binding of the analyte to the analyte-binding aptamer and the resulting conformational change of the analyte-binding aptamer can result in a change in the proximity between the redox-active molecule and the working electrode to which the aptamer is bound, and thus in the electron transfer characteristics. This corresponds to the analyte concentration. Due to the analyte-binding properties of the aptamer, changes in the electrode's electron transfer properties correspond to analyte concentration, and these can be investigated by various electrochemical techniques, such as voltammetry, potentiometry, chronoamperometry, and / or electrochemical impedance spectroscopy. Voltammetric techniques vary the potential as a function of time and plot the resulting current as a function of potential. For example, cyclic voltammetry (CV) sweeps the cell's potential linearly over a voltage range, while fast-scan CV (FSCV) techniques do this at a faster rate. Alternating current voltammetry (ACV) uses the application of a sinusoidal oscillating voltage to an electrochemical cell. Square wave voltammetry (SWV) uses a square wave superimposed on a step function to provide sweep measurements, providing two sampling instances per potential. As a result of this sampling technique, the contribution to the total current resulting from non-faradaic currents is minimized in SWV. In potentiometry, the open-circuit potential between the reference and working electrodes is measured. In chronoamperometry, the potential is stepped at the beginning of the measurement and then remains constant throughout the duration of the measurement, and the current resulting from this stimulus can be plotted as a function of time.In electrochemical impedance spectroscopy, the complex impedance of an electrode is determined at one or more frequencies. The contributions to the impedance (or admittance) from resistive and reactive circuit elements depend on the position of the redox probe tethered to the surface-bound aptamer and can be correlated to the analyte concentration. ii. Passivation element

[0196] In some variations, the devices described herein may include a passivation element to help reduce or prevent interference within the biorecognition layer and / or at the surface of the electrode material. More specifically, in some instances, functionalizing the available surface of the electrode material solely with biorecognition elements may result in biorecognition element molecules being too close to each other, thus interfering with each other's ability to function as intended. In these instances, occupying all of the available surface of the electrode material with biorecognition element molecules may be disadvantageous or suboptimal. On the other hand, the presence of exposed electrode material surfaces may cause an undesirable increase in electrochemical interactions unrelated to the binding of the biorecognition element with the analyte, resulting in increased noise in the signal generated by the electrode. Therefore, in some variations of the sensors described herein, the biorecognition layer may further include a passivation element, along with the biorecognition element, that acts to shield the exposed electrode material surface from undesired chemical reactions. The passivation element may include multiple passivation molecules that can be directly or indirectly chemisorbed onto the electrode material surface. In some variations, the passivation molecule may be a thiol-group-containing molecule (a "thiol-based passivation molecule"), e.g., a small molecule or a peptide. In some variations, the number of passivation molecules is based on the density of the passivation molecules on the surface of the electrode. For example, the density is about 200 molecules / nm 2 ~about 1000 molecules / nm 2 , about 300 molecules / nm 2 ~about 900 molecules / nm 2 , about 400 molecules / nm 2 ~about 800 molecules / nm 2 , and / or about 500 molecules / nm 2 ~about 700 molecules / nm 2In some variations, the passivating molecules may form a monolayer on the electrode material surface. In some variations, the passivating molecules may form a bilayer or other multilayer (e.g., 3, 4, 5, or more) structure on the electrode material surface. The passivating elements may function to prevent or reduce direct interactions between the electrode material surface and molecules dissolved in biological fluids without significantly interfering with the function of the biorecognition elements. In some variations, the passivating elements described herein may also function to provide or improve biocompatibility and to provide antifouling properties of the working electrode. In some embodiments, the passivating elements may also function to provide a desired surface density of biorecognition elements (e.g., aptamers) interspersed among the passivating molecules on the surface of the electrode material.

[0197] In some variations, the multiple passivating molecules of the passivating element may include thiol-group-containing small molecules (which may be interchangeably referred to herein as "small molecule thiols"). In some variations, the multiple passivating molecules may include 1, 2, 3, 4, 5, 6, 7, 8, or more different types of small molecule thiols. A passivating element that includes two or more types of passivating molecules may be referred to as a "multi-component passivating element." Each small molecule thiol may be tethered to an electrode material and may act to passivate the exposed electrode material (e.g., gold) surface from undesired chemical reactions, such as the reduction of oxygen. In some variations, each small molecule thiol may have the following structure: HS-CX-Z (Formula 1) where X has a value of 2 to 16 and constitutes a linear, unbranched hydrocarbon chain, and Z is a terminal functional group selected from the group consisting of hydrogen, hydroxyl, carboxyl, amine, trimethylammonium, phosphatidylcholine, sulfonate, sulfate ester, phosphonate, phosphate ester, and oligomers of polyethylene glycol (PEG) 1 to 50 units long (also known as PEG / polyethylene oxide (PEO)). For example, a small molecule thiol can contain a hydrophilic moiety at the end opposite the thiol group tethered to the electrode material. In some variations, the small molecule thiol is 1-hexanethiol, 6-mercapto-1-hexaneamine, 6-mercapto-1-phosphatidylcholine hexane, 6-mercapto-1-hexanol (MCH), 7-mercapto-1-heptanol, 8-mercapto-1-octanol (MCO), 9-mercapto-1-nonanol, 10-mercapto-1-decanol, 11-mercapto-1-undecanol, 6-amino-1-hexanethiol, 7-amino-1-heptanethiol, 8-amino-1-octanethiol, 9-amino- 1-nonanethiol, 10-amino-1-decanethiol, 11-amino-1-undecanethiol, (6-mercaptohexyl)-N,N,N-trimethylammonium bromide, (7-mercaptoheptyl)-N,N,N-trimethylammonium bromide, (8-mercaptooctyl)-N,N,N-trimethylammonium bromide, (9-mercaptononyl)-N,N,N-trimethylammonium bromide, (10-mercaptodecyl)-N,N,N-trimethylammonium bromide, (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide, 6-mercaptohexyl phosphate, 7-mercaptoheptyl phosphate, 8-mercaptooctyl phosphate, 9-mercaptononyl phosphate, 10-mercaptodecyl phosphate, 11-mercaptoundecyl phosphate, 6-mercaptohexanoic acid, 7-mercaptoheptanoic acid, 8-mercaptooctanoic acid, 9-mercaptononanoic acid, 10-mercaptodecanoic acid, 11-mercaptoundecanoic acid, 6-mercaptohexanesulfonate, 7-mercaptoheptanesulfonate, 8-mercaptooctane sulfonate, 9-mercaptononanesulfonate, 10-mercaptodecanesulfonate, 11-mercaptoundecanesulfonate, poly(ethylene glycol) dithiol, 1,6-hexanedithiol, 1,7-heptanedithiol, 1,8-octane Dithiols, 1,9-nonanedithiol, 1,10-decanedithiol, 1,11-undecanedithiol, 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate (PC), thiolated oligoethylene glycol (OEG), thiolated polyethylene glycol, (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyltrimethylammonium)chloride (AC), (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethylsulfonate) potassium (SP), and (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyldimethylammonio-(3-propanesulfonate)) (AP), 1,2-Bis-(11-sulfanylundecanoyl)-sn-glycero-3-phosphocholine, methacrylate alkyl thiol ester, methacrylate polyethylene glycol (PEG) thiol ester, acrylate alkyl thiol ester, acrylate PEG thiol ester, vinyl-terminated alkyl thiol, vinyl-terminated PEG thiol, acetylene-terminated alkyl thiol, acetylene-terminated PEG thiol, benzophenone-terminated alkyl thiol, benzophenone-terminated PEG thiol, azide-terminated alkyl thiol, azide-terminated PEG thiol, N-hydroxysulfonyl The thiol may be one selected from the group consisting of succinimide-terminated alkyl thiol, N-hydroxysuccinimide-terminated PEG thiol, ferrocene-terminated alkyl thiol, ferrocene-terminated PEG thiol, methylene blue-terminated alkyl thiol, methylene blue-terminated PEG thiol, anthraquinone-terminated alkyl thiol, anthraquinone-terminated PEG thiol, hydroquinone-terminated alkyl thiol, hydroquinone-terminated PEG thiol, RGD peptide-terminated thiol, YIGSR peptide-terminated thiol, and all homologous series of any of the above thiols having different carbon chain lengths.

[0198] In some variations, in the case of a multi-component passivation element, the plurality of passivation molecules of the passivation element may include a plurality of first small molecule thiols or first thiol-based passivation molecules and a plurality of second, different small molecule thiols or second, different thiol-based passivation molecules. Each of the first thiol-based passivation molecules and each of the second, different thiol-based passivation molecules may be selected from the above list of small molecule thiols. For example, the plurality of first thiol-based passivation molecules may be MCH, and the plurality of second, different thiol-based passivation molecules may be PC. In some variations, the passivation element may further include a plurality of third, different thiol-based passivation molecules, a plurality of fourth, different thiol-based passivation molecules, a plurality of fifth, different thiol-based passivation molecules, and / or a plurality of sixth, different thiol-based passivation molecules. For example, a first thiol-based passivating molecule can be MCH, a second different thiol-based passivating molecule can be PC, and a third different thiol-based passivating molecule can be a ferrocene-terminated alkyl thiol.

[0199] In some variations, the inclusion of a multi-component passivation element (e.g., comprising two or more small molecule thiols) extends sensor lifetime. For example, the sensor lifetime of the sensors described herein can be at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, and / or at least 14 days. Similarly, the sensor half-life, a measure of sensor lifetime, can be at least 3 days, at least 4 days, at least 5 days, at least 6 days, and / or at least 7 days.

[0200] In some variations, the distal end of each of the plurality of passivating molecules can include one or more of a hydrophilic moiety, a hydrophobic moiety, a charged moiety, and a zwitterionic moiety. For example, the distal end of at least one of the plurality of passivating molecules can include a zwitterionic moiety. In some variations, the zwitterionic moiety can be a zwitterionic phosphorylcholine head group.

[0201] In some variations, a pseudo-film may form on the electrode material surface in response to deposition of the passivation element, for example, by the methods described herein with reference to Figures 18A and 18B. The thickness of the pseudo-film may be based in part on whether a monolayer or a multilayer is formed and may therefore depend on the natural length of the small molecule thiol, but the thickness may also be predetermined and controllable during deposition. Nevertheless, the thickness of the pseudo-film formed by the passivating element can be about 0.001 nm to about 1000 nm, about 0.01 nm to about 100 nm, about 0.1 nm to about 10 nm, about 0.05 nm to about 500 nm, about 0.1 nm to about 200 nm, about 1 nm to about 1000 nm, about 1 μm to about 500 μm, about 2 μm to about 400 μm, about 3 μm to about 300 μm, about 4 μm to about 200 μm, about 5 μm to about 100 μm, about 10 μm to about 50 μm, or about 20 μm to about 40 μm. For example, the thickness of the pseudo-film formed by the passivating element can be about 0.1 nm to about 10 nm.

[0202] Without being bound by any particular theory, the antifouling properties provided by utilizing passivating elements on the surface of an electrode material can be improved by configuring the electrode material to have a low net surface charge, e.g., zero net surface charge. When used in biosensors, self-assembled monolayers with a net positive or net negative charge are prone to biofouling due to nonspecific adsorption of proteins such as fibrinogen and lysozyme. As described herein, one possible approach to reducing biofouling may be to use zwitterionic passivating elements, as introduced above. Thus, in some variations, the multiple passivating molecules of the passivating element may include zwitterions. Zwitterions, which may be referred to in the art as "inner salts" or "zwitterions," are molecules containing a mixture of positively charged (cationic) and negatively charged (anionic) functional groups. In some variations, a zwitterion may have an equal number of positively and negatively charged functional groups such that the net charge of the zwitterion is neutral (i.e., the positively and negatively charged functional groups are in balance with each other so that the molecule has a net charge of zero). Zwitterions are typically characterized by their isoelectric point, often expressed as pI, pH(I), or IEP, which is the pH value at which the molecule's net charge is zero. In some variations, the passivating element may be or include a zwitterion.

[0203] In some cases, the zwitterion may be a peptide, in which case it may be referred to as a "zwitterionic peptide." In some variations, the multiple passivating molecules of the passivating element may include zwitterionic peptides. In some variations, the passivating element may include a mixture of one or more types of small molecule thiols and one or more types of zwitterionic peptides. In some variations, the passivating element may consist of one or more types of zwitterionic peptides. The zwitterionic nature of a peptide may be based on the presence of multiple charged functional groups, which may be negatively charged (anionic) or positively charged (cationic). One or more of the charged functional groups may be the R groups (side chains) of the constituent amino acids of a given peptide. Certain amino acids have positively charged (cationic) or negatively charged (anionic) side chains. For example, glutamate or aspartate residues contain a carboxyl group as a negatively charged side chain, and lysine residues contain an amine group as a positively charged side chain. Furthermore, the N-terminus of a peptide is a free amine group, which is a positively charged functional group unless modified to be charge neutral, and the C-terminus of a peptide is a free carboxyl group, which is a negatively charged functional group unless modified to be charge neutral. As used herein, a zwitterionic peptide refers to a peptide of three or more amino acids containing at least one negatively charged functional group and at least one positively charged functional group. In some variations, the zwitterionic peptide contains at least one amino acid with a positively charged side chain, at least one amino acid with a negatively charged side chain, or a combination thereof. In some variations, the zwitterionic peptide may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some variations, the N-terminus may be an unmodified free amine group or a modified N-terminus, which is charge neutral. In some variations, the C-terminus can be an unmodified free carboxyl group or a modified C-terminus that is neutral in charge. For example, to neutralize the C-terminus of a zwitterionic peptide, the C-terminus can be modified to be an amide group, ester, methoxyester, or methoxide group.To render the N-terminus of a zwitterionic peptide neutral, it can be modified to be an amide (e.g., acetamide via N-terminal acetyl modification) or alkylamine (via N-terminal alkylamine modification). In some variations, the number of positive functional groups may exceed the number of negative functional groups by no more than two, or by no more than one. In some variations, a zwitterionic peptide can have an equal number of positively and negatively charged functional groups, resulting in a neutral net charge for the zwitterionic peptide. The charge of a functional group, side chain, or peptide depends on the pH of the surrounding environment. Thus, a functional group, side chain, or peptide is described herein as having a positive, negative, or neutral charge with respect to when the peptide is in an ambient environment at physiological pH (approximately pH 7.4). Using zwitterionic peptides as components of passivation elements in sensors, as described herein, can provide several advantages. These advantages include, but are not limited to, the following: (1) improving the hydrophilicity of the electrode surface; (2) better blocking nonspecific adsorption of proteins on the electrode surface in vivo; (3) reducing the need for biologically incompatible reagents in the manufacturing process; (4) being odorless, thereby eliminating the need for a fume hood during the manufacturing process; and (5) reducing the toxicity of electrodes functionalized therewith when applied to or inserted into a subject.

[0204] In some variations, the amino acids of the zwitterionic peptide can be selected such that the N-terminus, C-terminus, or both can be optionally modified to have a neutral charge, as needed, so that the net charge of the peptide is neutral. In some variations of the zwitterionic peptide, the number of amino acids with negatively charged R groups can be equal to the number of amino acids with positively charged R groups, resulting in a net neutral charge for the zwitterionic peptide. In some variations, the number of amino acids with negatively charged R groups can exceed the number of amino acids with positively charged R groups by one, and the N-terminus can be a positively charged free amine group, and the C-terminus (which in the unmodified state is a negatively charged free carboxyl group) can be a modified C-terminus that is a neutral functional group (i.e., an amide group), resulting in a net neutral charge for the zwitterionic peptide. In some variations, the number of amino acids with positively charged R groups can exceed the number of amino acids with negatively charged R groups by one, the C-terminus can be a negatively charged free carboxyl group, and the N-terminus (which in the unmodified state is a positively charged amine group) can be modified so that the N-terminus can have a neutral functional group such as an amide group, such that the zwitterionic peptide has a net neutral charge.

[0205] In some variations, the at least one negatively charged R group may be a carboxyl group. The carboxyl group may be the side chain of a glutamate or aspartate residue. In some variations, the at least one negatively charged functional group may be located at the N-terminal amino acid of the peptide. In some variations, the at least one negatively charged R group may be included in the N-terminal glutamate or aspartate residue.

[0206] In some variations, the at least one positively charged (cationic) functional group may be one or a combination of two or more of an amine group, a guanidine group, and an imidazole group. The amine group may be the side chain of a lysine residue or the side chain of an ornithine residue. The guanidine group may be the side chain of an arginine residue. The imidazole group may be the side chain of a histidine residue.

[0207] In some variations, the zwitterionic peptide may include at least one thiol group, which may be or may be capable of being chemisorbed to the surface of the electrode material. Thus, the at least one thiol group may form or be capable of forming a thiol linker that connects the zwitterionic peptide to the electrode material. The thiol group may be the side chain of a cysteine ​​residue. In some variations, the cysteine ​​residue may be a terminal cysteine ​​located at the C- or N-terminal position of the zwitterionic peptide. In some variations, the cysteine ​​residue may be a chemically modified cysteine ​​residue, e.g., having an extended carbon chain of 1 to 20 additional carbon atoms included in the thiol group, e.g., between the central alpha carbon atom of the cysteine ​​residue and the sulfur atom of the thiol group. In some variations, the N- or C-terminus of the terminal cysteine ​​residue may be modified to maintain charge neutrality. Without being bound by theory, a terminal cysteine ​​having a negatively charged free amine or a positively charged free carboxyl group at the N-terminus may, at least in some instances, prevent chemisorption of a thiol group to an electrode material. Therefore, it may be advantageous to neutralize either the N-terminus or the C-terminus at which the terminal cysteine ​​is located so as not to impair the peptide's ability to be chemisorbed onto an electrode material. For example, if the terminal cysteine ​​is a C-terminal cysteine, the C-terminus of the C-terminal cysteine ​​can be modified to be an amide group, an ester, a methoxyester, or a methoxide group. For another example, if the terminal cysteine ​​is an N-terminal cysteine, the N-terminus of the N-terminal cysteine ​​can be modified to be an amide (e.g., acetamide) or an alkylamine.

[0208] In some variations, the zwitterionic peptides may include at least one non-standard amino acid, for example, the at least one non-standard amino acid may be ornithine, beta-alanine, γ-aminobutyric acid (GABA), 4-aminobenzoic acid, taurine, or a combination thereof.

[0209] In some variations, the zwitterionic peptide is A-(BA) x -P y C or CP y -(BA) x -B, where A is an amino acid containing a negatively charged side chain, B is an amino acid containing a positively charged side chain, P is a proline residue, and C is a cysteine ​​residue. In some variations, A and / or B can be one of the 20 standard amino acids, a chemically modified standard amino acid, or a non-standard amino acid. In some variations, A can be a glutamic acid residue or an aspartic acid residue. In some variations, B can be a lysine residue, an arginine residue, a histidine residue, or an ornithine residue. In some variations, one or more amino acids can be chemically modified. In some variations, the proline residue can be a chemically modified proline residue. In some variations, the cysteine ​​residue may be a chemically modified cysteine ​​residue, e.g., having an extended carbon chain of 1-20 additional carbon atoms included in its thiol group, e.g., between the central alpha carbon atom of the cysteine ​​residue and the sulfur atom of the thiol group. In some variations, the sequence A-(BA) x -P y C-terminal to the C-terminal cysteine ​​in the case of C, or the sequence CP y -(BA) xIn the case of -B, the N-terminus of the N-terminal cysteine ​​can be modified to have a neutral functional group. For example, the C-terminus of the C-terminal cysteine ​​can be an amide group (via amidation), or the N-terminus of the N-terminal cysteine ​​can be an acetamide group (via acetylation). In some variations, x can be 0 to 5, 0 to 2, 0 to 3, 0 to 4, 1 to 5, 1 to 3, 1 to 4, or 1 to 2, or can be 0, 1, 2, 3, 4, or 5. In some variations, y can be 0 to 5, 1 to 5, 1 to 3, or can be 0, 1, 2, 3, 4, or 5.

[0210] In some variations, the zwitterionic peptide is (N-terminus to C-terminus) X-(KX) x -P y C or CP y -(KX) x X is a glutamic acid residue or an aspartic acid residue, K is a lysine residue, P is a proline residue, and C is a cysteine ​​residue. In some variations, the zwitterionic peptide comprises (from N-terminus to C-terminus): E-(KE) x -P y C or CP y -(KE) x The amino acid sequence X-(KX)-K, where E is a glutamic acid residue, K is a lysine residue, P is a proline residue, and C is a cysteine ​​residue. In some variations, one or more amino acids may be chemically modified. In some variations, the proline residue may be a chemically modified proline residue. In some variations, the cysteine ​​residue may be a chemically modified cysteine ​​residue, e.g., having an extended carbon chain of 1-20 additional carbon atoms included in its thiol group, e.g., between the central alpha carbon atom of the cysteine ​​residue and the sulfur atom of the thiol group. In some variations, the sequence X-(KX) x -P y C or E-(KE) x -P yC-terminal to the C-terminal cysteine ​​in the case of C, or the sequence CP y -(KX) x -K or CP y -(KX) x In the case of -K, the N-terminus of the N-terminal cysteine ​​can be modified to have a neutral functional group. For example, the C-terminus of the C-terminal cysteine ​​can be an amide group (via amidation), or the N-terminus of the N-terminal cysteine ​​can be an acetamide group (via acetylation). In some variations, x can be 0 to 5, 0 to 2, 0 to 3, 0 to 4, 1 to 5, 1 to 3, 1 to 4, or 1 to 2, or can be 0, 1, 2, 3, 4, or 5. In some variations, y can be 0 to 5, 1 to 5, 0 to 3, or 1 to 3, or can be 0, 1, 2, 3, 4, or 5.

[0211] In some variations, the zwitterionic peptide can consist of the amino acid sequence of EKEKEPPC (SEQ ID NO: 2). In some variations, the zwitterionic peptide can consist of EKEPPC (SEQ ID NO: 3), EKEKEPC (SEQ ID NO: 4), EKEPC (SEQ ID NO: 5), EPPC (SEQ ID NO: 6), EKEKEPPPC (SEQ ID NO: 7), EKEPPPC (SEQ ID NO: 8), or EPPPC (SEQ ID NO: 9). In some variations, the C-terminus of the C-terminal cysteine ​​residue of SEQ ID NOs: 2-9 can be modified to be charge neutral. As an example, as provided in SEQ ID NOs: 10-17, the C-terminus of the C-terminal cysteine ​​residue of SEQ ID NOs: 2-9 can be modified to be an amide group via amidation. An example of a zwitterionic peptide (SEQ ID NO: 10) consisting of the amino acid sequence of SEQ ID NO: 2 with a C-terminal cysteine ​​residue modified with a C-terminal amide group via amidation is shown in Figure 26. As shown therein, the C-terminal cysteine ​​provides a thiol group.

[0212] In some variations, the zwitterionic peptide may consist of the amino acid sequence of CPPKEKEK (SEQ ID NO: 18), CPPKEK (SEQ ID NO: 19), CPKEKEK (SEQ ID NO: 20), CPKEK (SEQ ID NO: 21), CPPK (SEQ ID NO: 22), CPPPKEKEK (SEQ ID NO: 23), CPPPKEK (SEQ ID NO: 24), or CPPPK (SEQ ID NO: 25). In some variations, the N-terminus of the N-terminal cysteine ​​residue of SEQ ID NOs: 18-25 may be modified to be charge neutral. By way of example, as provided in SEQ ID NOs: 26-33, the N-terminus of the N-terminal cysteine ​​residue of SEQ ID NOs: 18-25 may be modified to be an amide group (specifically, an acetamide group) via acetylation. iii. Biocompatible layer

[0213] In some variations, the working electrode may further include a biocompatible layer disposed on the biorecognition layer. The biocompatible layer may allow the passage of small molecular weight analytes (e.g., cortisol) therethrough, but may block the passage of relatively high molecular weight biological components (e.g., albumin). In this manner, the biocompatible layer may allow small molecular weight analytes to reach the analyte-binding aptamer while blocking unwanted high molecular weight components.

[0214] In some variations, the biocompatible layer can be a hydrogel comprising a hydrophilic polymer. The hydrophilic polymer can be selected from the group consisting of agarose, poly(urethane), poly(N-vinylpyrrolidone), poly(acrylamide), poly(acrylic acid), poly(methacrylic acid), poly(2-hydroxyethyl methacrylate), poly(acrylic acid-co-acrylamide), poly(N-isopropylacrylamide), poly(2-acrylamido-2-methylpropanesulfonic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(lactic acid), poly(glycolic acid), poly(glycolic acid-co-lactic acid), collagen, alginate, hyaluronic acid, heparin, glycosaminoglycan, chitosan, Nafion, carboxymethylcellulose, or cellulose acetate. In some embodiments, the hydrophilic polymer can be agarose. The agarose may have a sulfate content of less than about 0.5% w / w, less than about 0.4% w / w, less than about 0.3% w / w, less than about 0.2% w / w, and / or less than about 0.1% w / w. The agarose may be a low-electroosmosis agarose characterized by an electroosmosis of about 0.09 to about 0.14, about 0.09 to about 0.13, about 0.09 to about 0.12, or about 0.09 to about 0.11. In some variations, the agarose may have a melting point of about 75°C to about 97°C, about 80°C to about 95°C, or about 85°C to about 90°C. Agarose forms a hydrogel based on a temperature transition during which hydrogen bonds are formed and a physical structure emerges. This process is relatively mild compared to other polymers, which require chemical crosslinking with reactive molecules to form crosslinks between polymer chains. Chemical cross-linking and polymerization typically require harsh chemicals, initiators, and possibly heat or UV light, all of which can contribute to damage to the sensor architecture and result in a less robust and stable aptamer sensor. Therefore, using agarose advantageously reduces chemical wear and tear.

[0215] In some variations, the hydrogel may contain agarose at a concentration of about 0.5% w / w to about 4% w / w, or about 0.5% w / w to about 2% w / w, in buffered saline, e.g., PBS, at the time the hydrogel is applied onto the biorecognition layer. In some variations, the agarose may be dissolved in a buffered saline solution, e.g., PBS, at a temperature of about 85°C to about 95°C, or about 90°C. In some variations, the hydrogel may have a pore size of about 10 nm to about 1000 nm, about 100 nm to about 1000 nm, or about 10 nm to about 100 nm.

[0216] The hydrogel may further comprise an aqueous solvent in which the hydrophilic polymer is dissolved as the hydrogel is formed. The aqueous solvent may be water or a saline solution containing one or more salts, such as NaCl, KCl, NaHPO, KHPO, or a combination thereof. In some embodiments, the saline solution may be a buffered saline solution, such as phosphate buffered solution (PBS), acetate buffer, Tris buffer, citrate buffer, McIlvaine buffer, Tris-acetate-EDTA buffer, or Tris-EDTA buffer.

[0217] The biocompatible layer can be applied over the biorecognition layer and / or the diffusion-limiting layer (if present) by at least one of spray coating, dip coating, chemical vapor deposition, drop casting, plasma deposition, chemical vapor deposition, and electrodeposition.

[0218] Hydrogels are generally continuous, three-dimensional networks of hydrophilic polymers with water dispersed throughout. The degree of hydration of a hydrogel can range from fully hydrated to fully dried. A fully hydrated hydrogel can be fully hydrated so that its degree of hydration is in equilibrium with the aqueous environment in which it is placed. A partially hydrated (or partially dried) hydrogel will take up more water and thus "swell" when placed in an aqueous environment, whereas a fully hydrated hydrogel will not swell further. A partially dried hydrogel can be fully dried so that its degree of hydration is in equilibrium with the humidity of the air to which the hydrogel (e.g., on the working electrode) is exposed, for example, during storage. As used herein, a fully dried hydrogel is substantially free of water. Generally, a dried hydrogel can be rehydrated and reconstituted to a fully hydrated state when placed in an aqueous environment. The hydrogel of the biocompatible layer is generally fully hydrated when the sensor is used, but the hydrogel of the biocompatible layer may be fully or partially dried during storage, i.e., after the electrode (or sensor including one or more electrodes) is produced and before use on a subject.

[0219] In some variations, for example, during storage, the hydrogel of the biocompatible layer may be at least partially dried. In some variations, the at least partially dried hydrogel may contain about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% water compared to the fully hydrated state of the hydrogel. In some variations, the at least partially dried hydrogel may be completely dried.

[0220] In some variations, the at least partially dried hydrogel may have a thickness of about 0.001 μm to about 1000 μm, about 0.01 μm to about 100 μm, about 0.1 μm to about 10 μm, about 1 μm to about 50 μm, about 5 μm to about 30 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 200 μm, or about 1 μm to about 1000 μm (including all values ​​and subranges within any of the foregoing). It is understood that the thickness of the hydrogel will depend on the degree of drying. Working Electrode - Exemplary Architecture

[0221] 16A shows a schematic diagram of an exemplary set of layers for an analyte-sensing working electrode 1610A. For example, as described above, in some variations, the working electrode 1610A can include an electrode material 1612 and a biorecognition layer 1614 including a biorecognition element immobilized on the surface of the electrode material 1612. In some variations, the biorecognition layer 1614 further includes a passivation element, which can include one or more of the small molecule thiols described above, bound to the surface of the electrode material 1612 around the immobilized biorecognition element of the biorecognition layer 1614. In some variations, the biorecognition element can be an analyte-binding aptamer functionalized with a redox-active molecule. The electrode material 1612 can be used to electrically detect a change in electron transfer properties between the redox-active molecule bound to the analyte-binding aptamer included in the biorecognition layer 1614 and the electrode material 1612 caused by analyte binding to the analyte-binding aptamer. The electrode material 1612 also provides ohmic contact and routes the electrical signal from the electrocatalytic reaction to the processing circuitry. In some variations, the passivation element may include a multi-component passivation element (e.g., including two or more small molecule thiols). In some variations, the electrode material 1612 may be platinum or gold, as shown in FIG. 16A . In other variations, the electrode material 1612 may include, for example, silver, palladium, iridium, rhodium, ruthenium, titanium, nickel, alloys of the aforementioned metals, cobalt-chromium alloys, cobalt-chromium-molybdenum alloys, stainless steel 316L, iridium oxide, titanium nitride, carbon, doped diamond, boron-doped diamond, silicon, doped silicon, or other suitable materials. The carbon may be pyrolytic carbon, pyrolytic graphite, or glassy carbon.

[0222] In some variations, the electrode material 1612 may be coated with a highly porous electrocatalytic layer, such as a platinum black layer 1613, which may increase the electrode surface area for enhanced sensitivity (e.g., as shown in FIG. 16D). In some variations, the platinum black layer 1613 may be omitted (e.g., as shown in FIGS. 16A and 16G). In some variations, the electrode material 1612 may be or include unpolished electroplated gold, which may have a roughened surface and thereby increase the electrode surface area.

[0223] In some variations, the biorecognition layer 1614 may include a conductive polymer. The conductive polymer may be permselective to contribute to the robustness of the biorecognition layer to circulating endogenous electroactive species (e.g., ascorbic acid, vitamin C, etc.), fluctuations of which can adversely affect sensor sensitivity. Such permselective conductive polymers in the biorecognition layer may be more robust to pharmacological interferences in interstitial fluid (e.g., acetaminophen) that can affect sensor accuracy. The conductive polymer may be made permselective, for example, by removing excess charge carriers through an oxidative electropolymerization process or by intentionally peroxidizing the conductive polymer at a high potential after polymerization, disrupting its conjugated backbone and rendering it non-conductive. These oxidatively polymerized conductive polymers exhibit permselectivity and thus can exclude ions of similar charge polarity (net positive or negative) to the dopant ion or by size exclusion due to the conductive polymer's dense, compact morphology. In some variations, the conductive polymer can include one or more of aniline, pyrrole, pyrrole-3-carboxylic acid, pyrrole-1-propionic acid, acetylene, phenylene, phenylene vinylene, phenylenediamine, thiophene, 3-methylthiophene, 3-hexylthiophene, 3-thiophenecarboxylic acid, 3,4-ethylenedioxythiophene (EDOT), EDOT carboxylic acid, and aminophenylboronic acid. An example of a working electrode in which the biorecognition layer 1714 includes a film 1723 of a conductive polymer is shown in Figures 17A and 17B.

[0224] In some variations, the conductive polymer may exhibit self-sealing and / or self-healing properties. For example, the conductive polymer may undergo oxidative electropolymerization, in which the conductive polymer may lose its conductivity as the thickness of the conductive polymer deposited on the electrode increases until the lack of sufficient conductivity reduces the deposition of additional conductive polymer. If the conductive polymer suffers minor physical damage (e.g., during use), the polymer backbone may reassemble to neutralize free charges, thereby lowering the overall surface energy of the molecular structure, which may manifest as self-sealing and / or self-healing properties.

[0225] Examples of working electrodes including electrode materials and aptamer-based analyte-sensing biorecognition layers are shown in Figures 16J-M, 17A-C, 17E, 17G, and 17I.

[0226] Generally, an aptamer-based analyte sensor can include a working electrode containing an electrode material, a passivation element, and an analyte-sensing aptamer. The analyte-sensing aptamer and passivation element can be disposed on the electrode material. The aptamer can be functionalized with a redox reporter molecule and tethered directly or indirectly to the electrode material. In some variations, the passivation elements of Figures 16J, 16L, and 16M can act to passivate the exposed surface of the electrode material from undesired chemical reactions, such as oxygen reduction. The passivation element can also be used to provide a desired surface density of analyte-sensing aptamers on the surface of the electrode material.

[0227] 16J shows an aptamer-based analyte sensor including a working electrode 1610J including a biorecognition layer 1614J including a passivation element 1629J and an analyte-sensing aptamer 1625J functionalized with a redox reporter molecule 1626J. The passivation element 1629J may include a single type of thiol-based small molecule and thereby be tethered to the electrode material 1612J of the working electrode 1610J, and the analyte-sensing aptamer 1625J may be tethered to the electrode material 1612J, optionally via a linker 1628J. The redox reporter molecule 1626J may be, for example, methylene blue (MB), ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, exTTF, or carboxy-X-rhodamine. The linker 1628J may be any functional group or molecule (including a polymer) that provides a covalent bond between the terminus of the aptamer 1625J and the electrode material 1612J. The identity of the linker 1628J may vary based on the composition of the electrode material. For example, the linker chemistry available for a platinum electrode surface may be different from that for a gold electrode surface.

[0228] 16L shows an aptamer-based analyte sensor including a working electrode 1610L including a biorecognition layer 1614L that includes a multi-component passivation element and an analyte-sensing aptamer 1625L functionalized with a redox reporter molecule 1626L. The multi-component passivation element can include a first small molecule thiol 1630L and a second, different small molecule thiol 1631L, each of which can be tethered to an electrode material 1612L of the working electrode 1610L. The analyte-sensing aptamer 1625L can optionally be tethered to the electrode material 1612L via a linker 1628. The redox reporter molecule 1626L may be, for example, methylene blue (MB), ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, exTTF, or carboxy-X-rhodamine. The linker 1628L may be any functional group or molecule (including a polymer) that provides a covalent bond between the terminus of the aptamer 1625L and the electrode material 1612L. The identity of the linker 1628L may vary based on the composition of the electrode material. For example, the linker chemistry available for a platinum electrode surface may be different from that for a gold electrode surface.

[0229] 16M shows an aptamer-based analyte sensor including a working electrode 1610M including a biorecognition layer 1614M that includes a multi-component passivation element, and an analyte-sensing aptamer 1625M functionalized with a redox reporter molecule 1626M. The multi-component passivation element can include a first small molecule thiol 1630M, a second, different small molecule thiol 1631M (each of which can be tethered to an electrode material 1612M of the working electrode 1610M), and a zwitterion 1632M (which, in some variations, can be a zwitterionic peptide or a thiol-based small molecule with a zwitterionic phosphorylcholine head group). The analyte-sensing aptamer 1625M can be tethered to the electrode material 1612M, for example, via a linker 1628M. The redox reporter molecule 1626M may be, for example, methylene blue (MB), ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, exTTF, or carboxy-X-rhodamine. The linker 1628M may be any functional group or molecule (including a polymer) that provides a covalent bond between the terminus of the aptamer 1625M and the electrode material 1612M. The identity of the linker 1628M may vary based on the composition of the electrode material. For example, the linker chemistry available for a platinum electrode surface may be different from that for a gold electrode surface, as described in more detail herein.

[0230] Linker chemistries, which can generally include thiols, silanes, covalent chemistries, and oxidation-based amines, are selected according to the molecules to be linked and based on the particular electrode material used. For example, thiols can be attached to many electrode materials, including platinum electrodes. Silanes can be attached to oxide films on the surface of various electrode materials. Any carbon-containing electrode material can form direct covalent bonds with organic molecules. For example, carbon-based electrode materials can be oxidized to carboxylic acids, and then organic molecules can be coupled with amines to form amide bonds connecting them to the electrode material surface. Any of the electrode materials described herein can have a polymer deposited on its surface, which can be used as an insulating material and / or as a site for attachment of biorecognition elements. In these variations, the polymer can be a conductive polymer. Various examples of linker chemistries are described below with respect to Figures 17A-17K.

[0231] 17B, ​​the electrode material 1712 may be platinum, gold, silver, palladium, iridium, rhodium, ruthenium, titanium, nickel, alloys of the above metals, cobalt-chromium alloys, cobalt-chromium-molybdenum alloys, stainless steel 316L, iridium oxide, indium oxide, titanium nitride, carbon, boron-doped diamond, doped diamond, silicon, doped silicon, or other suitable material. The carbon may be pyrolytic carbon, pyrolytic graphite, or glassy carbon.

[0232] The biorecognition layer 1714 may optionally include a conductive polymer layer 1723 having an analyte-binding aptamer 1725 tethered to the conductive polymer layer 1723 via a linker. The linker may be an amide linker formed from a carboxyl group and a primary amine group. In some variations, the carboxyl group may be substituted with an NHS-ester, an isocyanate, an isothiocyanate, or a benzoyl fluoride. The present disclosure also provides aptamers tethered via other linkers, such as triazole linkers or thioether linkers. The thioether linker may be based on a combination of a maleimide moiety on the conductive polymer and a thiol moiety on the terminus of the aptamer, a combination of a vinyl surface attached to the conductive polymer layer and a thiol moiety on the terminus of the aptamer, or a combination of an epoxide moiety on the conductive polymer and a thiol moiety on the terminus of the aptamer.

[0233] The analyte-binding aptamer 1725 may be functionalized with a redox-active molecule 1726, such as methylene blue (MB), ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, exTTF, or carboxy-X-rhodamine.

[0234] The conductive polymer layer 1723 may have a thickness of about 1 nm to about 100 nm and includes a conductive polymer and, optionally, a counterion. Examples of conductive polymers include one or more of aniline, pyrrole, pyrrole-3-carboxylic acid, pyrrole-1-propionic acid, acetylene, phenylene, phenylene vinylene, phenylenediamine, thiophene, 3-methylthiophene, 3-hexylthiophene, 3-thiophenecarboxylic acid, EDOT, EDOT carboxylic acid, and aminophenylboronic acid. The conductive polymer may also be a copolymer of two or more of the listed monomers. Examples of counterions include chloride, phosphate, acetate, sulfate, bisulfate, nitrate, bromide, perchlorate, hexafluorophosphate, tetrafluoroborate, para-toluenesulfonate, benzenesulfonate, camphor-10-sulfonate, trifluoromethanesulfonate, bis(trifluoromethylsulfonyl)imide, dodecylbenzenesulfonate, poly(styrenesulfonate), poly(styrenesulfonate-co-acrylic acid), poly(acrylic acid), poly(methacrylic acid), poly(acrylic acid-co-acrylic acid), and poly(methacrylic acid-co-acrylic acid). Examples of polymers that may be used include but are not limited to: poly(2-acrylamido-2-methylpropanesulfonic acid), poly(styrenesulfonate-block-butylene-ran-ethylene-block-styrenesulfonate), ...2-acrylamido-2-methylpropanesulfonic acid), poly(styrenesulfonate-block-butylene-ran-ethylene-block-styrenesulfonate), alginate, glycosaminoglycans, hyaluronic acid, collagen, and any combination of the foregoing polymers as copolymers or block copolymers not explicitly mentioned.

[0235] 17C , electrode material 2712 may be gold, and biorecognition layer 2714 may include analyte-sensing aptamers 2725 functionalized with redox reporter molecules 2726, such as methylene blue (MB), ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, exTTF, or carboxy-X-rhodamine. The analyte-sensing aptamers may be tethered to electrode material 2712 via thiol linkers 2728. Optionally, biorecognition layer 2714 may further include a passivation element comprising passivation molecules 2729. Passivation molecules 2729 may provide a desired surface density of analyte-sensing aptamers 2725 on the surface of electrode material 2712. In some variations, passivating molecules 2729 may include small molecule thiols and / or zwitterions (e.g., zwitterionic peptides or thiol-based small molecules with zwitterionic phosphorylcholine head groups), which may be tethered to electrode material 2712 as described herein.

[0236] 17E, the electrode material 3712 may be optionally doped silicon. The biorecognition layer 3714 may include a monolayer of silane 3741 bound to the silicon surface and an analyte-sensing aptamer 3725 functionalized with a redox reporter molecule 3726 (e.g., methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, or carboxy-X-rhodamine) optionally tethered to the silicon electrode material via an amide linker.

[0237] 17G, the electrode material 4712 may be carbon, optionally pyrolytic carbon, pyrolytic graphite, or glassy carbon. The biorecognition layer 4714 may include an analyte-sensing aptamer 4725 functionalized with a redox reporter molecule 4726 (e.g., methylene blue (MB), ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, exTTF, or carboxy-X-rhodamine) optionally tethered to the carbon electrode material via an amide linker.

[0238] 17B, ​​17C, 17E, and 17G (and 17I-17K) show working electrodes having their respective biorecognition layers without a biocompatible layer, it will be understood that each of the exemplary working electrodes can further include a biocompatible layer applied and disposed thereon, as shown in FIG. 16K. The biocompatible layer can be a hydrogel biocompatible layer, as described below.

[0239] As shown in Figures 16A, 16D, 16G, and 16K, in some variations, the working electrode may further include a biocompatible layer 1616. The biocompatible layer 1616 may allow the passage of small molecular weight analytes (e.g., cortisol) to reach the analyte-binding aptamer 1625 while blocking the passage of relatively high molecular weight biological components, such as cells, cell debris, or macromolecular aggregates. The size selectivity of the biocompatible layer 1616 depends on the material comprising the layer, such as the pore size. Such selective passage can prevent or reduce nonspecific adsorption and accumulation of high molecular weight biological components on the biorecognition layer 1614. Adsorption and / or accumulation of relatively high molecular weight biological components may interfere with the conformational switching mechanism of the analyte-sensing aptamer 1625 and reduce the responsiveness of the working electrode. The biocompatible layer 1616 may also reduce foreign body reactions, for example.

[0240] The biocompatible layer 1616 may generally be applied over the biorecognition layer 1614 after it has been deposited (i.e., after it is already in place). As such, the biocompatible layer 1616 is shown in FIGS. 16A, 16D, and 16G diagrammatically as being disposed on and above the biorecognition layer 1614. That said, the biorecognition layer, as shown in FIGS. 17A-17K, for example, may be a nanoscale layer of certain molecular or macromolecular components and may be substantially thinner than the biocompatible layer. It will be understood, therefore, that the depictions of the biorecognition layer 1614 in FIGS. 16A, 16D, and 16G are schematic in nature and may not reflect their actual size.

[0241] In some variations, the biocompatible layer may be applied individually to each working electrode. In some variations, the biocompatible layer may be generally disposed over all or a portion of the electrode and / or microneedle array including multiple electrodes. As a result, the biocompatible layer may have a surface area substantially larger than that of an individual electrode. It will be understood that the biocompatible layer may actually extend laterally beyond the surface of a given electrode. Furthermore, in some variations, multiple electrodes may be covered with the same biocompatible layer, as shown, for example, in FIG. 21C.

[0242] In some variations, application of the biocompatible layer may be limited to the portion of the electrode array that includes the working electrode. Figures 16A-16C, for example, show a working electrode 1610A (Figure 16A) that includes a biocompatible layer 1616 disposed on a biorecognition layer 1614, as well as a counter electrode 1620A (Figure 16B) and a reference electrode 1630A (Figure 16C), each of which lacks a biocompatible layer. In other variations, the biocompatible layer may be applied generally to one or more surfaces (e.g., the skin-facing or skin-penetrating surface) of the electrode array or microneedle array, such that one or more of the counter and reference electrodes, including, for example, all of the electrodes in the array, include a biocompatible layer. Figures 16G-16I, for example, show a working electrode 1610C (Figure 16G), a counter electrode 1620C (Figure 16H), and a reference electrode 1630C (Figure 16I), each of which includes a biocompatible layer. Opposite

[0243] As described above, the counter electrode is the electrode that supplies or sinks the electrons (via current) necessary to sustain the electrochemical reaction at the working electrode. To avoid the current-carrying capacity of the counter electrode limiting the change in electron transfer properties between the redox-active molecules at the working electrode and the electrode material 1612, the number of counter electrode components can be increased to increase the surface area, in the form of a counter electrode array. Therefore, to avoid current-carrying capacity limitations, it may be desirable to have excess counter electrode area relative to the working electrode area. When the working electrode operates as an anode, the counter electrode functions as a cathode, and vice versa. Similarly, when an oxidation reaction occurs at the working electrode, a reduction reaction occurs at the counter electrode, and vice versa. Unlike the working or reference electrode, the counter electrode can dynamically swing to the potential necessary to sustain the change in the electron transport properties of the working electrode.

[0244] 16B, counter electrode 1620A can include electrode material 1622, similar to electrode material 1612. For example, similar to electrode material 1612, electrode material 1622 of counter electrode 1620A can include gold, platinum, palladium, iridium, rhodium, ruthenium, titanium, nickel, alloys of the aforementioned metals, cobalt chromium alloys, cobalt chromium molybdenum alloys, stainless steel 316L, iridium oxide, titanium nitride, carbon, doped diamond, silicon, doped silicon, or other suitable materials.

[0245] In some variations, the counter electrode may have little or no additional layer over the electrode material 1622 (e.g., counter electrode 1620A as shown in FIG. 16B). However, in some variations, the counter electrode 1620 may benefit from an increased surface area to increase the amount of current it can support. For example, the counter electrode material 1622 may be textured or otherwise roughened to increase the surface area of ​​the electrode material 1622 to enhance its current source or sink capability. Additionally or alternatively, the counter electrode may include a layer of platinum black 1624 as shown in FIG. 16E, as counter electrode 1620B, which may enhance the electrode surface as described above with respect to some variations of the working electrode. However, in some variations of the counter electrode, the platinum black layer may be omitted (e.g., counter electrodes 1620A and 1620C as shown in FIGS. 16B and 16H, respectively).

[0246] Additionally or alternatively, in some variations shown in Figure 16H, the counter electrode 1620C may include a biocompatible layer 1626 disposed over the electrode, e.g., to reduce foreign body reaction. The biocompatible layer 1626 may be similar in structure and composition to the biocompatible layer 1616 described above with respect to Figure 16A, for example. reference electrode

[0247] As noted above, the reference electrode functions to provide a reference potential for the system; that is, the potential to which the working electrode is biased is referenced to the reference electrode. A fixed or at least controlled potential relationship may be established between the working and reference electrodes, and, within practical limits, no current is sourced from or sunk by the reference electrode.

[0248] As shown in FIG. 16C , the reference electrode 1630A can include an electrode material 1632, similar to the electrode material 1612. In some variations, similar to the electrode material 1612, the electrode material 1632 of the reference electrode 1630A can include a metal salt or metal oxide that functions as a stable redox coupled to a known electrode potential. For example, the metal salt can include, for example, silver-silver chloride (Ag / AgCl), and the metal oxide can include iridium oxide (IrOx / Ir2O3 / IrO2). In other variations, noble and inert metal surfaces, as well as those coated with conductive polymers such as poly(3,4-ethylenedioxythiophene), can function as quasi-reference electrodes and can include gold, platinum, palladium, iridium, carbon, doped diamond, and / or other suitable catalytic and inert materials. Furthermore, in some variations, the reference electrode 1630A can be textured or otherwise roughened to enhance adhesion with any subsequent layers. Such subsequent layers on the electrode material 1632 can include a platinum black layer 1634 (as shown in FIG. 16F as reference electrode 1630B) and / or a biocompatible layer 1637 (as shown in FIG. 161 as reference electrode 1630C). The biocompatible layer 1637 can be similar to the biocompatible layer 1616 described above with respect to FIG. 16A, for example. In some variations, the platinum black layer can be omitted (e.g., as shown in FIGS. 16C and 161 as reference electrodes 1630A and 1630B, respectively).

[0249] The reference electrode 1630A, in some variations, may further include a redox couple layer 1636, which may include a surface-immobilized solid-state redox couple with a stable thermodynamic potential. For example, the reference electrode may operate at a stable standard thermodynamic potential relative to a standard hydrogen electrode (SHE). High electrode potential stability may be achieved by using a redox system in which the concentrations of each participant in the redox reaction are constant (e.g., buffered or saturated). For example, the reference electrode may include a metal with an Ag / AgCl salt film (E = +0.197 V vs. SHE) or IrOx (E = +0.177 V vs. SHE, pH = 7.00) within the redox couple layer 1636. In some variations, the reference electrode may be used as a half-cell to construct a complete electrochemical cell. Electrode-method

[0250] The various layers of the working, counter, and reference electrodes may be applied and / or functionalized, etc. to the microneedle array using suitable processes as described below.

[0251] In a microneedle array pretreatment step, the microneedle array can be plasma cleaned in an inert gas (e.g., an RF-generated inert gas such as argon) plasma environment to make the surfaces of materials, including electrode materials (e.g., electrode materials 1612, 1622, and 1632 as described above), more hydrophilic and chemically reactive. This pretreatment not only physically removes organic debris and contaminants, but also serves to clean and prepare the electrode surfaces to enhance adhesion of subsequently deposited films thereon. working electrode

[0252] Anodization: To configure the working electrode after the pretreatment step, in some variations, the electrode material 1612 may undergo an anodization process using an amperometric approach. For example, the electrode component assigned to the working electrode function may be subjected to a fixed, high anodic potential (e.g., +1.0 V to +1.3 V vs. an Ag / AgCl reference electrode) in a medium-strength acid solution (e.g., 0.1 M to 3 M H2SO4) for a suitable time (e.g., about 30 seconds to about 10 minutes). This process may produce a thin but stable native oxide layer on the electrode surface. Due to the low pH that occurs at the electrode surface, trace contaminants may be removed as well.

[0253] In an alternative embodiment using coulometric techniques, anodization can proceed until a specified amount of charge (measured in coulombs) has passed. As described above, an anodic potential may be applied, however, the duration of this can vary until the specified charge amount has elapsed.

[0254] Activation: Following the anodization process, the working electrode component may be subjected to a periodically scanned potential waveform in an activation process using cyclic voltammetry (CV), in some variations. In the activation process, which may occur in a moderately strong acidic solution (e.g., 0.1-3 M H2SO4), the applied potential may be varied over time with a suitable function (e.g., a sawtooth function or a trigonometric function). For example, the voltage may be linearly scanned between cathodic values ​​(e.g., between -0.3 V and -0.2 V vs. an Ag / AgCl reference electrode) and anodic values ​​(e.g., between +1.0 V and +1.3 V vs. an Ag / AgCl reference electrode) in an alternating function (e.g., between 15 and 50 linear sweep segments). In one example, the working electrode component may be subjected to a fast CV (at approximately 500 mV / s), followed by two rounds of slow CV (at approximately 100 mV / s), with the acid solution replaced between each step. The scan rate of this waveform can vary between 1 mV / s and 1000 mV / s. Note that the current peak occurring during the anodic sweep (positive sweep) corresponds to the oxidation of a species, and the current peak occurring during the subsequent cathodic sweep (negative sweep) corresponds to the reduction of that species.

[0255] Gold Electrodeposition: Following the activation and cleaning process, the working electrode component may, in some variations, be subjected to a cyclically scanned potential waveform or a constant potential to electrodeposit gold metal onto the surface. In the deposition process, which may occur with dilute concentrations of gold complex (e.g., 0.5 wt.% to 3 wt.% AuCl3 or Au(CN)2), the applied potential may be varied over time in a suitable manner. For example, the voltage may be linearly scanned between anodic values ​​(e.g., between +0.2 V and +1.2 V vs. an Ag / AgCl reference electrode) in an alternating function (e.g., 10 to 30 linear sweep segments). The scan rate for this waveform may range from 100 mV / s to 1000 mV / s. Note that the rise in current during the cathodic sweep (the sweep to the negative electrode) corresponds to the reduction of gold species in the solution and plating onto the working electrode surface. Note also that the sudden increase in current during the first scan is due to the formation of nucleation sites. In some variations, the gold may be deposited through physical vapor deposition.

[0256] Gold Surface Activation: Depending on the method of gold deposition on the working electrode (provided gold is used), the electrode may require electrochemical activation in some variations. Activation can begin with a basic solution (e.g., 0.1 M to 3 M NaOH), and the applied potential can be varied over time with a suitable function (e.g., a trigonometric function) such as cyclic voltammetry. For example, the voltage can be linearly scanned between cathodic values ​​(e.g., between -2.0 V and -1.2 V vs. an Ag / AgCl reference electrode) and anodic values ​​(e.g., between -1.1 V and -0.5 V vs. an Ag / AgCl reference electrode) in an alternating function (e.g., 50 to 1000 sweep segments). The scan rate for this waveform can range from 10 mV / s to 2000 mV / s. After potential cycling in alkaline medium, the gold electrode must be cycled in an acidic solution (e.g., 0.1 M to 3 M H2SO4). The potential can be varied over time according to a suitable function (e.g., a trigonometric function). For example, the voltage me is linearly scanned between cathodic values ​​(e.g., between −0.2 V and +0.2 V vs. an Ag / AgCl reference electrode) and anodic values ​​(e.g., between +1.2 V and +1.8 V vs. an Ag / AgCl reference electrode) in an alternating function (e.g., 50 to 1000 sweep segments). The scan rate for this waveform can range from 10 mV / s to 2000 mV / s. It should be noted that in both basic and acidic media, current peaks occurring during the anodic sweep (swirl toward the positive electrode) correspond to the oxidation of a chemical species, while current peaks occurring during the subsequent cathodic sweep (swirl toward the negative electrode) correspond to the reduction of that species.

[0257] Biorecognition Layer Functionalization: Following the activation process, the working electrode component may, in some variations, be functionalized with a biorecognition layer 1614 as described above. Assuming the working electrode associated with the microneedle array has undergone the above-described steps, such as deposition and activation, the biorecognition layer may be applied in a variety of ways. The biorecognition layer application process may depend on various factors, including what material is used for the working electrode material 1612. Various exemplary processes are described herein below.

[0258] In some variations, the electrode material 1612 is electrochemically coated with a conductive polymer film by oxidation of reactive monomers at the electrode surface, either potentiostatically (e.g., chronoamperometry), potentiodynamically (e.g., cyclic voltammetry), or galvanostatically (e.g., chronopotentiometry). The conditions required for polymerization of the monomer depend on the chemical properties of the conductive polymer used. By adjusting the current, potential, sweep rate, and / or duration of the electrolysis process, the final properties of the conductive polymer film can be controlled (e.g., thickness, conductivity, permselectivity). Optionally, the conductive polymer film may be overoxidized in a secondary oxidation step by potentiostatic, potentiodynamic, or galvanostatic electrochemical processes. This secondary oxidation step can be used to modify the conductivity and barrier properties of the conductive polymer film. The conductive polymer and / or counterions incorporated within the conductive polymer may provide pendant chemical moieties for covalent attachment to aptamers functionalized with compatibility moieties. By way of example, the pendant chemical moiety in the conductive polymer film may comprise a carboxyl group and the aptamer may be functionalized at the 3' or 5' end with a primary amine. Alternatively, the pendant chemical moiety in the conductive polymer film may be a primary amine and the aptamer may be functionalized at the 3' or 5' end with a carboxyl group.

[0259] In some variations, for example where the electrode material 1612 is gold, the aptamer may be functionalized at one end with a reactive organosulfur compound (e.g., thiol / mercaptan, disulfide) that spontaneously reacts and attaches to the surface of the gold working electrode material. The remaining gold surface of the working electrode may optionally be passivated, for example, with a small molecule containing a terminal thiol group to form a self-assembled monolayer.

[0260] 17A, the working electrode 1710 may be functionalized with an aqueous solution containing a monomer precursor of a conductive polymer and, optionally, a counterion. Functionalization may be performed by constant current electrodeposition (e.g., chronopotentiometry), where the anodic current density through the working electrode is 0.05 mA / cm. 2 ~5mA / cm 2 and the duration may be from about 1 minute to about 100 minutes. In another variation, the functionalization may be performed through chronocoulometric deposition, and the deposition charge may be about 1 mC / cm 2 ~10C / cm 2 The duration may be about 1 minute to about 100 minutes. The conductive polymer, the counterion, or both may contain carboxyl groups 1721. In this process, as shown in FIG. 17A, a thin film (e.g., about 10 nm to about 100 nm) of biorecognition layer 1714 including conductive polymer layer 1723 can be formed (e.g., by electrodeposition or electropolymerization) on the surface of electrode material 1712, which may include platinum. After conductive polymer layer 1723 is formed, analyte-sensing aptamer 1725 functionalized at one end with primary amine group 1727 and at the other end with redox-active molecule 1726 is introduced. Aptamer 1725 is covalently bound to carboxyl groups 1721 included in conductive polymer layer 1723 using a carbodiimide crosslinking method, for example, EDC / NHS coupling using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). An alternative method for linking primary amines and carboxylic acids to amide bonds is by DMTMM crosslinking with 4-(4,6-dimethoxy-1,3,5 triazin-2-yl)-4-methyl-morpholinium chloride. The remaining pendant activated carboxylic acid groups on the silane layer that are not bound to the aptamer can optionally be crosslinked to another amine-containing molecule (e.g., ethanolamine, glycine, lysine, PEG-amine) to alter the working electrode surface properties and biocompatibility. The resulting functionalized working electrode 1710 is shown in Figure 17B.

[0261] In some variations, the arrangement of the carboxyl and primary amine groups for carbodiimide crosslinking may be reversed, so that the conducting polymer, the counterion, or both may contain primary amine groups, and the aptamer may be functionalized with the carboxyl groups. The two moieties may then be linked by EDC / NHS coupling or DMTMM crosslinking, as described above with respect to Figure 17A. An example of a conducting polymer containing primary amine groups is a p-phenylenediamine (PPD)-based polymer.

[0262] While Figures 17A and 17B show aptamers tethered via an amide linker formed between a carboxyl group and a primary amine group, the present disclosure provides for the use of other moieties. For example, the carboxyl group may be substituted with an NHS-ester, an isocyanate, an isothiocyanate, or a benzoyl fluoride. The present disclosure also provides for aptamers tethered via other linkers, such as a triazole linker or a thioether linker. The thioether linker may be based on a combination of a maleimide moiety on the conductive polymer and a thiol moiety on the terminus of the aptamer, a combination of a vinyl surface attached to the conductive polymer layer and a thiol moiety on the terminus of the aptamer, or a combination of an epoxide moiety on the conductive polymer and a thiol moiety on the terminus of the aptamer.

[0263] In an exemplary variation shown in FIG. 17D, a working electrode 3710 comprising silicon electrode material 3712 may be functionalized with aptamer 3725 via silane chemistry. The silicon electrode material may comprise doped silicon. First, the surface of the silicon electrode material 3712 is activated by exposure to oxygen plasma at an operating power of about 50 W to about 500 W and a pressure of about 200 mTorr to about 700 mTorr for about 0.5 to about 10 minutes. The activated electrode material surface is then transferred to a vacuum chamber containing a small amount of a suitable reactive silane 3741 comprising a terminal primary amine (e.g., 4-aminobutyltriethoxysilane, shown in FIG. 17D) at a pressure of about 1 Torr to about 100 Torr for 10 to 90 minutes. The working electrode is then removed, rinsed with an organic solvent (e.g., methanol, ethanol, or isopropanol), and then baked at about 80°C to about 140°C for about 10 minutes to about 60 minutes to cure the attached silane 3741. Figure 17D schematically illustrates a silicon electrode material 3712 having a monolayer of attached silane 3741. Also shown in Figure 17D, the electrode material is treated with a solution containing an aptamer 3725 terminated at one end with a carboxyl group 3743 and at the other end with a redox-active molecule 3726. The solution can be an aqueous solution, such as a phosphate buffer solution, with an aptamer concentration of about 0.1 μM to about 2 μM. The carboxyl group 3743 forms an amide linker with the pendant amine group of the attached silane 3741, thereby tethering the aptamer 3725 to the electrode material 3712. The resulting biorecognition layer 3714 comprising the tethered aptamer 3725 and silane 3741 formed on the silicon electrode material 3712 is shown in Figure 17E. The amide linker can be formed by EDC / NHS crosslinking chemistry or DMTMM crosslinking chemistry. The remaining portion of the pendant amine groups on the silane layer (not shown) that are not bound to the aptamer can optionally be crosslinked to another carboxyl-containing molecule (e.g., glycolic acid, oxalic acid, glycine, PEG-carboxylic acid) to alter the working electrode surface properties and biocompatibility.

[0264] In an exemplary variation shown in FIG. 17F, a working electrode 4710 comprising a glassy carbon electrode material 4712 can be functionalized with an aptamer via activation of the carbon surface and formation of an amide bond tethering the aptamer to the carbon surface. In a first step, the surface of the glassy carbon electrode material 4712 is electrochemically activated by holding a constant potential of 1.2 V to 2.0 V in an acidic solution (e.g., a 0.1 M to 3 M HSO solution) for 1 to 60 minutes. The activation step forms terminal carboxyl groups 4745 on the surface of the carbon electrode material 4712. The activated surface of the electrode material 4712 is then functionalized with an aptamer 4725 having a primary amine group 4727 at its 3' or 5' end and a redox-active molecule 4726 at the other end. The carboxyl group 4745 forms an amide linker with the amine group 4727, thereby tethering the aptamer 4725 to the electrode material 4712. The resulting biorecognition layer 4714 including tethered aptamer 4725 formed on carbon electrode material 4712 is shown in Figure 17G. The amide linker can be formed by EDC / NHS crosslinking chemistry or DMTMM crosslinking chemistry.

[0265] While Figures 17D-17G show aptamers tethered via an amide linker formed between a carboxyl group and a primary amine group, the present disclosure provides for the use of other moieties. By way of example, the carboxyl group may be substituted with an NHS-ester, an isocyanate, an isothiocyanate, or a benzoyl fluoride. The present disclosure also provides for aptamers tethered via other linkers, such as a triazole linker or a thioether linker. The thioether linker may be based on a combination of a maleimide moiety on the conductive polymer and a thiol moiety on the terminus of the aptamer, a combination of a vinyl surface attached to the conductive polymer layer and a thiol moiety on the terminus of the aptamer, or a combination of an epoxide moiety on the conductive polymer and a thiol moiety on the terminus of the aptamer.

[0266] 17C, 17J, and 17K, functionalization of biorecognition layer 2714, 6714, 7714 can include deposition of each of aptamers 2725, 6725, 7725 and passivation elements comprising at least one thiol-based small molecule 2729, 6730, 6731, 7730, 7731 and / or zwitterion 7732 (which, in some variations, can be a zwitterionic peptide or a thiol-based small molecule with a zwitterionic phosphorylcholine head group). In this way, a portion of the surface of electrode material 2712, 6712, 7712 includes the aptamer, and another portion includes the passivation element, which can include a thiol-based small molecule and / or zwitterion (e.g., a zwitterionic peptide or a thiol-based small molecule with a zwitterionic phosphorylcholine head group) to passivate the surface and / or to attach biocompatible functional groups. For example, as shown in FIG. 17C, small molecule thiols 2729 fill the surface of electrode material 2712 that is not occupied by aptamers 2725.

[0267] To this end, Figures 18A and 18B provide flow diagrams of methods for depositing a passivation element comprising at least one thiol-based small molecule on the surface of an electrode material. After completion of pretreatment, anodization, and activation of the electrode material, which may each be performed as described above under either Method 1810A or Method 1810B, the working electrode component may be functionalized with a biorecognition layer. While described below in one exemplary order, it should be understood that the biorecognition element (e.g., an aptamer) and the passivation element may be deposited simultaneously or sequentially. For example, as described below, the biorecognition element may be deposited before the passivation element, or, if desired, the passivation element may be deposited before the biorecognition element.

[0268] It should also be understood that the functionalization described herein can include polymerization of a polymer that captures a biorecognition element therein. In some cases, when the biorecognition layer includes a conductive polymer, the voltage can be linearly scanned (e.g., 10 linear sweep segments) between cathodic values ​​(e.g., between −0.5 V and 0.0 V relative to an Ag / AgCl reference electrode) and anodic values ​​(e.g., between 0.5 V and +1.5 V relative to an Ag / AgCl reference electrode). In an exemplary variation, the scan rate of this waveform can range from about 1 mV / sec to about 1,000 mV / sec in an aqueous solution composed of a monomer precursor to the capture conductive polymer and a biorecognition element (e.g., an aptamer). The biorecognition element can be present in the aqueous solution at a concentration of 0.1 μM to about 2 μM. This process can produce (e.g., by electrodeposition or electropolymerization) a thin film (e.g., about 10 nm to about 1,000 nm) of a polymer containing a biorecognition element on the surface of the working electrode.

[0269] However, methods 1810A and 1810B are described with reference to a biorecognition layer lacking a polymerized polymer network. To this end, in variations in which the biorecognition layer does not include a polymer, depositing a biorecognition element (e.g., an aptamer) in step 1820A of method 1810A and step 1820B of method 1810B includes immersing the electrode material in a solution containing the aptamer. The electrode material may be immersed for a predetermined period of time ranging from about 3 hours to about 30 hours, and the concentration of the aptamer in the solution may be from about 0.1 μM to about 2 μM, from about 0.2 μM to about 1 μM, from about 0.3 μM to about 0.75 μM, or from about 0.4 μM to about 0.6 μM.

[0270] Referring now to FIG. 18A , following deposition of the biorecognition element in step 1810A, a first thiol-based small molecule can be deposited (i.e., applied) in step 1830A of method 1810A. The first thiol-based small molecule can be any one of the thiol-based small molecules described herein and can be deposited according to any suitable method, including, but not limited to, drop casting, printing, spray coating, dipping, spin coating, and chemical vapor deposition. For example, the first thiol-based small molecule can be deposited by dipping or immersing the electrode in a first solution. In some variations, the first solution can include the first thiol-based small molecule in a first solvent. The first solvent can include an organic solvent or an inorganic solvent. For example, the first solvent can be one or more solvents selected from the group consisting of ethanol, methanol, propanol, isopropanol, butanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, propylene carbonate, ethylene carbonate, deionized water, phosphate-buffered saline (PBS), and a buffered salt solution. In one example, the first solvent can be ethanol. In one example, the first solvent can be a mixture of methanol or ethanol with deionized water, PBS, methanol with a buffered salt solution, ethanol with deionized water, ethanol with PBS, or an ethanol-buffered salt solution. The first thiol-based small molecule can be present in the first solvent at a concentration of about 0.1 mM to about 50 mM, about 0.5 mM to about 40 mM, about 1 mM to about 30 mM, about 2 mM to about 20 mM, or about 5 mM to about 15 mM. In some variations, the electrode may be immersed in the first solution for a predetermined period of time. The predetermined period of time may be about 4 hours to about 48 hours, about 12 hours to about 24 hours, or about 16 hours to about 20 hours. In some variations, depositing the first thiol-based small molecule may be performed at a predetermined temperature. The predetermined temperature may be adjusted to control the packing density of the first thiol-based small molecule. Lowering the temperature may increase the packing density.The predetermined temperature may be from about -20°C to about 37°C, from about 1°C to about 30°C, or from about 3°C ​​to about 23°C.

[0271] Following deposition of the first thiol-based small molecule in step 1830A, a second, different thiol-based small molecule can be deposited (i.e., applied) onto the biorecognition layer in step 1840A of method 1810A. The second, different thiol-based small molecule can be any one of the thiol-based small molecules described herein and can be deposited according to any suitable method, including, but not limited to, drop casting, printing, spray coating, dipping, spin coating, and chemical vapor deposition. For example, the second, different thiol-based small molecule can be deposited by immersing or dipping the electrode in a second solution. In some variations, the second solution can include the second, different thiol-based small molecule in a second solvent. The second solvent can include an organic solvent or an inorganic solvent. For example, the second solvent can be one or more solvents selected from the group consisting of ethanol, methanol, propanol, isopropanol, butanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, propylene carbonate, ethylene carbonate, deionized water, PBS, and a buffered salt solution. In one example, the second solvent can be ethanol. In another example, the second solvent can be a mixture of methanol or ethanol and deionized water, PBS, or a buffered salt solution. The second, different thiol-based small molecule can be present in the second solvent at a concentration of about 0.1 mM to about 50 mM, about 0.5 mM to about 40 mM, about 1 mM to about 30 mM, about 2 mM to about 20 mM, or about 5 mM to about 15 mM. In some variations, the electrode can be immersed in the second solution for a predetermined period of time. The predetermined period of time may be about 4 hours to about 48 hours, about 12 hours to about 24 hours, or about 16 hours to about 20 hours. In some variations, depositing the second, different thiol-based small molecule can be carried out at a predetermined temperature. The predetermined temperature can be adjusted to control the packing density of the second, different thiol-based small molecule. Lowering the temperature can increase the packing density. The predetermined temperature can be about -20°C to about 37°C, about 1°C to about 30°C, or about 3°C ​​to about 23°C.

[0272] In some variations, subsequent different thiol-based small molecules can be deposited in step 1850A of method 1810A. Any number of thiol-based small molecules can be arranged to achieve the desired passivation quality.

[0273] Referring now to Figure 18B, a variation of method 1810A is described in which a first thiol-based small molecule and a second, different thiol-based small molecule are simultaneously deposited. Following deposition of the biorecognition element in step 1820B, as described above, the first thiol-based small molecule and the second, different thiol-based small molecule can be deposited in step 1860B of method 1810B according to any suitable method, including, but not limited to, drop casting, printing, spray coating, dipping, spin coating, and chemical deposition. For example, the thiol-based small molecule can be deposited by dipping or immersing an electrode in a solution. In some variations, the solution can include a first thiol-based small molecule in a first solvent and a second, different thiol-based small molecule in a second solvent. The first solvent and the second solvent can include an organic solvent or an inorganic solvent. For example, the first solvent and the second solvent may be one selected from the group consisting of ethanol, methanol, propanol, isopropanol, butanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, propylene carbonate, ethylene carbonate, deionized water, phosphate buffered saline, and buffered salt solutions. In some variations, the first solvent and the second solvent may be the same. In some variations, the first solvent and the second solvent may be different.

[0274] In some variations, the first thiol-based small molecule can be present in the first solvent at a concentration of about 0.1 mM to about 50 mM, about 0.5 mM to about 40 mM, about 1 mM to about 30 mM, about 2 mM to about 20 mM, or about 5 mM to about 15 mM. The second thiol-based small molecule can be present in the second solvent at a concentration of about 0.1 mM to about 50 mM, about 0.5 mM to about 40 mM, about 1 mM to about 30 mM, about 2 mM to about 20 mM, or about 5 mM to about 15 mM.

[0275] In some variations, the electrode may be immersed in the combined first and second solutions for a predetermined period of time. The predetermined period of time may be about 4 hours to about 48 hours, about 12 hours to about 24 hours, or about 16 hours to about 20 hours. In some variations, the first thiol-based small molecule and the second, different thiol-based small molecule may be deposited at a predetermined temperature. The predetermined temperature may be about -20°C to about 37°C, about 1°C to about 30°C, or about 3°C ​​to about 23°C.

[0276] In some variations, the solution combining the first thiol-based small molecule and the second, different thiol-based small molecule comprises organic solvent in water at a concentration of at least about 5% v / v, at least about 10% v / v, at least about 15% v / v, at least about 20% v / v, at least about 25% v / v, at least about 30% v / v, at least about 35% v / v, at least about 40% v / v, at least about 45% v / v, at least about 50% v / v, at least about 55% v / v, at least about 60% v / v, at least about 65% v / v, at least about 70% v / v, at least about 75% v / v, at least about 80% v / v, at least about 85% v / v, at least about 90% v / v, or at least about 95% v / v.

[0277] In some variations, the combined solution of the first thiol-based small molecule and the second, different thiol-based small molecule comprises the first thiol-based passivating molecule and the second, different thiol-based passivating molecule in a ratio of about 1 mM to about 30 mM to about 2.5 mM to about 30 mM. For example, if the first thiol-based small molecule is MCH and the second, different thiol-based small molecule is PC, the combined solution comprises the first thiol-based passivating molecule and the second, different thiol-based passivating molecule in a ratio of about 1 mM to about 30 mM MCH to about 2.5 mM to about 30 mM PC. In one example, the combined solution comprises the first thiol-based passivating molecule and the second, different thiol-based passivating molecule in a ratio of 15 mM MCH to 10 mM PC.

[0278] In some variations, the composition of the passivation element can be selected according to the desired functionality of the resulting device. For example, it is shown herein that sensors having only MCH as the passivation element have improved signal strength but reduced long-term stability. It is also shown herein that sensors having only PC as the passivation element have excellent long-term stability but reduced signal strength. Therefore, in some variations, it may be beneficial to utilize a passivation element that includes both MCH and PC. In these variations, the desired composition, or ratio of MCH to PC, depends on the desired functional characteristics. The composition of the passivation element can be tailored to provide different sensor characteristics, such as different levels of long-term stability, different levels of signal strength, etc., depending on the passivation molecule selected.

[0279] In some variations, the passivation elements can be deposited until a predetermined density of passivation elements is achieved. In some variations, subsequent thiol-based small molecules can be deposited together with the first and second different thiol-based small molecules, or can be deposited in a subsequent step as shown in Figure 18A. Any number of thiol-based small molecules can be arranged to achieve the desired passivation quality.

[0280] In some variations, method 1810A and method 1810B may further include depositing a zwitterion (e.g., a zwitterionic peptide or a thiol-based small molecule with a zwitterionic phosphorylcholine head group) as part of the passivation element.

[0281] In some variations, at least one of the first thiol-based passivating molecule and the second thiol-based passivating molecule in solution has improved solubility. The improved solubility can be provided by including a surfactant and / or emulsifier in the solution or by using a passivating molecule with these properties. For example, some of the thiol-based small molecules described above can be sufficiently amphiphilic to exhibit the same properties as surfactants and / or emulsifiers. This is the case, for example, with MCH and PC.

[0282] In some variations, the passivating element may be deposited on the surface of the electrode material at a concentration of about 1 mM to about 30 mM, about 1 mM to about 15 mM, about 5 mM to about 20 mM, about 5 mM to about 10 mM, about 2 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 10 mM, about 15 mM, and about 20 mM. In some variations, a mixture of the biorecognition element and the passivating element may be applied to the surface of the electrode material. The biorecognition element and the passivating element may be mixed at appropriate relative concentrations to achieve a desired surface density of the biorecognition element. The appropriate relative concentrations may depend on the particular biorecognition element and the passivating element being mixed, but the ratio of biorecognition element to passivating element may be about 1:100 to about 1:10.

[0283] In some variations, the working electrode surface can be electrochemically roughened to enhance adhesion of the biorecognition layer to the electrode material 1612 surface (and / or the Pt black layer). The roughening process can involve cathodic treatment (e.g., cathodic deposition, a subset of amperometry) in which the electrode is subjected to a fixed cathodic potential (e.g., between −0.4 V and +0.2 V vs. an Ag / AgCl reference electrode) for a specified time (e.g., 5 seconds to 10 minutes) in an acidic solution (e.g., 0.01 mM to 100 mM HPtCl) containing the desired dissolved metal cations. Alternatively, the electrode is subjected to a fixed cathodic potential (e.g., about −0.4 V to about +0.2 V versus an Ag / AgCl reference electrode) in an acidic solution (e.g., 0.01 mM to 100 mM HPtCl) containing the desired metal cation dissolved therein until a certain amount of charge (e.g., 0.1 mC to 100 mC) is passed. In this process, a thin but highly porous layer of metal may be produced on the electrode surface, thereby dramatically increasing the electrode surface area. Additionally or alternatively, in some variations of the above, elemental platinum metal may be deposited on the electrode to form or deposit a platinum black layer 1613. Opposite

[0284] Anodization: In some variations, the counter electrode material can be anodized using an amperometric approach, in which the electrode component assigned to the counter electrode function is exposed to a fixed high anodic potential or a moderately strong acidic solution for a suitable time. Exemplary parameters and other details of the counter electrode anodization process can be similar to those described above for the working electrode. Similarly, the counter electrode anodization can alternatively use a coulometric technique as described above.

[0285] Activation: In some variations, following the anodization process, the counter electrode component may be subjected to a periodically scanned potential waveform in an activation process using cyclic voltammetry. In some variations, the activation process may be similar to the process described above for the working electrode.

[0286] Roughening: Additionally, in some variations, the surface of the counter electrode may be electrochemically roughened to enhance the current sinking or current supplying capabilities of this electrode. The electrochemical roughening process may be similar to that described above for the working electrode. Additionally or alternatively, in some variations described above, elemental platinum metal may be deposited on the electrode to form or deposit a platinum black layer 1624. reference electrode

[0287] Anodization: Similar to the working and counter electrodes described above, the reference electrode can be anodized, in some variations, using an amperometric approach in which the electrode component assigned to the counter electrode function is exposed to a fixed high anodic potential or a moderate strength acid solution for a suitable time. Exemplary parameters and other details of the anodization process for the counter electrode can be similar to those described above for the working electrode. Similarly, the anodization of the counter electrode can be performed by:

[0288] Activation: Following the anodization process, the reference electrode component may, in some variations, be subjected to a periodically scanned potential waveform in an activation process using cyclic voltammetry, which may be similar to the process described above for the working electrode.

[0289] Functionalization: Following the activation process, in some variations, the reference electrode component can be functionalized. In some variations, the reference electrode can be functionalized by silver plating, e.g., AgNO3, followed by oxidation in a chloride-containing medium, e.g., KCl. Assuming the reference electrode, conditioned on the microneedle array, has undergone the above steps, a fixed anodic potential (e.g., between +0.4 and +1.0 V relative to an Ag / AgCl reference electrode) can be applied in aqueous solution for a suitable period of time (e.g., about 10 seconds to about 10 minutes). Alternatively, the reference electrode can be subjected to a fixed anodic potential (e.g., between about +0.4 and +1.0 V relative to an Ag / AgCl reference electrode) until a certain amount of charge passes through the aqueous solution (e.g., 0.01 mC to 10 mC). In some variations, the aqueous solution can contain a monomer precursor of a conductive polymer and a charged dopant counterion or material (e.g., poly(styrene sulfonate)) with an opposite charge. In this process, a thin film (e.g., about 10 nm to about 10,000 nm) of conductive polymer with dispersed counterions or materials can be produced on the reference electrode surface. This results in a surface-immobilized solid-state redox coupled with a stable thermodynamic potential. In some variations, the conductive polymer can include one or more of aniline, pyrrole, acetylene, phenylene, phenylene vinylene, phenylenediamine, thiophene, 3,4-ethylenedioxythiophene, and aminophenylboronic acid.

[0290] In some alternative embodiments, a native iridium oxide film (e.g., IrO2 or Ir2O3 or IrO4) can be electrochemically grown on the iridium electrode surface in an oxidation process, which also generates a stable redox couple as described above.

[0291] Additionally, in some variations, the reference electrode surface can be electrochemically roughened to enhance adhesion of the surface-immobilized redox couple. The electrochemical roughening process can be similar to that described above for the working electrode. Additionally or alternatively, in some variations described above, elemental platinum metal can be deposited on the electrode to form or deposit a platinum black layer 1633. Sensor Warm-up

[0292] Warm-up: Many implanted electrochemical sensors require a "warm-up" period, or time for the sensor to achieve a stable signal value after implantation. This process stems from both physiology and sensor kinetics. However, various embodiments of the analyte monitoring devices described herein are configured to mitigate factors that contribute to warm-up time. For example, the analyte monitoring devices described herein may have a warm-up time of about 30 minutes or less (e.g., about 10 minutes to about 30 minutes, about 15 minutes to about 30 minutes, about 20 minutes to about 30 minutes, about 25 minutes to about 30 minutes), about 45 minutes or less, about 60 minutes or less, about 90 minutes or less, or about 120 minutes or less. In some variations, following the warm-up period, the analyte monitoring device can be calibrated during a calibration period.

[0293] Wound response: For example, implantation of a sensor results in a wound response due to localized tissue disruption, displacement, and destruction. The larger the sensor or the deeper the implantation, the more vigorous the wound response. Thus, there is a compelling rationale for miniaturizing the sensor and coating one or more electrodes (and / or sensors, sensor arrays, microneedles, microneedle arrays, or devices containing electrodes) with a biocompatible layer to induce an attenuation of the wound response, thereby resulting in a more rapid warm-up.

[0294] Protein adsorption: Furthermore, following sensor implantation, a foreign body response is immediately triggered. The foreign body response involves a complex biochemical cascade aimed at encapsulating the foreign body with cellular material. Hydrophobic surfaces tend to undergo endogenous protein adsorption very quickly after implantation, a process known as biofouling. Hydrophilic surfaces, on the other hand, resist biofouling due to their high water content. Human serum albumin (HSA) is the major protein in skin interstitial fluid, comprising approximately 60% of the total protein and maintaining a negative charge at physiological pH. When the sensor is polarized at a positive potential (as in some variations of analyte monitoring devices), endogenous HSA undergoes electrical drift and charge attraction to the sensor's positive (working) electrode. This can result in an increased tendency of the sensor surface to biofouling. This is the rationale behind the implementation of a biocompatible layer to effectively mask the sensor from being recognized as a foreign body, as explained in further detail above.

[0295] As described herein, the analyte monitoring device reduces the impact of the above physiological factors on warm-up time due to, for example, the shallow nature of the implant, the minimal volume of tissue displaced, the minimal amount of trauma to that tissue during implantation, and the lack of permeability of the deeper vasculature in the reticular dermis, which, when disturbed, can elicit a more proliferative wound response that would result in an accelerated effort to encapsulate the implant.

[0296] Achieving Equilibrium: One example of the effect of sensor dynamics on warm-up time concerns achieving equilibrium. Electrochemical sensors require a finite amount of time to achieve equilibrium when used in a new environment. This is typically associated with the establishment of thermodynamic equilibrium due to an adsorbed surface layer of ions at the electrode. Because the reference electrode in most implantable electrochemical sensors does not use an internal fill solution with a redox couple sealed from the rest of the electrochemical cell, this reference electrode must achieve equilibrium with its surroundings to establish a stable reference potential.

[0297] Hydration of the sensor layer: The electrode sensor layer must be immersed in an aqueous environment to function properly. The resulting hydration process can activate the electrode's polymeric layers, such as the biocompatible layer, as well as the biorecognition elements, linkers, and / or passivation elements in the biorecognition layer, allowing them to rearrange and return to their native, active tertiary structure, which is primarily responsible for their activity or intrinsic properties. This process, often known as sensor "wetting," allows the medium in which sensing occurs to intercalate the sensor layer to a sufficient degree. Therefore, the rationale behind the implementation of a biocompatible layer is to minimize or mitigate the degree of rearrangement of one or more components of the biorecognition layer during the hydration process, returning them to their native tertiary structure. Electronic Equipment Systems

[0298] 2A schematic of analyte monitoring device 110, electronics system 120 may be integrated within housing 112 such that electronics system 120 may be combined with a sensing element (e.g., a microneedle array) as part of a single unit. Further details of exemplary variations of electronics system 120 are described below. Analog Front End

[0299] In some variations, the electronics system of the analyte monitoring device may include an analog front end. The analog front end may include a sensor circuit (e.g., sensor circuit 124 as shown in FIG. 2A) that converts analog current measurements into digital values ​​that can be processed by a microcontroller. The analog front end may include, for example, a programmable analog front end suitable for use with electrochemical sensors. For example, the analog front end may include MAX30131, MAX30132, or MAX30134 components (which have one, two, and four channels, respectively) available from Maxim Integrated (San Jose, California), which are ultra-low-power programmable analog front ends for use with electrochemical sensors. The analog front end may include an EmStat® Pico Module or EmStat® Pico Core from PalmSens (Houten, Netherlands) for performing SWV. The analog front end may also include the AD5940, ADuCM355, or AD5941 components available from Analog Devices (Norwood, MA), which are high-precision, impedance, and electrochemical front ends. Similarly, the analog front end may also include the LMP91000 available from Texas Instruments (Dallas, TX), which is a configurable analog front-end potentiostat for low-power chemical sensing applications. The analog front end can provide bias and a complete measurement path, including an analog-to-digital converter (ADC). Ultra-low power can enable continuous biasing of the sensor to maintain accuracy and fast response when long-term (e.g., 7-day) measurements are required using a body-worn, battery-operated device.

[0300] In some variations, the analog front-end device may be compatible with both two- and three-terminal electrochemical sensors, such as to enable both DC current measurement, AC current measurement, and electrochemical impedance spectroscopy (EIS) measurement capabilities. Additionally, the analog front-end may include an internal temperature sensor and programmable voltage reference, support external temperature monitoring and external reference sources, and integrate voltage monitoring of bias and supply voltages for safety and compliance.

[0301] In some variations, the analog front end may include a multi-channel potentiostat for multiplexing the sensor inputs and processing multiple signal channels.

[0302] In some variations, the analog front end and peripheral electronics may be integrated into an application-specific integrated circuit (ASIC), which may help to reduce costs, for example. This integrated solution may, in some variations, include a microcontroller, as described below. Microcontroller

[0303] In some variations, the electronics system of the analyte monitoring device may include at least one microcontroller (e.g., controller 122 shown in FIG. 2A). The microcontroller may include, for example, a processor with integrated flash memory. In some variations, the microcontroller within the analyte monitoring device may be configured to perform analytics that correlate sensor signals to analyte measurements. For example, the microcontroller may execute programmed routines in firmware to interpret digital signals (e.g., from the analog front end), perform any associated algorithms and / or other analytics, and route processed data to and from a communications module. By keeping the analytics on-board the analyte monitoring device, for example, the analyte monitoring device may broadcast analyte measurements in parallel to multiple devices (e.g., mobile computing devices such as smartphones or smartwatches, therapy delivery systems such as insulin pens or pumps, etc.) while ensuring that each connected device has the same information.

[0304] In some variations, the microcontroller may be configured to activate and / or deactivate the analyte monitoring device upon one or more detected conditions. For example, the device may be configured to power on the analyte monitoring device upon insertion of the microneedle array into the skin. This may enable, for example, a power-saving feature where the battery is disconnected until the microneedle array is placed on the skin, at which point the device may begin broadcasting sensor data. Such a feature may help, for example, to improve the shelf life of the analyte monitoring device and / or simplify the analyte monitoring device-external device pairing process for the user.

[0305] Additionally or alternatively, the microcontroller may be configured to actively confirm insertion of the microneedle array into the skin based on sensor measurements made with the microneedle array. For example, after two or more microneedles in the microneedle array are presumed to be inserted into the skin, a fixed or time-varying potential or current can be applied to the microneedles. Successful insertion of the microneedle array into the skin is confirmed by measuring the signal (e.g., potential or current value) generated between the electrodes of the inserted microneedles and comparing it to a known reference value. The reference value may include, for example, voltage, current, resistance, conductance, capacitance, inductance, and / or impedance.

[0306] In some variations, the microcontroller may utilize 8-bit, 16-bit, 32-bit, or 64-bit data structures. Suitable microcontroller architectures include ARM® and RISC® architectures, and flash memory may be embedded in the microcontroller or external to the microcontroller as suitable data storage. In some variations, the microcontroller may be a single-core microcontroller, while in some variations, the microcontroller may be a multi-core (e.g., dual-core) microcontroller, which may allow for flexible architectures to optimize power and / or performance within a system. For example, the cores within a microcontroller may include similar or different architectures. For example, in one exemplary variation, the microcontroller may be a dual-core microcontroller including a first core having a high-performance and high-power architecture and a second core having a low-performance and low-power architecture. The first core may function as a “workhorse” in that it may be used to process higher-performance functions (e.g., sensor measurements, algorithmic calculations, etc.), while the second core may be used to perform lower-performance functions (e.g., background routines, data transmission, etc.). Thus, different cores of the microcontroller can operate at different duty cycles optimized for their respective functions (e.g., a second core for a lower performance function can operate at a higher duty cycle), thereby improving overall power efficiency. Additionally or alternatively, in some variations, the microcontroller may include embedded analog circuitry, such as for interfacing with additional sensors and / or microneedle arrays. In some variations, the microcontroller may be configured to operate using a 0.8V to 5V power supply, such as a 1.2V to 3V power supply. Method for producing and drying biocompatible layer

[0307] As shown by way of example in Figures 16A-16M, the biorecognition layer 1614 may have a relatively complex molecular structure with various macromolecular components, such as aptamers, arranged in an ordered manner. Therefore, the layer is susceptible to degradation and damage. Furthermore, the biorecognition layer 1614 may be configured to perform its function in an aqueous environment; for example, exposure to air during dry storage may accelerate degradation due to removal of component molecules from the aqueous environment. Unexpectedly and advantageously, it has been found that a partially or fully dried hydrogel biocompatible layer 1616 disposed on the biorecognition layer 1614 protects the biorecognition layer 1614 from degradation during dry storage. Methods for applying a hydrogel biocompatible layer

[0308] In some variations, the biocompatible layer 1616 may be applied onto the biorecognition layer 1614 by at least one of spray coating, dip coating, drop casting, chemical vapor deposition, plasma deposition, and electrodeposition. In some variations where the hydrogel biocompatible layer is applied by, for example, drop casting or spray coating, the hydrogel may be drop cast onto the biorecognition layer at a temperature of about 45°C to about 75°C. Method for drying a hydrogel biocompatible layer

[0309] A working electrode (e.g., working electrode 1610A) including a biorecognition layer (e.g., biorecognition layer 1614) can be prepared for dry storage by applying a hydrogel biocompatible layer (e.g., biocompatible layer 1616) over the biorecognition layer and drying the hydrogel to a desired level of dryness (e.g., to at least partially dry). The hydrogel biocompatible layer can be applied using a variety of deposition techniques and dried under a variety of conditions, such as, for example, different temperatures and relative humidity, to achieve different levels of dryness.

[0310] After application, the hydrogel may be dried under controlled conditions until the hydrogel achieves a desired level of dryness. In some variations, the hydrogel may be dried at an ambient temperature of about 15°C to about 30°C and an ambient humidity of about 10% to about 80% relative humidity. In some variations, the ambient temperature may be about 15°C to about 25°C, about 18°C ​​to about 22°C, about 15°C to about 20°C, or room temperature. In some variations, the ambient humidity may be about 50% to about 80% relative humidity, about 70% to about 80% relative humidity, about 20% to about 60% relative humidity, about 30% to about 70% relative humidity, about 50% relative humidity, about 60% relative humidity, about 70% relative humidity, and about 80% relative humidity.

[0311] As described above, the hydrogel may be dried under controlled conditions until the hydrogel achieves a desired level of dryness. In some variations, the hydrogel may be dried until the at least partially dried hydrogel contains about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% water compared to the hydrogel's fully hydrated state. In some variations, the at least partially dried hydrogel may be completely dried.

[0312] In some variations, after the drying step, one or more electrodes (e.g., a working electrode and / or a sensor, sensor array, microneedle, microneedle array, or device including a working electrode) can be stored exposed to air. The storage period can be about 1 day to about 1 week, about 1 day to 1 month, about 3 days to 1 week, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. When a hydrogel biocompatible layer is disposed on the biorecognition layer, the rate of the biorecognition layer degrades at a rate of about 1% per day to about 5% per day, about 3% or less per day, about 2% or less per day, about 1% or less per day, and about 5% or less per day. Storage conditions may be an ambient temperature of about 15°C to about 30°C and an ambient humidity of about 10% to about 80% relative humidity, about 10% to about 50% relative humidity, about 30% to about 80% relative humidity, or about 20% to about 50% relative humidity, or both.

[0313] In some variations, after the drying step, one or more electrodes (e.g., a working electrode and / or a sensor, sensor array, microneedle, microneedle array, or device including a working electrode) may be packaged for storage. For storage, one or more electrodes (e.g., a working electrode and / or a sensor, sensor array, microneedle, microneedle array, or device including a working electrode) may be packaged in a sealed disposable package, the interior of the package having an ambient humidity of about 50% to about 80% relative humidity. In some variations, the interior of the package may have about the same ambient humidity as the drying step. In some variations, the interior of the package may have about the same ambient humidity, about 10% higher, 20% higher, or 30% higher than the ambient humidity of the drying step. The interior humidity may be controlled or maintained within a specific range by including a silica packet. Protection of biorecognition from degradation during sterilization using hydrogel biorecognition layers

[0314] As described in detail herein, one or more electrodes (e.g., a working electrode 1610A, a counter electrode 1620A, and a reference 1630A as shown in FIGS. 16A-16C ) may be used to monitor an analyte in a physiological fluid within a subject and, therefore, may be placed within the subject, e.g., within the viable epidermis or dermis. Accordingly, one or more electrodes (e.g., a working electrode and / or a sensor, a sensor array, a microneedle, a microneedle array, or a device including the working electrode) may be sterilized prior to storage and / or use. During sterilization, one or more electrodes (e.g., a working electrode and / or a sensor, a sensor array, a microneedle, a microneedle array, or a device including the working electrode) may be exposed to radiation. However, as shown by way of example in FIGS. 6A-6D , the biorecognition layer of the working electrode has a relatively complex molecular structure with various macromolecules, such as aptamers, arranged in an ordered manner. Radiation exposure is known to modify or degrade macromolecules and their sequences (e.g., but not limited to, small molecule thiols in the biorecognition layer). Therefore, the biorecognition layer may be susceptible to degradation and damage due to radiation exposure. As shown in Example 2 herein below, the presence of a hydrogel biocompatible layer unexpectedly and advantageously reduces degradation of the biorecognition layer due to radiation exposure.

[0315] In some variations, a working electrode (and / or a sensor, sensor array, microneedle, microneedle array, or device including a working electrode) including a biorecognition layer and a hydrogel biocompatible layer applied over the biorecognition layer can be sterilized by exposure to radiation. The present disclosure provides a method of sterilizing a sensor configured to generate a signal indicative of the concentration of an analyte in a fluid. The method can include providing a working electrode including a biorecognition layer disposed on an electrode material, applying a hydrogel biocompatible layer over the biorecognition layer, and sterilizing the working electrode by exposure to radiation.

[0316] The radiation may include ultraviolet radiation, gamma radiation, X-ray radiation, or electron beam radiation (E-beam). In some variations in which E-beam radiation is used, the E-beam radiation may be applied to the working electrode (and / or sensor, sensor array, microneedle, microneedle array, or device comprising the working electrode) at a dose of about 2 Megarads to about 4 Megarads, about 2.5 Megarads to about 3.5 Megarads, or about 2.7 Megarads to about 3.1 Megarads.

[0317] As described above, the biocompatible layer can protect the biorecognition layer from degradation, such as exposure to radiation during sterilization. In some variations, the biorecognition layer, when including a hydrogel biocompatible layer 1616 disposed thereon, can degrade by about 2% or less, about 5% or less, about 10% or less, about 15% or less, or about 20% or less after radiation exposure (e.g., during sterilization). Degradation of the biorecognition layer can be determined by placing a working electrode in a fluid containing an analyte, collecting a signal from the working electrode indicative of the concentration of the analyte in the fluid, and comparing the signal strength to a reference signal strength. In some variations, the reference signal strength can be based on an initial signal measured by the same or an equivalent working electrode that is not sterilized and placed in an equivalent measurement environment, e.g., the same fluid, within a certain time after generation (which can be when the hydrogel is applied to the working electrode). The certain time period can be about 1 hour to about 10 hours, about 1 hour to about 5 hours, about 1 hour, about 2 hours, about 4 hours, or about 5 hours to about 10 hours. In some variations, the baseline signal strength may be based on a comparative signal measured by an equivalent working electrode stored for an equivalent period of time and placed in an equivalent fluid, the equivalent sensor lacking a hydrogel disposed on the biorecognition layer.

[0318] In some variations, degradation of the biorecognition layer can be detected or quantified as a loss of passivation, which can be detected as a change in one or more electrical or electrochemical properties of the working electrode, such as one or more square wave voltammetry characteristics, to more closely resemble a bare electrode surface containing little or no passivating elements. Use of the Analyte Monitoring System

[0319] The following provides an overview of various aspects of methods of use and operation of the analyte monitoring system, including the analyte monitoring device and peripherals. Applications of Analyte Monitoring Devices

[0320] As described above, the analyte monitoring device is applied to a user's skin such that the microneedle array within the device penetrates the skin and the electrodes of the microneedle array are positioned in the upper dermis for access to cutaneous interstitial fluid. For example, in some variations, the microneedle array may be geometrically configured to penetrate the outer layer of skin, the stratum corneum, penetrate the epidermis, and rest within the papillary or upper reticular dermis. The sensing area, limited to the electrodes in the distal extent of each microneedle element of the array (as described above), may be configured to remain resting and seated in the papillary or upper reticular dermis after application to ensure adequate exposure to circulating cutaneous interstitial fluid (ISF) without risk of bleeding or undue influence from nerve endings.

[0321] In some variations, the analyte monitoring device may include a wearable housing or patch having an adhesive layer configured to adhere to the skin and secure the microneedle array in place. While the analyte monitoring device may be applied manually (e.g., by removing a protective film on the adhesive layer and manually pressing the patch onto the skin at the desired wear site), in some variations, the analyte monitoring device may be applied to the skin using a suitable applicator.

[0322] The analyte monitoring device may be applied in any suitable location, although in some variations it may be desirable to avoid anatomical regions of thick or raw skin (e.g., palmar and plantar regions), or regions that undergo significant flexion (e.g., olecranon or patella). Suitable application sites may include, for example, the arms (e.g., upper arms, lower arms), shoulders (e.g., over the deltoid muscles), backs of the hands, neck, face, scalp, torso (e.g., on the back, such as the pectoral region, lumbar region, sacral region, or on the chest or abdomen), buttocks, legs (e.g., upper legs, lower legs, etc.), and / or tops of the feet.

[0323] As mentioned above, in some variations, the analyte monitoring device may be configured to automatically activate upon insertion and / or to confirm proper insertion into the skin, details of these features are described in more detail above. Pairing with peripheral devices

[0324] In some variations, the analyte monitoring device may be paired with at least one peripheral device such that the peripheral device receives broadcast or otherwise transmitted data from the analyte monitoring device, including measurement data. Suitable peripheral devices include, for example, mobile computing devices (e.g., smartphones, smartwatches) that may be running mobile applications. Additionally or alternatively, the analyte monitoring device may be paired (or otherwise associated) with a therapy delivery device.

[0325] As previously mentioned, pairing may be accomplished by a suitable wireless communication module (e.g., implementing Bluetooth®). In some variations, pairing may occur after the analyte monitoring device is applied and inserted into the user's skin (e.g., after the analyte monitoring device is activated). Additionally or alternatively, pairing may occur before the analyte monitoring device is applied and inserted into the user's skin.

[0326] Thus, a paired mobile or other device may receive data broadcast or transmitted from the analyte monitoring device. The peripheral device may display, store, and / or transmit the measurement data to the user and / or healthcare provider and / or support network. Additionally, in some variations, the paired mobile or wearable device may perform algorithmic processing on the data to improve signal fidelity, accuracy, and / or calibration, etc. In some variations, the measurement data and / or other user information may additionally or alternatively be communicated and / or stored via a network (e.g., a cloud network).

[0327] For example, in some variations, a mobile computing device or other computing device (e.g., a smartphone, smartwatch, tablet, etc.) may be configured to run a mobile application that provides an interface for displaying estimated analyte-based values, trend information, historical data, etc.

[0328] In some variations, the mobile application may use the Bluetooth framework of the mobile computing device to scan for analyte monitoring devices. As shown in FIG. 20 , the analyte monitoring device may power on or initialize as soon as it is applied to the skin, and the analyte monitoring device may begin the notification process. The mobile application may then connect to the analyte monitoring device and begin priming the sensor for measurement. If the mobile application detects multiple analyte monitoring devices, the mobile application may detect the analyte monitoring device that is closest to itself and / or may request the user (e.g., via a user interface on the mobile device) to confirm disambiguation. In some variations, the mobile application may also be able to connect to multiple analyte monitoring devices simultaneously. This may be useful, for example, to replace sensors that are reaching the end of their life.

[0329] In some variations, the Bluetooth® Low Energy™ (BLE) protocol may be used for the connection. For example, the sensor implements a custom BLE peripheral profile for the analyte monitoring system. Data may be exchanged after establishing a standard, secure BLE connection between the analyte monitoring device and a smartphone, smartwatch, or tablet running a mobile application. The BLE connection may be maintained permanently for the life of the sensor. If the connection is lost for any reason (e.g., a weak signal), the analyte monitoring device may begin advertising itself again, and the mobile application may re-establish the connection at the earliest opportunity, for example, when within range based on physical proximity.

[0330] In some variations, one or more additional layers of security may be implemented on top of the BLE connection to ensure authorized access consisting of a combination of one or more techniques such as passcode protection, shared secrets, encryption, and multi-factor authentication.

[0331] The mobile application can guide the user through starting a new analyte monitoring device. Once this process is complete, the mobile application is not required for the analyte monitoring device to operate and record measurements. A secondary display device, such as a smartwatch, can be authorized by the mobile application to receive analyte readings directly from the sensor.

[0332] Further, in some variations, the mobile application may additionally or alternatively help calibrate the analyte monitoring device, for example, the analyte monitoring device may indicate a calibration request to the mobile application, and the mobile application may request calibration input from the user to calibrate the sensor. Sensor Measurement

[0333] Once the analyte monitoring device is inserted and warm-up and any calibration are completed, the analyte monitoring device may be ready to provide a sensor measurement of the analyte from the biological environment, through the biocompatible and diffusion-limiting layer on the working electrode, to the biorecognition layer containing the biorecognition element.

[0334] In some embodiments, a bias potential can be applied between the working and reference electrodes of the analyte monitoring device, and a current can be applied from the counter electrode to maintain a fixed potential relationship between the working and reference electrodes. This causes the electroactive product to be oxidized or reduced, resulting in a current flow between the working and counter electrodes. The current value is proportional to the proximity of the redox reporter molecule functionalized to the analyte-binding aptamer to the electrode material of the working electrode, as described in more detail above, and specifically, proportional to the concentration of the analyte in the skin interstitial fluid or some derivative thereof according to the Cottrell relationship.

[0335] The current may be converted to a voltage signal by a transimpedance amplifier and quantized into a digital bitstream by an analog-to-digital converter (ADC). Alternatively, the current may be quantized directly into a digital bitstream by a current-mode ADC. The digital representation of the current may be processed within an embedded microcontroller within the analyte monitoring device and relayed to a wireless communication module for broadcast or transmission (e.g., to one or more peripheral devices). In some variations, the microcontroller may perform additional algorithmic processing on the data to improve signal fidelity, accuracy, and / or calibration, etc.

[0336] In some variations, the digital representation of the current or sensor signal can be correlated with the analyte measurement by the analyte monitoring device. For example, a microcontroller can execute programmed routines in firmware to interpret the digital signal and perform any associated algorithms and / or other analyses. Interpreting the digital signal can include translating the digital signal into a user state based on the analyte measurement associated with the user or caregiver. Examples of a user state include analyte concentration in bodily fluids, e.g., skin interstitial fluid or blood, % change in analyte concentration, whether the analyte concentration is above, within, or below a threshold, and the user's state of mind, e.g., degree of stress and / or whether the stress is acute or chronic, or tracking diurnal variations in analyte levels. Keeping the analytics on-board the analyte monitoring device can, for example, enable the analyte monitoring device to broadcast analyte measurements to multiple devices in parallel, ensuring each connected device has the same information. Thus, in general, analyte-based user values ​​can be estimated, stored in the analyte monitoring device, and communicated to one or more peripheral devices.

[0337] Data exchange can be initiated by either the mobile application or the analyte monitoring device. For example, the analyte monitoring device can notify the mobile application of new analyte data as it becomes available. The frequency of updates can vary, for example, from about 5 seconds to about 5 minutes and can depend on the type of data. Additionally or alternatively, the mobile application can request data from the analyte monitoring device (e.g., if the mobile application identifies a gap in collected data due to a disconnection, etc.).

[0338] When the mobile application is not connected to the analyte monitoring device, the mobile application may not receive data from the sensor electronics. However, the electronics in the analyte monitoring device may store each actual and / or estimated analyte data point. When the mobile application reconnects to the analyte monitoring device, the mobile application can request the data that was missed during the disconnection period, and the electronics on the analyte monitoring device can transmit that data set as well (e.g., backfill).

[0339] Generally, the mobile application may be configured to provide a real-time or near-real-time display of analyte measurement data, such as on a display of a mobile computing device executing the mobile application. In some variations, the mobile application may communicate via a user interface regarding an analysis of the analyte measurements, such as alerts, alarms, trend insights, etc., to notify the user of analyte measurements requiring attention or follow-up action (e.g., high analyte measurements, low analyte measurements, high rates of change, analyte measurements outside of preset ranges, etc.). In some variations, the mobile application may additionally or alternatively facilitate communication of the measurement data to a cloud for storage and / or archiving for later retrieval. Interpretation of the Analyte Monitoring Device User Interface

[0340] In some variations, analyte measurement data and / or information about the analyte monitoring device may be communicated via a user interface of the analyte monitoring device. In some variations, the user interface of the analyte monitoring device may be used to communicate information to the user in addition to, or instead of, communicating such information via a peripheral device, such as via a mobile application on a computing device. Thus, the user and / or those around the user can easily and intuitively look at the analyte monitoring device itself to evaluate analyte measurement data (e.g., analyte measurement status, such as current and / or trending analyte measurement levels) and / or device status, without having to look at a separate device (e.g., a peripheral or other device remote from and in communication with the analyte monitoring device). Having such information available directly on the analyte monitoring device itself can also allow the user and / or those around the user to be more quickly alerted to any concerns (e.g., analyte measurements above or below the target range and / or analyte measurements increasing or decreasing at an alarming rate), thereby allowing the user to take appropriate corrective action more quickly. Numbered Embodiments of the Invention

[0341] Without limiting the scope of the appended claims, the present disclosure describes the following numbered embodiments.

[0342] Embodiment I-1. A working electrode, an electrode material; a biorecognition element disposed on the electrode material and configured to selectively and reversibly bind to an analyte in the fluid; a first thiol-based passivation molecule disposed on the electrode material; a second, different thiol-based passivation molecule disposed on the electrode material.

[0343] Embodiment I-2. The first thiol-based passivating molecule and the second, different thiol-based passivating molecule are 1-hexanethiol, 6-mercapto-1-hexanamine, 6-mercapto-1-phosphatidylcholinehexane, 6-mercapto-1-hexanol (MCH), 7-mercapto-1-heptanol, 8-mercapto-1-octanol (MCO), 9-mercapto-1-nonanol, 10-mercapto-1-decanol, 11-mercapto-1-undecanol, 6-amino-1-hexanol, 12-mercapto-1-pentanol, 13-mercapto-1-pentanol, 14-mercapto-1-pentanol, 15-mercapto-1-pentanol, 16-mercapto-1-pentanol, 17-mercapto-1-pentanol, 18-mercapto-1-pentanol, 19-mercapto-1-pentanol, 20-mercapto-1-pentanol, 21-mercapto-1-pentanol, 22-mercapto-1-pentanol, 23-mercapto-1-pentanol, 24-mercapto-1-pentanol, 25-mercapto-1-pentanol, 26-mercapto-1-pentanol, 27-mercapto-1-pentanol, 28-mercapto-1-pentanol, 29-mercapto-1-pentanol, 30-mercapto-1-pentanol, 31-mercapto-1-pentanol, 32-mercapto-1-pentanol, 33-mercapto-1-pentanol, 34-mercapto-1-pentanol, 35-mercapto-1-pentanol, 36-mercap Santhiol, 7-amino-1-heptanethiol, 8-amino-1-octanethiol, 9-amino-1-nonanethiol, 10-amino-1-decanethiol, 11-amino-1-undecanethiol, (6-mercaptohexyl)-N,N,N-trimethylammonium bromide, (7-mercaptoheptyl)-N,N,N-trimethylammonium bromide, (8-mercaptooctyl)-N,N,N-trimethylammonium bromide, (9-mercaptononyl)-N,N,N-trimethylammonium bromide ammonium bromide, (10-mercaptodecyl)-N,N,N-trimethylammonium bromide, (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide, 6-mercaptohexyl phosphate, 7-mercaptoheptyl phosphate, 8-mercaptooctyl phosphate, 9-mercaptononyl phosphate, 10-mercaptodecyl phosphate, 11-mercaptoundecyl phosphate, 6-mercaptohexanoic acid, 7-mercaptoheptanoic acid, 8-mercaptooctanoic acid, 9-mercaptononanoic acid, 10-Mercaptodecanoic acid, 11-mercaptoundecanoic acid, 6-mercaptohexanesulfonate, 7-mercaptoheptanesulfonate, 8-mercaptooctane sulfonate, 9-mercaptonanolsulfonate, 10-mercaptodecanesulfonate, 11-mercaptoundecanesulfonate, poly(ethylene glycol) dithiol, 1,6-hexanedithiol, 1,7-heptanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol, 1,10-decanedithiol, 1,11-Undecanedithiol, 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate (PC), thiolated oligoethylene glycol (OEG), thiolated polyethylene glycol, (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyltrimethylammonium)chloride (AC), (3-((6-mercapto (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethylsulfonate) potassium (SP), and (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyldimethylammonio-(3-propanesulfonate)) (AP), 1,2-bis-(11-sulfanylundecanoyl)-sn-glycero-3-phosphocholine, methacrylate alkyl thiol ester, methacrylate PEG thiol ester, acrylate The working electrode of embodiment I-1, wherein the thiol is selected from the group consisting of acrylate alkyl thiol esters, acrylate PEG thiol esters, vinyl-terminated alkyl thiols, vinyl-terminated PEG thiols, acetylene-terminated alkyl thiols, acetylene-terminated PEG thiols, benzophenone-terminated alkyl thiols, benzophenone-terminated PEG thiols, azide-terminated alkyl thiols, azide-terminated PEG thiols, N-hydroxysuccinimide-terminated alkyl thiols, N-hydroxysuccinimide-terminated PEG thiols, ferrocene-terminated alkyl thiols, ferrocene-terminated PEG thiols, methylene blue-terminated alkyl thiols, methylene blue-terminated PEG thiols, anthraquinone-terminated alkyl thiols, anthraquinone-terminated PEG thiols, hydroquinone-terminated alkyl thiols, hydroquinone-terminated PEG thiols, RGD peptide-terminated thiols, and YIGSR peptide-terminated thiols.

[0344] Embodiment I-3. The working electrode of embodiment I-2, wherein the first thiol-based passivating molecule is MCH and the second, different thiol-based passivating molecule is PC.

[0345] Embodiment I-4. The working electrode of embodiment I-1, wherein the working electrode comprises a third, different thiol-based passivating molecule.

[0346] Embodiment I-5. The working electrode of embodiment I-1, wherein the first thiol-based passivating molecule and the second, different thiol-based passivating molecule each comprise a plurality of passivating molecules.

[0347] Embodiment I-6. The working electrode of embodiment I-5, wherein the distal end of the first thiol-based passivation molecule, the second, different thiol-based passivation molecule, or both, comprises one or more of a hydrophilic moiety, a hydrophobic moiety, a charged moiety, and a zwitterionic moiety.

[0348] Embodiment I-7. The working electrode of embodiment I-6, wherein the distal end of the first thiol-based passivating molecule, the second, different thiol-based passivating molecule, or both, comprises a zwitterionic moiety, and the zwitterionic moiety is a zwitterionic phosphorylcholine head group.

[0349] Embodiment I-8. The working electrode of embodiment I-5, wherein the first thiol-based passivating molecule, the second, different thiol-based passivating molecule, or both, is a zwitterionic peptide.

[0350] Embodiment I-9. The working electrode of embodiment I-8, wherein the zwitterionic peptide comprises a cysteine ​​residue.

[0351] Embodiment I-10. The working electrode of embodiment I-9, wherein the zwitterionic peptide comprises a thiol group as a side chain of the cysteine ​​residue.

[0352] Embodiment I-11. The working electrode of embodiment I-1, wherein the electrode material comprises platinum, silver, palladium, iridium, rhodium, gold, ruthenium, titanium, nickel, carbon, glassy carbon, pyrolytic carbon, doped diamond, boron-doped diamond, or a combination thereof.

[0353] Embodiment I-12. The working electrode of embodiment I-1, wherein the pseudo-film formed by the first thiol-based passivation molecule and the second, different thiol-based passivation molecule on the electrode material has a thickness of about 0.001 nm to about 1000 nm, about 0.01 nm to about 100 nm, about 0.1 nm to about 10 nm, about 0.05 nm to about 500 nm, about 0.1 nm to about 200 nm, about 1 nm to about 1000 nm, about 1 μm to about 500 μm, about 2 μm to about 400 μm, about 3 μm to about 300 μm, about 4 μm to about 200 μm, about 5 μm to about 100 μm, about 10 μm to about 50 μm, or about 20 μm to about 40 μm.

[0354] Embodiment I-13. The working electrode of embodiment I-1, wherein the biorecognition element is functionalized with a redox-active molecule and configured to undergo a conformational change upon binding to an analyte to move the redox-active molecule closer to or farther from the electrode material.

[0355] Embodiment I-14. The working electrode of embodiment I-13, wherein the redox-active molecule comprises one selected from the group consisting of methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, hydroquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, carboxy-X-rhodamine, and π-extended tetrathiafulvalene (exTTF).

[0356] Embodiment I-15. The working electrode of embodiment I-13, wherein the biorecognition element is an aptamer.

[0357] Embodiment I-16. The working electrode of embodiment I-15, wherein the aptamer comprises a thiol group at the 3' or 5' end.

[0358] Embodiment I-17. The working electrode of embodiment I-1, wherein the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array.

[0359] Embodiment I-18. An analyte sensor configured to generate a signal indicative of a concentration of an analyte in a fluid, the analyte sensor comprising the working electrode of embodiment I-1.

[0360] Embodiment I-19. The sensor of embodiment I-18, further comprising a microneedle, wherein the working electrode is coupled to the microneedle.

[0361] Embodiment I-20. The sensor of embodiment I-19, further comprising a reference electrode and a counter electrode.

[0362] Embodiment I-21. A method of manufacturing a working electrode for an analyte sensor configured to generate a signal indicative of a concentration of an analyte in a fluid, the method comprising: providing a working electrode comprising an electrode material and a biorecognition element deposited on the electrode material, the biorecognition element being configured to selectively and reversibly bind to an analyte; applying a first thiol-based passivating molecule to the electrode material for a first predetermined period of time; applying a second, different thiol-based passivating molecule to the electrode material for a second predetermined period of time.

[0363] Embodiment I-22. The method of embodiment I-21, wherein applying comprises spray coating one or more of the first thiol-based passivation molecule and the second, different thiol-based passivation molecule onto the electrode material.

[0364] Embodiment I-23. The method of embodiment I-21, wherein applying comprises immersing the electrode material in a solution comprising one or more of a first thiol-based passivation molecule and a second, different thiol-based passivation molecule.

[0365] Embodiment I-24. The first thiol-based passivating molecule and the second different thiol-based passivating molecule are, respectively, 1-hexanethiol, 6-mercapto-1-hexanamine, 6-mercapto-1-phosphatidylcholinehexane, 6-mercapto-1-hexanol (MCH), 7-mercapto-1-heptanol, 8-mercapto-1-octanol (MCO), 9-mercapto-1-nonanol, 10-mercapto-1-decanol, 11-mercapto-1-undecanol, 6-amino-1 -Hexanethiol, 7-amino-1-heptanethiol, 8-amino-1-octanethiol, 9-amino-1-nonanethiol, 10-amino-1-decanethiol, 11-amino-1-undecanethiol, (6-mercaptohexyl)-N,N,N-trimethylammonium bromide, (7-mercaptoheptyl)-N,N,N-trimethylammonium bromide, (8-mercaptooctyl)-N,N,N-trimethylammonium bromide, (9-mercaptononyl)-N,N,N-trimethylammonium bromide ammonium bromide, (10-mercaptodecyl)-N,N,N-trimethylammonium bromide, (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide, 6-mercaptohexyl phosphate, 7-mercaptoheptyl phosphate, 8-mercaptooctyl phosphate, 9-mercaptononyl phosphate, 10-mercaptodecyl phosphate, 11-mercaptoundecyl phosphate, 6-mercaptohexanoic acid, 7-mercaptoheptanoic acid, 8-mercaptooctanoic acid, 9-mercaptononanoic acid , 10-mercaptodecanoic acid, 11-mercaptoundecanoic acid, 6-mercaptohexanesulfonate, 7-mercaptoheptanesulfonate, 8-mercaptooctane sulfonate, 9-mercaptonanolsulfonate, 10-mercaptodecanesulfonate, 11-mercaptoundecanesulfonate, poly(ethylene glycol) dithiol, 1,6-hexanedithiol, 1,7-heptanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol, 1,10-decanedithiol, 1,11-Undecanedithiol, 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate (PC), thiolated oligoethylene glycol (OEG), thiolated polyethylene glycol, (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyltrimethylammonium)chloride (AC), (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyltrimethylammonium)ethyl)phosphate (PC), (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethylsulfonate) potassium (SP), and (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyldimethylammonio-(3-propanesulfonate)) (AP), 1,2-bis-(11-sulfanylundecanoyl)-sn-glycero-3-phosphocholine, methacrylate alkyl thiol ester, methacrylate PEG thiol ester, and The method of embodiment I-21, wherein the alkyl thiol is selected from the group consisting of acrylate alkyl thiol esters, acrylate PEG thiol esters, vinyl-terminated alkyl thiols, vinyl-terminated PEG thiols, acetylene-terminated alkyl thiols, acetylene-terminated PEG thiols, benzophenone-terminated alkyl thiols, benzophenone-terminated PEG thiols, azide-terminated alkyl thiols, azide-terminated PEG thiols, N-hydroxysuccinimide-terminated alkyl thiols, N-hydroxysuccinimide-terminated PEG thiols, ferrocene-terminated alkyl thiols, ferrocene-terminated PEG thiols, methylene blue-terminated alkyl thiols, methylene blue-terminated PEG thiols, anthraquinone-terminated alkyl thiols, anthraquinone-terminated PEG thiols, hydroquinone-terminated alkyl thiols, hydroquinone-terminated PEG thiols, RGD peptide-terminated thiols, and YIGSR peptide-terminated thiols.

[0366] Embodiment I-25. The method of embodiment I-24, wherein the first thiol-based passivating molecule is MCH and the second, different thiol-based passivating molecule is PC.

[0367] Embodiment I-26. The method of embodiment I-25, wherein applying the first thiol-based passivating molecule and the second thiol-based passivating molecule comprises contacting the electrode material with a solution comprising the first thiol-based passivating molecule and a second, different thiol-based passivating molecule in a ratio of MCH to PC of about 1 mM to about 30 mM MCH to about 2.5 mM to about 30 mM PC.

[0368] Embodiment I-27. The method of embodiment I-25, wherein applying the first thiol-based passivating molecule and the second thiol-based passivating molecule comprises contacting the electrode material with a solution comprising the first thiol-based passivating molecule and a second, different thiol-based passivating molecule in a ratio of MCH to PC of about 15 mM MCH to about 10 mM PC.

[0369] Embodiment I-28. The method of embodiment I-21, wherein at least one of the first predetermined period of time and the second predetermined period of time is from about 4 hours to about 48 hours, from about 12 hours to about 24 hours, or from about 16 hours to about 20 hours.

[0370] Embodiment I-29. The method of embodiment I-21, wherein a first thiol-based passivating molecule and a second, different thiol-based passivating molecule are applied to the electrode material to a predetermined density of the passivation layer.

[0371] Embodiment I-30. The method of embodiment I-21, wherein applying the first thiol-based passivating molecule and applying the second, different thiol-based passivating molecule are carried out at a temperature of from about -20°C to about 37°C, from about 1°C to about 30°C, or from about 3°C ​​to about 23°C.

[0372] Embodiment I-31. The method of embodiment I-21, wherein the biorecognition element is functionalized with a redox-active molecule and configured to undergo a conformational change upon binding to the analyte to move the redox-active molecule closer to or farther from the electrode material.

[0373] Embodiment I-32. The method of embodiment I-31, wherein the biorecognition element is an aptamer.

[0374] Embodiment I-33. The method of embodiment I-31, wherein the redox active molecule comprises one selected from the group consisting of methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, hydroquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, carboxy-X-rhodamine, and π-extended tetrathiafulvalene (exTTF).

[0375] Embodiment I-34. The method of embodiment I-21, further comprising depositing cysteine ​​residues on the electrode material by chemisorption.

[0376] Embodiment I-35. Dissolving a first thiol-based passivating molecule in a first solvent; The method of embodiment I-21, further comprising dissolving a second, different thiol-based passivating molecule in the second solvent.

[0377] Embodiment I-36. The method of embodiment I-35, wherein the first solvent and the second solvent are each independently an organic solvent or an inorganic solvent.

[0378] Embodiment I-37. The method of embodiment I-35, wherein the first solvent and the second solvent are each independently selected from the group consisting of ethanol, methanol, propanol, isopropanol, butanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, propylene carbonate, ethylene carbonate, deionized water, phosphate buffered saline, and buffered salt solutions.

[0379] Embodiment I-38. The method of embodiment I-35, wherein at least one of the first solvent and the second solvent comprises an organic solvent in water at a concentration of at least about 5% v / v, at least about 10% v / v, at least about 15% v / v, at least about 20% v / v, at least about 25% v / v, at least about 30% v / v, at least about 35% v / v, at least about 40% v / v, at least about 45% v / v, at least about 50% v / v, at least about 55% v / v, at least about 60% v / v, at least about 65% v / v, at least about 70% v / v, at least about 75% v / v, a...

Claims

1. A working electrode, an electrode material; a biorecognition element disposed on the electrode material and configured to selectively and reversibly bind to an analyte in a fluid; a first thiol-based passivation molecule disposed on the electrode material; and a second, different thiol-based passivation molecule disposed on the electrode material.

2. The first thiol-based passivating molecule and the second different thiol-based passivating molecule are selected from the group consisting of 1-hexanethiol, 6-mercapto-1-hexanamine, 6-mercapto-1-phosphatidylcholine hexane, 6-mercapto-1-hexanol (MCH), 7-mercapto-1-heptanol, 8-mercapto-1-octanol (MCO), 9-mercapto-1-nonanol, 10-mercapto-1-decanol, 11-mercapto-1-undecanol, 6-amino-1-hexanethiol, 1-hexanethiol, 6-mercapto-1-hexaneamine, 6-mercapto-1-phosphatidylcholine hexane, 6-mercapto-1-hexanol (MCH), 7-mercapto-1-heptanol, 8-mercapto-1-octanol (MCO), 9-mercapto-1-nonanol, 10-mercapto-1-decanol, 11-mercapto-1-undecanol, 6-amino-1-hexanethiol, ... ol, 7-amino-1-heptanethiol, 8-amino-1-octanethiol, 9-amino-1-nonanethiol, 10-amino-1-decanethiol, 11-amino-1-undecanethiol, (6-mercaptohexyl)-N,N,N-trimethylammonium bromide, (7-mercaptoheptyl)-N,N,N-trimethylammonium bromide, (8-mercaptooctyl)-N,N,N-trimethylammonium bromide, (9-mercaptononyl)-N,N,N-trimethylammonium bromide ammonium bromide, (10-mercaptodecyl)-N,N,N-trimethylammonium bromide, (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide, 6-mercaptohexyl phosphate, 7-mercaptoheptyl phosphate, 8-mercaptooctyl phosphate, 9-mercaptononyl phosphate, 10-mercaptodecyl phosphate, 11-mercaptoundecyl phosphate, 6-mercaptohexanoic acid, 7-mercaptoheptanoic acid, 8-mercaptooctanoic acid, 9-mercaptononanoic acid, 1 0-mercaptodecanoic acid, 11-mercaptoundecanoic acid, 6-mercaptohexanesulfonate, 7-mercaptoheptanesulfonate, 8-mercaptooctane sulfonate, 9-mercaptonanolsulfonate, 10-mercaptodecanesulfonate, 11-mercaptoundecanesulfonate, poly(ethylene glycol)dithiol, 1,6-hexanedithiol, 1,7-heptanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol, 1,10-decanedithiol, 1,11-Undecanedithiol, 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate (PC), thiolated oligoethylene glycol (OEG), thiolated polyethylene glycol, (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyltrimethylammonium)chloride (AC), (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyltrimethylammonium)ethyl)phosphate (PC), (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethylsulfonate) potassium (SP), and (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyldimethylammonio-(3-propanesulfonate)) (AP), 1,2-bis-(11-sulfanylundecanoyl)-sn-glycero-3-phosphocholine, methacrylate alkyl thiol ester, methacrylate PEG thiol ester, 2. The working electrode of claim 1, wherein the thiol is selected from the group consisting of acrylate alkyl thiol esters, acrylate PEG thiol esters, vinyl-terminated alkyl thiols, vinyl-terminated PEG thiols, acetylene-terminated alkyl thiols, acetylene-terminated PEG thiols, benzophenone-terminated alkyl thiols, benzophenone-terminated PEG thiols, azide-terminated alkyl thiols, azide-terminated PEG thiols, N-hydroxysuccinimide-terminated alkyl thiols, N-hydroxysuccinimide-terminated PEG thiols, ferrocene-terminated alkyl thiols, ferrocene-terminated PEG thiols, methylene blue-terminated alkyl thiols, methylene blue-terminated PEG thiols, anthraquinone-terminated alkyl thiols, anthraquinone-terminated PEG thiols, hydroquinone-terminated alkyl thiols, hydroquinone-terminated PEG thiols, RGD peptide-terminated thiols, and YIGSR peptide-terminated thiols.

3. 3. The working electrode of claim 2, wherein the first thiol-based passivation molecule is MCH and the second, different thiol-based passivation molecule is PC.

4. The working electrode of claim 1 , wherein the working electrode comprises a third, different thiol-based passivation molecule.

5. 10. The working electrode of claim 1, wherein the first thiol-based passivation molecule and the second, different thiol-based passivation molecule each comprise a plurality of passivation molecules.

6. 6. The working electrode of claim 5, wherein a distal end of the first thiol-based passivation molecule, the second, different thiol-based passivation molecule, or both, comprises one or more of a hydrophilic moiety, a hydrophobic moiety, a charged moiety, and a zwitterionic moiety.

7. 7. The working electrode of claim 6, wherein the distal end of the first thiol-based passivation molecule, the second, different thiol-based passivation molecule, or both, comprises a zwitterionic moiety, the zwitterionic moiety being a zwitterionic phosphorylcholine head group.

8. 6. The working electrode of claim 5, wherein the first thiol-based passivating molecule, the second different thiol-based passivating molecule, or both are zwitterionic peptides.

9. The working electrode of claim 8 , wherein the zwitterionic peptide comprises a cysteine ​​residue.

10. The working electrode of claim 9 , wherein the zwitterionic peptide comprises a thiol group as a side chain of the cysteine ​​residue.

11. 10. The working electrode of claim 1, wherein the electrode material comprises platinum, silver, palladium, iridium, rhodium, gold, ruthenium, titanium, nickel, carbon, glassy carbon, pyrolytic carbon, doped diamond, boron-doped diamond, or a combination thereof.

12. 2. The working electrode of claim 1, wherein the pseudo-film formed by the first thiol-based passivation molecule and the second, different thiol-based passivation molecule on the electrode material has a thickness of about 0.001 nm to about 1000 nm, about 0.01 nm to about 100 nm, about 0.1 nm to about 10 nm, about 0.05 nm to about 500 nm, about 0.1 nm to about 200 nm, about 1 nm to about 1000 nm, about 1 μm to about 500 μm, about 2 μm to about 400 μm, about 3 μm to about 300 μm, about 4 μm to about 200 μm, about 5 μm to about 100 μm, about 10 μm to about 50 μm, or about 20 μm to about 40 μm.

13. 2. The working electrode of claim 1, wherein the biorecognition element is functionalized with a redox-active molecule and configured to undergo a conformational change upon binding to the analyte to move the redox-active molecule toward or away from the electrode material.

14. 14. The working electrode of claim 13, wherein the redox-active molecule comprises one selected from the group consisting of methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, hydroquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, carboxy-X-rhodamine, and π-extended tetrathiafulvalene (exTTF).

15. The working electrode of claim 13 , wherein the biorecognition element is an aptamer.

16. The working electrode of claim 15 , wherein the aptamer comprises a thiol group at the 3′ or 5′ end.

17. The working electrode of claim 1 , wherein the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array.

18. 10. An analyte sensor configured to generate a signal indicative of a concentration of an analyte in a fluid, the analyte sensor comprising the working electrode of claim 1.

19. 20. The sensor of claim 18, further comprising a microneedle, the working electrode being coupled to the microneedle.

20. 20. The sensor of claim 19, further comprising a reference electrode and a counter electrode.

21. 1. A method of manufacturing a working electrode for an analyte sensor configured to generate a signal indicative of a concentration of an analyte in a fluid, the method comprising: providing a working electrode comprising an electrode material and a biorecognition element deposited on the electrode material, the biorecognition element configured to selectively and reversibly bind to the analyte; applying a first thiol-based passivating molecule to the electrode material for a first predetermined period of time; applying a second, different thiol-based passivation molecule to the electrode material for a second predetermined period of time.

22. 22. The method of claim 21 , wherein applying comprises spray coating one or more of the first thiol-based passivation molecule and the second, different thiol-based passivation molecule onto the electrode material.

23. 22. The method of claim 21 , wherein applying comprises immersing the electrode material in a solution comprising one or more of the first thiol-based passivation molecule and the second, different thiol-based passivation molecule.

24. The first thiol-based passivating molecule and the second different thiol-based passivating molecule are, respectively, 1-hexanethiol, 6-mercapto-1-hexanamine, 6-mercapto-1-phosphatidylcholine hexane, 6-mercapto-1-hexanol (MCH), 7-mercapto-1-heptanol, 8-mercapto-1-octanol (MCO), 9-mercapto-1-nonanol, 10-mercapto-1-decanol, 11-mercapto-1-undecanol, 6-amino-1-hexanol, 12-mercapto-1-pentanol, 13-mercapto-1-pentanol, 14-mercapto-1-pentanol, 15-mercapto-1-pentanol, 16-mercapto-1-pentanol, 17-mercapto-1-pentanol, 18-mercapto-1-pentanol, 19-mercapto-1-pentanol, 20-mercapto-1-pentanol, 21-mercapto-1-pentanol, 22-mercapto-1-pentanol, 23-mercapto-1-pentanol, 24-mercapto-1-pentanol, 25-mercapto-1-pentanol, 26-mercapto-1-pentanol, 27-mercapto-1-pentanol, 28-mercapto-1-pentanol, 29-mercapto-1-pentanol, 30-mercapto-1-pentanol, 31-mercapto-1-pentanol, 32-mercapto-1-pentanol, 33-mercapto-1-pentanol, 34-mercapto-1-pentanol, 35-mercapto-1-pentanol, 36-mercapto- 1-mercaptohexyl, 7-amino-1-heptanethiol, 8-amino-1-octanethiol, 9-amino-1-nonanethiol, 10-amino-1-decanethiol, 11-amino-1-undecanethiol, (6-mercaptohexyl)-N,N,N-trimethylammonium bromide, (7-mercaptoheptyl)-N,N,N-trimethylammonium bromide, (8-mercaptooctyl)-N,N,N-trimethylammonium bromide, (9-mercaptononyl)-N,N,N-trimethylammonium bromide ammonium bromide, (10-mercaptodecyl)-N,N,N-trimethylammonium bromide, (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide, 6-mercaptohexyl phosphate, 7-mercaptoheptyl phosphate, 8-mercaptooctyl phosphate, 9-mercaptononyl phosphate, 10-mercaptodecyl phosphate, 11-mercaptoundecyl phosphate, 6-mercaptohexanoic acid, 7-mercaptoheptanoic acid, 8-mercaptooctanoic acid, 9-mercaptononanoic acid, 1 0-mercaptodecanoic acid, 11-mercaptoundecanoic acid, 6-mercaptohexanesulfonate, 7-mercaptoheptanesulfonate, 8-mercaptooctane sulfonate, 9-mercaptonanolsulfonate, 10-mercaptodecanesulfonate, 11-mercaptoundecanesulfonate, poly(ethylene glycol)dithiol, 1,6-hexanedithiol, 1,7-heptanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol, 1,10-decanedithiol, 1,11-Undecanedithiol, 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate (PC), thiolated oligoethylene glycol (OEG), thiolated polyethylene glycol, (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyltrimethylammonium)chloride (AC), (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyltrimethylammonium)ethyl)phosphate (PC), (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethylsulfonate) potassium (SP), and (3-((6-mercaptohexyl)thio)-2-methylpropanoate-(2-ethyldimethylammonio-(3-propanesulfonate)) (AP), 1,2-bis-(11-sulfanylundecanoyl)-sn-glycero-3-phosphocholine, methacrylate alkyl thiol ester, methacrylate PEG thiol ester, 22. The method of claim 21, wherein the thiol is selected from the group consisting of acrylate alkyl thiol esters, acrylate PEG thiol esters, vinyl-terminated alkyl thiols, vinyl-terminated PEG thiols, acetylene-terminated alkyl thiols, acetylene-terminated PEG thiols, benzophenone-terminated alkyl thiols, benzophenone-terminated PEG thiols, azide-terminated alkyl thiols, azide-terminated PEG thiols, N-hydroxysuccinimide-terminated alkyl thiols, N-hydroxysuccinimide-terminated PEG thiols, ferrocene-terminated alkyl thiols, ferrocene-terminated PEG thiols, methylene blue-terminated alkyl thiols, methylene blue-terminated PEG thiols, anthraquinone-terminated alkyl thiols, anthraquinone-terminated PEG thiols, hydroquinone-terminated alkyl thiols, hydroquinone-terminated PEG thiols, RGD peptide-terminated thiols, and YIGSR peptide-terminated thiols.

25. 25. The method of claim 24, wherein the first thiol-based passivating molecule is MCH and the second, different thiol-based passivating molecule is PC.

26. 26. The method of claim 25, wherein applying the first thiol-based passivating molecule and the second thiol-based passivating molecule comprises contacting the electrode material with a solution comprising the first thiol-based passivating molecule and the second, different thiol-based passivating molecule in a ratio of MCH to PC of about 1 mM to about 30 mM MCH to about 2.5 mM to about 30 mM PC.

27. 26. The method of claim 25, wherein applying the first thiol-based passivating molecule and the second thiol-based passivating molecule comprises contacting the electrode material with a solution comprising the first thiol-based passivating molecule and the second, different thiol-based passivating molecule in an MCH to PC ratio of about 15 mM MCH to about 10 mM PC.

28. 22. The method of claim 21, wherein at least one of the first predetermined period of time and the second predetermined period of time is from about 4 hours to about 48 hours, from about 12 hours to about 24 hours, or from about 16 hours to about 20 hours.

29. 22. The method of claim 21, wherein the first thiol-based passivation molecule and the second, different thiol-based passivation molecule are applied to the electrode material to a predetermined density of a passivation layer.

30. 22. The method of claim 21, wherein applying the first thiol-based passivating molecule and applying the second, different thiol-based passivating molecule are carried out at a temperature of from about −20° C. to about 37° C., from about 1° C. to about 30° C., or from about 3° C. to about 23° C.

31. 22. The method of claim 21 , wherein the biorecognition element is functionalized with a redox active molecule and configured to undergo a conformational change upon binding to the analyte to move the redox active molecule toward or away from the electrode material.

32. 32. The method of claim 31 , wherein the biorecognition element is an aptamer.

33. 32. The method of claim 31 , wherein the redox active molecule comprises one selected from the group consisting of methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, hydroquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, carboxy-X-rhodamine, and π-extended tetrathiafulvalene (exTTF).

34. 22. The method of claim 21, further comprising depositing cysteine ​​residues on the electrode material by chemisorption.

35. dissolving the first thiol-based passivating molecule in a first solvent; 22. The method of claim 21, further comprising: dissolving the second, different thiol-based passivating molecule in a second solvent.

36. 36. The method of claim 35, wherein the first solvent and the second solvent are each independently an organic solvent or an inorganic solvent.

37. 36. The method of claim 35, wherein the first solvent and the second solvent are each independently selected from the group consisting of ethanol, methanol, propanol, isopropanol, butanol, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, propylene carbonate, ethylene carbonate, deionized water, phosphate buffered saline, and buffered salt solutions.

38. 36. The method of claim 35, wherein at least one of the first solvent and the second solvent comprises an organic solvent in water at a concentration of at least about 5% v / v, at least about 10% v / v, at least about 15% v / v, at least about 20% v / v, at least about 25% v / v, at least about 30% v / v, at least about 35% v / v, at least about 40% v / v, at least about 45% v / v, at least about 50% v / v, at least about 55% v / v, at least about 60% v / v, at least about 65% v / v, at least about 70% v / v, at least about 75% v / v, at least about 80% v / v, at least about 85% v / v, at least about 90% v / v, or at least about 95% v / v.

39. 36. The method of claim 35, wherein the first solvent and the second solvent are the same.

40. 22. The method of claim 21, wherein the first thiol-based passivation molecule and the second, different thiol-based passivation molecule are applied simultaneously.

41. 41. The method of claim 40, wherein the first thiol-based passivating molecule and the second, different thiol-based passivating molecule are applied simultaneously in the same solution.

42. 22. The method of claim 21 , wherein applying the first thiol-based passivating molecule and the second thiol-based passivating molecule comprises contacting the electrode material with a solution comprising the first thiol-based passivating molecule and the second, different thiol-based passivating molecule.

43. 43. The method of claim 42, wherein at least one of the first thiol-based passivating molecule and the second thiol-based passivating molecule in the solution has enhanced solubility.

44. A wearable device, 1. A wearable device comprising: an electrochemical aptamer-based sensor comprising a first thiol-based passivation molecule and a second, different thiol-based passivation molecule on an electrode material, wherein the first thiol-based passivation molecule comprises 6-mercapto-1-hexanol (MCH) and the second, different thiol-based passivation molecule comprises 2-((3-((6-mercaptohexyl)thio)-2-methylpropanoyl)oxy)ethyl(2-trimethylammonium)ethyl)phosphate (PC).

45. 45. The wearable device of claim 44, wherein the MCH and the PC are applied to the electrode material in a ratio of MCH to PC of about 1 mM to about 30 mM MCH to about 2.5 mM to about 30 mM PC.

46. 45. The wearable device of claim 44, wherein the MCH and the PC are applied to the electrode material in an MCH to PC ratio of 15 mM MCH to 10 mM PC.

47. 45. The wearable device of claim 44, wherein the first thiol-based passivation molecule and the second, different thiol-based passivation molecule on the electrode material maintain stability of the sensor for more than 150 hours, stability being determined by comparing a current based on an oxygen reduction reaction to a predetermined threshold.

48. 1. A sensor configured to generate a signal indicative of a concentration of an analyte in a fluid, the sensor comprising: an electrode material; a biorecognition layer at least partially disposed on the electrode material and including a biorecognition element that selectively and reversibly binds to an analyte; and an at least partially dried hydrogel disposed on the biorecognition layer.

49. 49. The sensor of claim 48, wherein the hydrogel comprises about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% water compared to a fully hydrated state of the hydrogel.

50. 49. The sensor of claim 48, wherein the hydrogel is completely dried.

51. 49. The sensor of claim 48, wherein the hydrogel comprises one hydrophilic polymer or a combination of two or more hydrophilic polymers selected from the group consisting of agarose, poly(urethane), poly(N-vinylpyrrolidone), poly(acrylamide), poly(acrylic acid), poly(methacrylic acid), poly(2-hydroxyethyl methacrylate), poly(acrylic acid-co-acrylamide), poly(N-isopropylacrylamide), poly(2-acrylamido-2-methylpropanesulfonic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(lactic acid), poly(glycolic acid), poly(glycolic acid-co-lactic acid), collagen, alginate, hyaluronic acid, heparin, glycosaminoglycans, chitosan, Nafion, carboxymethyl cellulose, and cellulose acetate.

52. 49. The sensor of claim 48, wherein the hydrogel has a thickness of about 0.001 μm to about 1000 μm, about 0.01 μm to about 100 μm, about 0.1 μm to about 10 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 200 μm, or about 1 μm to about 1000 μm.

53. 49. The sensor of claim 48, wherein the working electrode is configured as a planar microelectrode.

54. 54. The sensor of claim 53, wherein the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array.

55. 49. The sensor of claim 48, wherein the sensor further comprises a microneedle, the working electrode being coupled to the microneedle.

56. 56. The sensor of claim 55, wherein the sensor further comprises a microneedle array, and the microneedle is part of the microneedle array.

57. 49. The sensor of claim 48, wherein the biorecognition element is an aptamer.

58. 1. A method of manufacturing a sensor configured to generate a signal indicative of a concentration of an analyte in a fluid, the method comprising: (a) providing a working electrode comprising an electrode material and a biorecognition layer at least partially disposed on the electrode material, the biorecognition layer comprising a biorecognition element that selectively and reversibly binds to an analyte; (b) applying a hydrogel onto the biorecognition layer; (c) drying the hydrogel to an at least partially dry state.

59. 60. The method of claim 58, wherein the biorecognition layer further comprises a passivation element at least partially disposed on the electrode material.

60. 59. The method of claim 58, wherein the hydrogel in the at least partially dried state comprises about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% water compared to the hydrogel in its fully hydrated state.

61. 59. The method of claim 58, wherein the hydrogel is completely dried.

62. 59. The method of claim 58, wherein the hydrogel comprises one or a combination of two or more hydrophilic polymers selected from the group consisting of agarose, poly(urethane), poly(N-vinylpyrrolidone), poly(acrylamide), poly(acrylic acid), poly(methacrylic acid), poly(2-hydroxyethyl methacrylate), poly(acrylic acid-co-acrylamide), poly(N-isopropylacrylamide), poly(2-acrylamido-2-methylpropanesulfonic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(lactic acid), poly(glycolic acid), poly(glycolic acid-co-lactic acid), collagen, alginate, hyaluronic acid, heparin, glycosaminoglycans, chitosan, Nafion, carboxymethylcellulose, and cellulose acetate.

63. 59. The method of claim 58, wherein the working electrode is configured as a planar microelectrode.

64. 64. The method of claim 63, wherein the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array.

65. 59. The method of claim 58, wherein the working electrode is coupled to a microneedle.

66. 66. The method of claim 65, wherein the sensor further comprises a microneedle array, and the microneedle is part of the microneedle array.

67. 60. The method of claim 58, wherein the hydrogel in the at least partially dried state has a thickness of about 0.001 μm to about 1000 μm, about 0.01 μm to about 100 μm, about 0.1 μm to about 10 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 200 μm, or about 1 μm to about 1000 μm.

68. 59. The method of claim 58, wherein the hydrogel is applied by drop casting, spin coating, spray coating, chemical vapor deposition (CVD), or dip coating.

69. 59. The method of claim 58, wherein the hydrogel comprises agarose.

70. 59. The method of claim 58, wherein the agarose is a low electroosmosis agarose characterized by an electroosmosis of about 0.09 to about 0.

14.

71. 59. The method of claim 58, wherein the hydrogel comprises agarose at a concentration of about 0.5% w / w to about 4% w / w in a buffered saline solution when the hydrogel is applied onto the biorecognition layer.

72. 72. The method of claim 71, wherein the buffered saline is phosphate buffered saline.

73. 73. The method of claim 72, wherein the buffered saline is acetate buffer, Tris buffer, citrate buffer, McIlvaine buffer, Tris-acetate-EDTA buffer, or Tris-EDTA buffer.

74. 60. The method of claim 58, wherein the hydrogel is drop-cast onto the biorecognition layer at a temperature of about 45°C to about 75°C.

75. 75. The method of claim 74, wherein the agarose has a melting point of about 75°C to about 97°C.

76. 76. The method of claim 75, wherein the hydrogel is applied by dip coating or spin coating.

77. 77. The method of claim 76, wherein the agarose has a sulfate content of less than about 0.20% w / w.

78. 59. The method of claim 58, wherein the hydrogel is dried at an ambient temperature of about 15°C to about 30°C and an ambient humidity of about 10% to about 80% relative humidity for at least 10 hours.

79. 79. The method of claim 78, wherein the ambient humidity is from about 50% to about 80% relative humidity.

80. 80. The method of claim 79, further comprising packaging the sensor in a sealed disposable package, the interior of the package having an ambient humidity of about 50% to about 80% relative humidity.

81. 59. The method of claim 58, wherein the biorecognition element is an aptamer.

82. A sensor, a working electrode comprising an electrode material, a biorecognition layer at least partially disposed on the electrode material and including a biorecognition element that selectively and reversibly binds to an analyte, and a hydrogel disposed on the biorecognition layer; The sensor is sterilized by exposure to radiation, the sterilized sensor being configured to generate a signal indicative of a concentration of an analyte in a fluid.

83. 83. The sensor of claim 82, wherein the biorecognition layer further comprises a passivation element at least partially disposed on the electrode material.

84. 83. The sensor of claim 82, wherein the radiation is ultraviolet radiation, gamma radiation, x-ray radiation, or electron beam radiation.

85. 85. The sensor of claim 84, wherein the radiation is electron beam radiation.

86. 86. The sensor of claim 85, wherein the electron beam radiation is applied to the working electrode at a dose of about 2 megarads to about 4 megarads, about 2.5 megarads to about 3.5 megarads, or about 2.7 megarads to about 3.1 megarads.

87. 83. The sensor of claim 82, wherein the hydrogel comprises one or a combination of two or more hydrophilic polymers selected from the group consisting of agarose, poly(urethane), poly(N-vinylpyrrolidone), poly(acrylamide), poly(acrylic acid), poly(methacrylic acid), poly(2-hydroxyethyl methacrylate), poly(acrylic acid-co-acrylamide), poly(N-isopropylacrylamide), poly(2-acrylamido-2-methylpropanesulfonic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(lactic acid), poly(glycolic acid), poly(glycolic acid-co-lactic acid), collagen, alginate, hyaluronic acid, heparin, glycosaminoglycans, chitosan, Nafion, carboxymethyl cellulose, and cellulose acetate.

88. 83. The sensor of claim 82, wherein the working electrode is a planar microelectrode.

89. 89. The sensor of claim 88, wherein the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array.

90. 83. The sensor of claim 82, wherein the sensor comprises a microneedle, the working electrode being coupled to the microneedle.

91. 91. The sensor of claim 90, wherein the sensor comprises a microneedle array, and the microneedle is part of the microneedle array.

92. 83. The sensor of claim 82, wherein the hydrogel has a thickness of about 0.001 μm to about 1000 μm, about 0.01 μm to about 100 μm, about 0.1 μm to about 10 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 200 μm, or about 1 μm to about 1000 μm.

93. 83. The sensor of claim 82, wherein the biorecognition layer is degraded by the radiation exposure by about 2% or less, about 5% or less, about 10% or less, about 15% or less, or about 20% or less.

94. Decomposition of the biorecognition layer placing the working electrode in a fluid containing an analyte and collecting a signal from the working electrode indicative of the concentration of the analyte in the fluid; and 94. The sensor of claim 93, wherein the strength of the signal is determined by comparing it to a reference signal strength.

95. 95. The sensor of claim 94, wherein the reference signal strength is based on an initial signal measured with an identical or identical working electrode placed in an identical fluid, and wherein the identical or identical working electrode has not been sterilized by exposure to radiation.

96. 83. The method of claim 82, wherein the biorecognition element is an aptamer.

97. 1. A method of sterilizing a sensor configured to generate a signal indicative of a concentration of an analyte in a fluid, the method comprising: (a) providing a working electrode comprising an electrode material and a biorecognition layer comprising a biorecognition element that selectively and reversibly binds to an analyte, the biorecognition layer being at least partially disposed on the electrode material; (b) applying a hydrogel onto the biorecognition layer; (c) sterilizing the working electrode by exposure to radiation.

98. 98. The method of claim 97, wherein the biorecognition layer further comprises a passivation element at least partially disposed on the electrode material.

99. 98. The method of claim 97, wherein the radiation is ultraviolet radiation, gamma radiation, x-ray radiation, or electron beam radiation.

100. 100. The method of claim 99, wherein the radiation is electron beam radiation.

101. 101. The method of claim 100, wherein the electron beam radiation is applied to the working electrode at a dose of about 2 megarads to about 4 megarads, about 2.5 megarads to about 3.5 megarads, or about 2.7 megarads to about 3.1 megarads.

102. 98. The method of claim 97, wherein the hydrogel comprises one or a combination of two or more hydrophilic polymers selected from the group consisting of agarose, poly(urethane), poly(N-vinylpyrrolidone), poly(acrylamide), poly(acrylic acid), poly(methacrylic acid), poly(2-hydroxyethyl methacrylate), poly(acrylic acid-co-acrylamide), poly(N-isopropylacrylamide), poly(2-acrylamido-2-methylpropanesulfonic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(lactic acid), poly(glycolic acid), poly(glycolic acid-co-lactic acid), collagen, alginate, hyaluronic acid, heparin, glycosaminoglycans, chitosan, Nafion, carboxymethylcellulose, and cellulose acetate.

103. 98. The method of claim 97, wherein the working electrode is a planar microelectrode.

104. 104. The method of claim 103, wherein the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array.

105. 98. The method of claim 97, wherein the working electrode is coupled to a microneedle.

106. 106. The method of claim 105, wherein the microneedles are part of a microneedle array.

107. 98. The method of claim 97, wherein the hydrogel has a thickness of about 0.001 μm to about 1000 μm, about 0.01 μm to about 100 μm, about 0.1 μm to about 10 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 200 μm, or about 1 μm to about 1000 μm.

108. 98. The method of claim 97, wherein the biorecognition layer is degraded by the radiation exposure by about 2% or less, about 5% or less, about 10% or less, about 15% or less, or about 20% or less.

109. Decomposition of the biorecognition layer placing the working electrode in a fluid containing an analyte and collecting a signal from the working electrode indicative of the concentration of the analyte in the fluid; and 109. The method of claim 108, wherein the strength of the signal is determined by comparing it to a reference signal strength.

110. 110. The method of claim 109, wherein the reference signal strength is based on an initial signal measured with an identical or identical working electrode placed in an identical fluid, and wherein the identical or identical working electrode has not been sterilized by exposure to radiation.

111. 98. The method of claim 97, wherein the biorecognition element is an aptamer.

112. 1. A sensor configured to generate a signal indicative of a concentration of an analyte in a fluid, the sensor comprising: an electrode material; a biorecognition layer at least partially disposed on the electrode material and including a biorecognition element that selectively and reversibly binds to an analyte; a hydrogel disposed on the biorecognition layer; A sensor wherein the biorecognition layer degrades at a rate of about 3% or less per day when stored at an ambient temperature of about 15° C. to about 30° C. and an ambient humidity of about 10% to about 80% relative humidity.

113. 113. The sensor of claim 112, wherein the biorecognition layer further comprises a passivation element at least partially disposed on the electrode material.

114. 113. The sensor of claim 112, wherein the hydrogel is in an at least partially dried state.

115. 115. The sensor of claim 114, wherein the hydrogel in the at least partially dried state contains about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% water compared to the hydrogel in a fully hydrated state.

116. 115. The sensor of claim 114, wherein the hydrogel is in a completely dried state.

117. 116. The sensor of claim 115, wherein the degradation of the biorecognition layer is measured based on a decrease in the signal generated by the working electrode when placed in a liquid containing the analyte, compared to a baseline signal intensity.

118. The reference signal strength is the initial signal measured by the same or an equivalent working electrode placed in an equivalent fluid within four hours after the hydrogel is applied onto the biorecognition layer; or The sensor of claim 117, wherein the sensor is based on a comparative signal measured by an equivalent working electrode stored for an equivalent period of time and placed in an equivalent fluid, the equivalent sensor lacking a hydrogel placed on the biorecognition layer.

119. 113. The sensor of claim 112, wherein the hydrogel comprises one or a combination of two or more hydrophilic polymers selected from the group consisting of agarose, poly(urethane), poly(N-vinylpyrrolidone), poly(acrylamide), poly(acrylic acid), poly(methacrylic acid), poly(2-hydroxyethyl methacrylate), poly(acrylic acid-co-acrylamide), poly(N-isopropylacrylamide), poly(2-acrylamido-2-methylpropanesulfonic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(lactic acid), poly(glycolic acid), poly(glycolic acid-co-lactic acid), collagen, alginate, hyaluronic acid, heparin, glycosaminoglycans, chitosan, Nafion, carboxymethyl cellulose, and cellulose acetate.

120. 115. The sensor of claim 114, wherein the hydrogel in the at least partially dried state has a thickness of about 0.01 μm to about 100 μm, about 0.1 μm to about 10 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 200 μm, or about 1 μm to about 1000 μm.

121. 119. The sensor of claim 118, wherein the working electrode is a planar microelectrode.

122. 122. The sensor of claim 121, wherein the sensor further comprises a microelectrode array, and the working electrode is part of the microelectrode array.

123. 113. The sensor of claim 112, wherein the sensor further comprises a microneedle, the working electrode being coupled to the microneedle.

124. 124. The sensor of claim 123, wherein the sensor further comprises a microneedle array, and the microneedle is part of the microneedle array.

125. 113. The sensor of claim 112, wherein the biorecognition element is an aptamer.

126. A working electrode configured to generate a signal indicative of a concentration of an analyte in a fluid, the working electrode comprising: an electrode material; a biorecognition element disposed on the electrode material that selectively binds to the analyte, the biorecognition element being functionalized with a redox active molecule and configured to undergo a conformational change upon binding to the analyte to move the redox active molecule toward or away from the electrode material; a passivation element comprising a zwitterionic peptide disposed on the electrode material.

127. 127. The working electrode of claim 126, wherein the zwitterionic peptide comprises at least one amino acid having a carboxyl group as a side chain.

128. 128. The working electrode of claim 127, wherein the at least one amino acid having a carboxyl group as a side chain comprises a glutamate residue, an aspartate residue, or a combination thereof.

129. 129. The working electrode of any one of claims 126 to 128, wherein the zwitterionic peptide comprises at least one amino acid having a positively charged side chain.

130. 130. The working electrode of claim 129, wherein the positively charged side chain is an amine group, a guanidino group, or an imidazole group.

131. 131. The working electrode of claim 129 or 130, wherein the at least one amino acid having a positively charged side chain is one or a combination of two or more of a lysine residue, a histidine residue, an ornithine residue, and an arginine residue.

132. 132. The working electrode of any one of claims 126 to 131, wherein the zwitterionic peptide comprises a thiol group.

133. 133. The working electrode of claim 132, wherein the zwitterionic peptide comprises a cysteine ​​residue and the thiol group is a side chain of the cysteine ​​residue.

134. 134. The working electrode of claim 133, wherein the thiol group comprises an extended carbon chain.

135. 135. The working electrode of claim 134, wherein the extended carbon chain is a chain of 1 to 20 carbon atoms.

136. 134. The working electrode of claim 133, wherein the cysteine ​​residue is a C-terminal cysteine ​​residue or an N-terminal cysteine.

137. 137. The working electrode of claim 136, wherein the C-terminus of the C-terminal cysteine ​​is a free carboxyl group.

138. 138. The working electrode of claim 137, wherein the C-terminus of the C-terminal cysteine ​​is a modified C-terminus having a neutral charge.

139. 139. The working electrode of claim 138, wherein the modified C-terminus having the neutral charge is an amide group, an ester, a methoxyester, or a methoxide group.

140. 137. The working electrode of claim 136, wherein the N-terminus of the N-terminal cysteine ​​is a free amine group.

141. 141. The working electrode of claim 140, wherein the N-terminus of the N-terminal cysteine ​​is a modified N-terminus having a neutral charge.

142. 142. The working electrode of claim 141, wherein the modified N-terminus having the neutral charge is an amide group or an alkylamine group.

143. 143. The working electrode of claim 142, wherein the amide group is an acetamide group.

144. 144. The working electrode of any one of claims 126 to 143, wherein the zwitterionic peptide comprises at least one non-standard amino acid.

145. 145. The working electrode of claim 144, wherein the at least one non-standard amino acid is selected from ornithine, beta-alanine, gamma-aminobutyric acid (GABA), 4-aminobenzoic acid, taurine, or a combination thereof.

146. The zwitterionic peptide is X-(KX) ｘ -P ｙ C or CP ｙ -(KX) ｘ -K, where x is 1 to 5, X is a glutamic acid residue or an aspartic acid residue, K is a lysine residue, P is a proline residue, and C is a cysteine ​​residue.

147. 147. The working electrode of claim 146, wherein y is 5 or less.

148. 147. The working electrode of claim 146, wherein y is 3 or less.

149. 149. The working electrode of any one of claims 133 to 148, wherein the cysteine ​​residue is chemisorbed to the electrode material.

150. 150. The working electrode of any one of claims 126 to 149, wherein the biorecognition element is an aptamer.

151. 151. The working electrode of claim 150, wherein the aptamer comprises a thiol group at the 3' or 5' end, and the thiol group is chemisorbed to the electrode material.

152. 152. The working electrode of any one of claims 126 to 151, wherein the electrode material is gold or glassy carbon.

153. 153. The working electrode of any one of claims 126 to 152, wherein the redox active molecule is selected from the group consisting of methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, carboxy-X-rhodamine, and pi-extended tetrathiafulvalene (exTTF).

154. 154. The working electrode of any one of claims 126 to 153, wherein the zwitterionic peptide is deposited on the surface of the electrode material at a concentration of about 1 mM to about 30 mM.

155. 155. An analyte sensor comprising the working electrode of any one of claims 126 to 154, a counter electrode, and a reference electrode.

156. 156. The analyte sensor of claim 155, wherein the electrode material is bonded to a microneedle.

157. 157. The analyte sensor of claim 156, wherein the analyte sensor further comprises a microneedle array, and the microneedle is a first microneedle of the microneedle array.

158. 158. The analyte sensor of claim 157, wherein the counter electrode is coupled to a second microneedle of the microneedle array and the reference electrode is coupled to a third microneedle of the microneedle array.

159. 159. The analyte sensor of claim 157 or 158, wherein the first microneedle includes a tapered distal portion having an insulated distal apex, and the working electrode is located on a surface of the tapered distal portion proximal to the insulated distal apex.

160. 160. The analyte sensor of claim 159, wherein the working electrode is an annular electrode including a proximal edge and a distal edge, the distal edge of the annular working electrode being adjacent to the proximal edge of the insulated distal tip.

161. A working electrode configured to generate a signal indicative of a concentration of an analyte in a fluid, the working electrode comprising: an electrode material; a biorecognition element disposed on the electrode material that selectively binds to the analyte; X-(KX) ｘ -P ｙ C or CP ｙ -(KX) ｘ and a passivation element comprising a zwitterionic peptide disposed on the electrode material, the zwitterionic peptide comprising an amino acid sequence of -K, wherein x is 0, 1, or 2, X is a glutamic acid residue or an aspartic acid residue, K is a lysine residue, P is a proline residue, and C is a cysteine ​​residue.

162. 162. The working electrode of claim 161, wherein the cysteine ​​residue comprises a thiol group comprising an extended carbon chain.

163. 163. The working electrode of claim 162, wherein the extended carbon chain is a chain of 1 to 20 carbon atoms.

164. The amino acid sequence of the zwitterionic peptide is X-(KX) containing a C-terminal cysteine. ｘ -P ｙ C, and the C-terminus of the C-terminal cysteine ​​is a free carboxyl group.

165. 164. The working electrode of any one of claims 161 to 163, wherein the C-terminus of the C-terminal cysteine ​​is a modified C-terminus having a neutral charge.

166. 166. The working electrode of claim 165, wherein the modified C-terminus having the neutral charge is an amide group, an ester, a methoxyester, or a methoxide group.

167. The amino acid sequence of the zwitterionic peptide is a CP containing an N-terminal cysteine. ｙ -(KX) ｘ -K, and the N-terminus of the N-terminal cysteine ​​residue is a free amine group.

168. The amino acid sequence of the zwitterionic peptide is a CP containing an N-terminal cysteine. ｙ -(KX) ｘ -K, and the N-terminus of the N-terminal cysteine ​​is a modified N-terminus having a neutral charge.

169. 169. The working electrode of claim 168, wherein the modified N-terminus having the neutral charge is an amide group or an alkylamine group.

170. 170. The working electrode of claim 169, wherein the amide group is an acetamide group.

171. 171. The working electrode of any one of claims 161 to 170, wherein y is 3 or less.

172. 172. The working electrode of claim 171, wherein the zwitterionic peptide consists of the amino acid sequence XKXKXPPC.

173. 172. The working electrode of claim 171, wherein the zwitterionic peptide consists of the amino acid sequence XKXPPC.

174. 162. The working electrode of claim 161, wherein the zwitterionic peptide consists of the amino acid sequence of any one of SEQ ID NOs: 2-33.

175. 175. The working electrode of any one of claims 161 to 174, wherein the cysteine ​​residue is chemisorbed to the electrode material.

176. 176. The working electrode of any one of claims 161 to 175, wherein the electrode material is gold or glassy carbon.

177. 177. The working electrode of any one of claims 161 to 176, wherein the biorecognition element is an aptamer.

178. 178. The working electrode of claim 177, wherein the aptamer comprises a thiol group at the 3' or 5' end, and the thiol group is chemisorbed to the electrode material.

179. 179. The working electrode of claim 178, wherein the biorecognition layer is substantially free of 6-mercaptohexanol.

180. 180. The working electrode of any one of claims 161 to 179, wherein the biorecognition element is functionalized with a redox active molecule and configured such that upon binding to the analyte, the redox active molecule undergoes a conformational change such that it moves toward or away from the electrode material.

181. 181. The working electrode of claim 180, wherein the redox active molecule is selected from the group consisting of methylene blue, ferrocene, pentamethylferrocene, C5-ferrocene, Nile blue, thionine, anthraquinone, C5-anthraquinone, gallocyanine, indophenol, neutral red, dabcyl, carboxy-X-rhodamine, and pi-extended tetrathiafulvalene (exTTF).

182. 182. The working electrode of any one of claims 161 to 181, wherein the zwitterionic peptide is deposited on the surface of the electrode material at a concentration of about 1 mM to about 30 mM.

183. 183. An analyte sensor comprising the working electrode of any one of claims 161 to 182, a counter electrode, and a reference electrode.

184. 184. The analyte sensor of claim 183, further comprising a microneedle, wherein the working electrode is coupled to the microneedle.

185. 184. The analyte sensor of claim 183, wherein the sensor further comprises a microneedle array, and the microneedle is a first microneedle of the microneedle array.

186. 186. The analyte sensor of claim 185, wherein the counter electrode is coupled to a second microneedle of the microneedle array and the reference electrode is coupled to a third microneedle of the microneedle array.

187. 187. The analyte sensor of claim 185 or 186, wherein the first microneedle includes a tapered distal portion having an insulated distal apex, and the working electrode is located on a surface of the tapered distal portion proximal to the insulated distal apex.

188. 188. The analyte sensor of claim 187, wherein the working electrode is an annular electrode including a proximal edge and a distal edge, the distal edge of the annular working electrode being adjacent to the proximal edge of the insulated distal tip.

189. X-(KX) ｘ -P ｙ C or CP ｙ -(KX) ｘ -K, where x is 0, 1, or 2, X is a glutamic acid residue or an aspartic acid residue, K is a lysine residue, P is a proline residue, and C is a cysteine ​​residue.

190. 190. The zwitterionic peptide of claim 189, wherein the cysteine ​​residue comprises a thiol group comprising an extended carbon chain.

191. 191. The zwitterionic peptide of claim 190, wherein the extended carbon chain is a chain of 1 to 20 carbon atoms.

192. The amino acid sequence of the zwitterionic peptide is X-(KX) containing a C-terminal cysteine. ｘ -P ｙ 192. The zwitterionic peptide of any one of claims 189 to 191, wherein the C-terminus of the C-terminal cysteine ​​is a free carboxyl group.

193. The amino acid sequence of the zwitterionic peptide is X-(KX) containing a C-terminal cysteine. ｘ -P ｙ 192. The zwitterionic peptide of any one of claims 189 to 191, wherein the C-terminus of the C-terminal cysteine ​​is a modified C-terminus having a neutral charge.

194. 194. The zwitterionic peptide of claim 193, wherein the modified C-terminus having a neutral charge is an amide group, an ester, a methoxyester, or a methoxide group.

195. The amino acid sequence of the zwitterionic peptide is a CP containing an N-terminal cysteine. ｙ -(KX) ｘ 192. The zwitterionic peptide of any one of claims 189 to 191, wherein the N-terminus of the N-terminal cysteine ​​is -K and the N-terminus of the N-terminal cysteine ​​is a free amine group.

196. The amino acid sequence of the zwitterionic peptide is a CP containing an N-terminal cysteine. ｙ -(KX) ｘ -K, and the N-terminus of the N-terminal cysteine ​​is a modified N-terminus having a neutral charge.

197. 197. The zwitterionic peptide of claim 196, wherein the modified N-terminus having a neutral charge is an amide group or an alkylamine group.

198. 198. The zwitterionic peptide of claim 197, wherein the amide group is an acetamide group.

199. 199. The zwitterionic peptide of any one of claims 189 to 198, wherein y is 3 or less.

200. 200. The zwitterionic peptide of claim 199, wherein the zwitterionic peptide consists of the amino acid sequence XKXKXPPC or XKXPPC.

201. 200. The zwitterionic peptide of claim 199, wherein the zwitterionic peptide consists of the amino acid sequence XKXPPC.

202. The zwitterionic peptide of claim 189, wherein the zwitterionic peptide consists of the amino acid sequence of any one of SEQ ID NOs: 2-33.

203. 203. The zwitterionic peptide of any one of claims 189-193 and 195-202, wherein the cysteine ​​residue is modified with an amide group, an ester, a methoxyester, or a methoxide group.

204. 1. A method for manufacturing a working electrode, comprising: depositing gold onto a substrate to create a gold surface; depositing a biorecognition element on the gold surface, the biorecognition element being functionalized with a redox active molecule and configured to undergo a conformational change upon binding to an analyte to move the redox active molecule toward or away from the gold surface; and depositing a passivating element comprising a zwitterionic peptide on said gold surface.

205. 205. The method of claim 204, wherein the gold surface is not polished prior to depositing the zwitterionic peptide and the biorecognition element.

206. 205. The method of claim 204, further comprising polishing the gold surface prior to depositing the passivation element and the biorecognition element.

207. 207. The method of claim 206, wherein the polishing comprises electropolishing.

208. 205. The method of claim 204, wherein the gold is deposited by electrodeposition, chemical vapor deposition, electroless plating, or physical vapor deposition.

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