Devices and methods for aptamer-assisted microneedle-based monitoring of biomarkers

Abstract

To provide a solution to the problem of being unable to provide real-time information regarding the concentrations of biomarkers.SOLUTION: Methods, apparatus, and systems, are described that relate to microneedle-assisted aptamer-based electrochemical sensing for label-free, continuous real-time monitoring of biomarkers in a biofluid. One exemplary device for electrochemical monitoring of one or more analytes in a biofluid includes a substrate and at least two microneedles coupled to the substrate. Each microneedle in the at least two microneedles includes a protruding needle structure and an electrode probe structure. The electrode probe structure of a first microneedle in the at least two microneedles includes an aptamer sequence which is specific for a first analyte, and the electrode probe structure of the first microneedle is operable as a working electrode for detection of the first analyte using a first electrochemical detection technique.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to provisional application No. 62 / 947,399, entitled "DEVICES AND METHODS FOR APTAMER-ASSISTED MICRONEEDLE-BASED MONITORING OF PROTEIN BIOMARKERS," filed December 12, 2019. The entire contents of the aforementioned provisional application are incorporated by reference as part of the disclosure of this document.

[0002] The subject matter of this application relates generally to electrochemical sensing, and more particularly to methods, systems, materials and devices for label-free, continuous, real-time monitoring of biomarkers in biological fluids. [Background technology]

[0003] Currently, there are no available devices for minimally or noninvasive continuous real-time monitoring of protein biomarkers, for example, in interstitial fluid (ISF). The only available methods for measuring the levels of these critically important diagnostic markers are based on traditional methods of blood sampling using a hypodermic needle and subsequent biomarker quantification using external instruments / techniques in a centralized laboratory. The ELISA method typically used for biomarker quantification is very time-consuming and expensive. Traditional approaches to biomarker quantification have significant disadvantages, including patient discomfort, the need for relatively high sample volumes, and the generation of sharps waste. More importantly, they cannot track trends and fluctuations in biomarker levels in real time and therefore cannot provide real-time information regarding biomarker concentrations. Therefore, there remains a need to create low-cost systems, devices, materials, and methods for continuous, real-time monitoring of biomarker levels in ISF. Summary of the Invention

[0004] The techniques disclosed herein can be implemented in various embodiments to achieve devices, systems, and methods for aptamer-assisted microneedle-based electrochemical sensing for label-free, continuous, real-time monitoring of biomarkers in biological fluids.

[0005] One aspect of the disclosed technology relates to a device including a substrate. The device also includes at least two microneedles connected to the substrate. Each microneedle of the at least two microneedles includes a protruding needle structure and an electrode probe structure. The protruding needle structure includes an outer wall extending outward from a surface of the substrate, the outer wall surrounding an interior volume of the protruding needle structure and forming an apex at the end of the outer wall. The electrode probe structure is configured to generate a signal in response to one or more chemical or biological substances in a biological fluid contacting the electrode probe structure. The electrode probe structure of a first microneedle of the at least two microneedles includes an aptamer sequence specific to a first analyte, and the electrode probe structure of the first microneedle is operable as a working electrode for detection of the first analyte using a first electrochemical detection technique. The electrode probe structure of a second microneedle of the at least two microneedles is operable as an electrochemical counter electrode or an electrochemical reference electrode.

[0006] Another aspect of the disclosed technology relates to a method for manufacturing an electrochemical sensing device, comprising providing a substrate. The method further comprises coupling at least two microneedles to the substrate. Each microneedle of the at least two microneedles comprises a protruding needle structure and an electrode probe structure. The protruding needle structure includes an outer wall extending outward from a surface of the substrate, the outer wall surrounding an interior volume of the protruding needle structure and forming an apex at the end of the outer wall. The electrode probe structure is configured to generate a signal in response to one or more chemical or biological substances in a biological fluid contacting the electrode probe structure. The electrode probe structure of a first microneedle of the at least two microneedles comprises an aptamer sequence specific to a first analyte, and the electrode probe structure of the first microneedle is operable as a working electrode for detection of the first analyte using a first electrochemical detection technique. The electrode probe structure of a second microneedle of the at least two microneedles is operable as an electrochemical counter electrode or an electrochemical reference electrode. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows a block diagram illustrating an example embodiment of an electrochemical microneedle sensor device having at least two microneedle-based working electrodes. [Figure 2] 2 shows a block diagram illustrating an example embodiment of the electrochemical microneedle sensor device of FIG. 1 in communication with an electronic device. [Figure 3] 3 shows a block diagram of an example embodiment of the electronic device shown in FIG. 2. [Figure 4] FIG. 1 shows a block diagram representing an example embodiment of an electrochemical microneedle sensor device having at least two groups of microneedle-based working electrodes. [Figure 5] FIG. 1 shows a block diagram illustrating an example embodiment of an electrochemical microneedle sensor device having at least three microneedle-based working electrodes. [Figure 6]FIG. 1 shows a block diagram illustrating an example embodiment of an electrochemical microneedle sensor device having at least four microneedle-based working electrodes. [Figure 7] FIG. 1 shows a diagram of an example embodiment of an electrochemical microneedle sensor device having at least four microneedle-based working electrodes. [Figure 8] 1 illustrates an example embodiment of a method for fabricating an electrochemical sensing device. DETAILED DESCRIPTION OF THE INVENTION

[0008] This application describes the design and fabrication of a wearable microneedle-based device for label-free, continuous, real-time monitoring of biomarkers, including protein biomarkers, as a non-invasive diagnostic tool to provide in vivo analytical information of clinically important biomarkers in, for example, interstitial fluid (ISF). The developed diagnostic microneedle-based device can be easily worn on an individual's skin and can measure the concentration of various biomarkers, including, for example, protein biomarkers, directly in ISF in a real-time manner.

[0009] The analysis is performed at an aptamer-modified electrode, which is in the form of a microneedle (e.g., a polymer hollow microneedle) and acts as an electrochemical sensor. Aptamers functionalized with redox reporter molecules (e.g., methylene blue or anthraquinone) specifically and reversibly bind to the analyte of interest, resulting in conformational folding of the electrode-bound aptamer. This binding-induced folding leads to a change in the electron transfer properties of the redox molecule that corresponds to the concentration of the target biomarker and is detected using electrochemical techniques, such as square wave voltammetry (SWV).

[0010] Although this document describes the application of the disclosed technology to the real-time aptamer-assisted measurement of cortisol and insulin biomarkers as model analytes, the technology disclosed in this application is readily extendable to the continuous monitoring of any biomarker, such as hormones and proteins of interest, by coupling to their relevant aptamer-based biorecognition elements.

[0011] Stable in vivo electrochemical detection of target analytes present in ISF can be achieved by the techniques disclosed herein without surface attachment by using effective antibiofouling self-assembled monolayers on the surface of microneedle electrodes or hydrogel coatings. Furthermore, aptamers can be rationally engineered in vitro to perform analyte detection over a desired concentration range.

[0012] Unlike microneedle-based enzyme sensing, the disclosed technology extends the capabilities of microneedle sensors to detect a wider range of biomarkers, the sensing of which typically relies on immunoassays that are not always feasible to perform on microneedle tips.

[0013] Another very important aspect of the disclosed technology is the integration of aptamer-based electrochemical sensing and enzymatic sensing in a single microneedle array device for simultaneous, continuous, real-time, and reversible monitoring of multiple biomarkers.

[0014] Currently, there are no available devices for minimally or noninvasive continuous monitoring of various biomarkers, and the only available methods for measuring biomarkers are based on traditional methods of blood sampling using a hypodermic needle followed by quantification using external instruments / techniques in centralized laboratories (mostly using ELISA methods, which are very time-consuming and expensive). Such methods have significant disadvantages, including patient discomfort, the need for relatively high sample volumes, and the generation of sharps waste. More importantly, they cannot track trends and fluctuations in biomarker levels and therefore cannot provide real-time information on biomarker concentrations.

[0015] Currently, there is no viable technology to provide real-time on-body information on the concentrations of different biomarkers, and the development of wearable multiplexed sensors that can simultaneously and continuously monitor the dynamically changing levels of different disease markers would be of great importance for disease diagnosis, prognosis, and treatment.

[0016] While there are many examples of sensors that detect physiologically relevant biomarkers in vitro, there are no examples of biosensors for the in vivo detection of ISF biomarkers. Wearable, on-body biosensing has the potential to revolutionize healthcare by enabling personalized medicine. Providing clinically relevant health information on a continuous basis using wearable, noninvasive sensors can fully understand an individual's health status and provide a platform for disease diagnosis, prognosis, and management. This document presents an example of a wearable, on-body sensor device for continuous, real-time monitoring of various biomarkers, eliminating the cost and complexity of traditional immunoassay-based approaches. An example microneedle device based on the technology disclosed in this application can be used for multiplexed monitoring of different biomarkers, such as cortisol, insulin, ketone bodies, and glucose for diabetic patients. Such a device can be in the form of a fully integrated, wearable microneedle patch that can be used for self-monitoring and / or by healthcare providers. The aptamer-based microneedle sensing protocol described herein can be easily extended to the detection of other biomarkers of interest. This represents a breakthrough in the field of disease diagnosis and personalized healthcare.

[0017] The microneedle aptamer-based electrochemical sensing device described herein is the first demonstration of combining electrochemical aptamer-based sensing with a microneedle device for diagnostic applications. This novel electrochemical platform is also the first example of a wearable, on-body patch capable of noninvasively monitoring different biomarkers in ISF.

[0018] Various embodiments of the microneedle-based sensor device according to the technology disclosed in this application integrate aptamer-based biomarker sensing with other sensing modalities and / or strategies, including but not limited to, enzymatic detection of important metabolite biomarkers on a single microneedle array platform, as shown herein for the example of simultaneous detection of four important diabetes-related analytes, namely, cortisol, insulin, ketone bodies, and glucose.

[0019] The application of microneedles for diagnostic and monitoring purposes has attracted much attention over the past decade due to the wealth of molecular information contained in interstitial fluid. However, direct sensing of ISF markers at the microneedle tip is usually performed using enzyme-modified microneedle electrode transducers, which rely on reversible biocatalytic reactions of corresponding substrates.

[0020] In contrast, performing conventional bioaffinity assays, such as immunoassays, on microneedle tips faces obstacles due to the strong interaction between the target antigen and the capture antibody, which makes regeneration of the antibody receptor extremely difficult, along with the need for several washing and incubation steps. Such assays often also require an additional incubation step with the relevant tagging reagent to perform competitive or sandwich assays, which is not always feasible to perform on the tip of a microneedle.

[0021] The only reports on microneedle-assisted antibody-based biomarker analysis are based on sampling ISF containing biomarkers or selectively capturing specific biomarkers by binding them to functionalized antibodies on the surface of microneedles, followed by an off-body analysis procedure. These reports lack the ability to accommodate continuous monitoring of biomarkers directly on the microneedle tip. As a result, the use of microneedle ISF sensors has not been reported for in vivo bioaffinity monitoring of ISF biomarkers, and realizing in situ real-time bioaffinity assays on microneedle tips for ISF monitoring of various markers remains a significant challenge.

[0022] This document describes the use of reversibly binding conformation-dependent aptamers to enable real-time bioaffinity assays on microneedle-tip electrodes.

[0023] Aptamers have recently been shown to be highly effective for real-time, continuous monitoring of therapeutic drugs in living animals using electrochemical techniques. However, these aptamers have only been reported for the continuous monitoring of drugs and other small analytes; they have not been used for the in vivo monitoring of higher molecular weight analytes (e.g., proteins). Also, they have been applied by invasive methods, such as using an 18-gauge catheter.

[0024] By combining microneedle and aptamer-based electrochemical sensing in the disclosed technology, we demonstrate the first example of in situ bioaffinity-based monitoring of biomarkers (including protein biomarkers) in a non-invasive manner. The disclosed technology can be used for highly sensitive and stable real-time monitoring of any biomarker of interest in association with the corresponding aptamer biorecognition element.

[0025] Another unique feature of the technology disclosed herein is the simultaneous multiplexed detection of biomarkers (e.g., metabolites) using non-aptamer substances (e.g., enzyme substrates) within a single microneedle array platform. The combination of different surface chemistries and sensing modalities for on-body continuous monitoring of various markers has been a long-standing goal. The sophisticated construction of different surface chemistries used in devices according to the technology disclosed herein enables a user-friendly approach to the multiplexed, wearable combination of these various sensing formats. The devices and methods reported herein for the multiplexed detection of cortisol, insulin, glucose, and ketone bodies can be applied / extended to cover other biomarkers of interest (e.g., proteins / metabolites / drugs / electrolytes). The technology disclosed herein also makes it possible to design closed-loop systems capable of continuously monitoring the concentrations of various biomarkers (disease-related or otherwise) and to adjust the delivery of therapeutic agents based on the measured biomarker concentrations.

[0026] Examples of these and other features of devices, systems, and methods according to the technology disclosed in this application are described below.

[0027] FIG. 1 shows a block diagram illustrating an example embodiment of an electrochemical microneedle sensor device 100 having at least two microneedle-based working electrodes for sensing two different analytes according to the present technology. In the device 100, the at least two microneedle-based working electrodes are disposed on a substrate in proximity to microneedle-based reference and / or counter electrodes. As shown in the block diagram in FIG. 1, the device 100 can include a first microneedle electrode 110 configured as a reference electrode RE and / or a counter electrode CE for electrochemical sensing of both a first analyte and a second analyte. The electrode 110 is disposed on a substrate 105. The device 100 can include a second microneedle electrode 120 configured as a first working electrode WE-1 and a third microneedle electrode 130 configured as a second working electrode WE-2. The electrodes 120 and 130 are similarly disposed on the substrate 105. The microneedle electrodes 110, 120, and 130 of the electrochemical microneedle sensor device 100 include a microneedle structure and an electrode probe structure. FIG. 1 shows microneedle structures 113, 123, and 133 of the microneedle electrodes 110, 120, and 130, respectively. The microneedle structures may include, for example, an outer wall extending outward from a base surface (e.g., the base surface may be located on the surface of the substrate 105) and forming an apex at the end of the outer wall. For example, a portion of the outer wall may have an opening that leads into a hollow interior (or cavity) of the needle structure defined by the inner wall. In some embodiments, the microneedle structures may have a pyramidal shape (e.g., a triangular pyramid with three outer walls or a square pyramid with four outer walls). In other embodiments, the microneedle structures may have a conical shape. In yet other example embodiments, at least two microneedle structures of the device 100 may have different shapes.For example, in an embodiment of device 100, the microneedle structure of electrode 110 may have a conical shape, the microneedle structure of electrode 120 may have a triangular pyramidal shape, and the microneedle structure of electrode 130 may have a square pyramidal shape.

[0028] In various example embodiments, the electrode probe structures are disposed (fully or partially) within, for example, the interior or cavity of the respective microneedle structure. In other example embodiments, the electrode probe structures are incorporated into or attached to the respective microneedle structure and are located (fully or partially) outside the interior or cavity of the respective microneedle structure. In still other example embodiments, the electrode probe structures are incorporated into or attached to the respective microneedle structure, and the respective microneedle structure does not have a reference interior or cavity. FIG. 1 shows electrode probe structures 117, 127, and 137 of microneedle electrodes 110, 120, and 130, respectively. For example, the electrode probe structures of the electrodes of microneedle sensor device 100 may comprise silver (Ag), gold (Au), or platinum (Pt) wire or film, and / or carbon paste (CP).

[0029] The substrate 105 used in example embodiments of the electrochemical microneedle sensor device 100 may comprise an electrically insulating material, such as a plastic material (e.g., polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), polyethylene naphthalate (PEN), polyimide (PI), etc.) The substrate 105 may be flexible and / or bendable and / or stretchable, and / or the substrate 105 may comprise an adhesive on at least one side of the substrate 105 to allow attachment of the device 100 to a subject, for example, via attachment to the subject's skin.

[0030] In some implementations, the height of the microneedle structures (e.g., base to apex) may range from 1 mm to 2 mm, for example, preferably about 1.5 mm. In some implementations, the diameter or width of the microneedle structures may range from 200 μm to 500 μm. The microneedle structures may be spaced at various intervals and configurations, which may be selected based on the application of the microneedle sensor device 100. In some implementations, the microneedle structures are spaced tens or hundreds of microns from base to base; for example, in some implementations, the microneedle structures are spaced about 1 mm from apex to apex. In some implementations, the microneedle structures are arranged on the substrate 105 in a linear array, while in some implementations, the microneedle structures are arranged on the substrate 105 in a circular array, a rectangular array, a triangular array, or any other pattern or patternless array.

[0031] The microneedle structures and electrodes can be configured in other configurations, for example, as described in US Pat. No. 9,737,247, which is incorporated herein by reference in its entirety as part of the disclosure of this application.

[0032] In some example embodiments, the second microneedle electrode 120 and the third microneedle electrode 130 may include one or more functionalizing materials, depending on the electrochemical sensing technique applied at the particular working electrode. In FIG. 1 , the second microneedle electrode 120 includes a functionalizing material 125 disposed on or integrated with at least a portion of an electrode probe structure 127. In some embodiments, the electrode probe structure 127 functionalized with the functionalizing material 125 may be located (e.g., at least partially) within an opening or cavity of the microneedle structure 123. Similarly, in some embodiments, the third microneedle electrode 130 includes a functionalizing material 135 disposed on or integrated with at least a portion of an electrode probe structure 137. In some embodiments, the electrode probe structure 137 functionalized with the functionalizing material 135 may be located within an opening or cavity of the microneedle structure 133.

[0033] For example, in some embodiments, the same or different types of materials can be used to fabricate the electrode probe structures 117, 127, and 137 of the microneedle electrodes 110, 120, and 130, respectively, e.g., for different detection purposes. For example, the electrode probe structure 117 can include a metallic Ag / AgCl wire (e.g., approximately 500 micron diameter) embedded within the interior of the microneedle structure 113 of the microneedle electrode 110 and can function as an RE. For example, the electrode probe structure 127 can include a gold wire (e.g., approximately 500 micron diameter) embedded within the cavity of the microneedle structure 123 of the microneedle electrode 120 and can be used as a WE, e.g., for the detection of protein biomarkers. The gold wire of the electrode probe structure 127 can be insulated with a conformal coating of, e.g., parylene (poly(para-xylylene) polymer). The parylene coating can produce a highly uniform, pinhole-free, and chemically resistant coating for the Au wire, e.g., approximately 30 microns thick. The parylene coating can serve multiple purposes, including, for example, electrical insulation, chemical isolation, and mechanical protection of the wire. The parylene coating of the gold wire of the electrode probe structure 127 can be configured to expose a reproducible surface area of ​​the wire for conduction of electrochemical measurements.

[0034] A high-affinity aptamer sequence, developed using, for example, systematic evolution of ligands by exponential enrichment (SELEX) techniques, that is specific for a target analyte (e.g., capable of binding to, for example, a target analyte (e.g., a biomarker, e.g., insulin) in a reversible manner) can be tethered to the Au wire of the electrode probe structure 127, for example, via a 5'-thiol linkage. The aptamer sequence can be functionalized with a redox reporter molecule, e.g., methylene blue or anthraquinone, at the 3' or 5' end of the aptamer sequence, such that selective binding of the target biomarker to the aptamer sequence will bring the redox reporter closer to the surface of the Au wire of the electrode probe structure 127, facilitating electron transfer between the reporter molecule and the electrode probe structure 127.

[0035] Aptamers functionalized with redox reporter molecules (e.g., methylene blue or anthraquinone) can specifically and reversibly bind to compounds of interest, resulting in conformational folding of the electrode-bound aptamer. This binding-induced folding can result in a change in the electron transfer properties of the redox molecule that corresponds to the concentration of the target compound of interest and is detected using electrochemical detection techniques, such as square wave voltammetry (SWV), implemented on the aptamer-functionalized electrode of sensor device 100. Multiplexed detection of different biomarkers can be achieved by labeling aptamers with different redox reporters with different redox properties or by spatially localized measurement of the current signal of a single redox tag.

[0036] Electrochemical detection modalities or measurement techniques other than square wave voltammetry can be used for the detection of target analytes at either the microneedle working electrodes 120 or 130 of sensor device 100. Among the potential electrochemical measurement techniques that may be used by sensor device 100 are the following:

[0037] Potentiometric techniques involve direct measurement of the open circuit potential of an electrochemical cell, which may be measured between a reference electrode (e.g., reference electrode 110 of sensor device 100) and a working electrode (e.g., working electrode 120 or working electrode 130 of sensor device 100).

[0038] Potentiometric techniques are contrasted with the amperometric family of measurements, in which the current is measured while controlling the cell's potential. For example, chronoamperometry is a powerful tool for measuring diffusion-limited reactions. In chronoamperometry, the potential is stepped at the beginning of the measurement and then held constant throughout the measurement. The current resulting from this stimulus is plotted as a function of time.

[0039] Voltammetric techniques vary the potential as a function of time. The resulting current is plotted as a function of potential. For example, cyclic voltammetry (CV) sweeps the cell's potential linearly over a voltage range, while the fast-scan CV (FSCV) technique does this at high speed. Square-wave voltammetry (SWV) uses square waves superimposed over a step function to provide a scanning measurement that provides two sampling instances per potential. As a result of this sampling technique, the contribution to the total current arising from non-sensitive currents is minimized. Like CV, the current is plotted as a function of potential.

[0040] In some embodiments, the electrode probe structure 137 of the microneedle electrode 130 can be structured in the same or similar manner as the electrode probe structure 127 of the microneedle electrode 120. The aptamer sequence tethered to the Au wire of the electrode probe structure 137 can be configured to bind to a target analyte different from the target analyte for which the aptamer sequence tethered to the Au wire of the electrode probe structure 127 is specific. For example, the aptamer sequence tethered to the Au wire of the electrode probe structure 137 can be configured to bind to cortisol. Note that, like insulin, cortisol is an important diabetes-related biomarker. Thus, the microneedle electrode 120 of the device 100 can be configured to detect a first analyte (e.g., insulin), while the microneedle electrode 130 of the device 100 can be configured to detect a second analyte (e.g., cortisol) that is different from the first analyte.

[0041] In certain embodiments of device 100, the aptamer sequence of electrode probe structure 127 may be functionalized with a first type of redox reporter molecule (e.g., methylene blue), while the aptamer sequence of electrode probe structure 137 may be functionalized with a second type of redox reporter molecule (e.g., anthraquinone) that is different from the first type of redox reporter molecule. In other embodiments, the aptamer sequence of electrode probe structure 127 and the aptamer sequence of electrode probe structure 137 may both be functionalized with the same type of redox reporter molecule (e.g., one of methylene blue or anthraquinone). The different types of reporter molecules used to functionalize the aptamer sequences may provide different redox properties that can be used to perform multiplex (simultaneous or otherwise) measurements of different target compounds.

[0042] Potentially high biofouling of the device 100 when operating in complex media, such as ISF, can be avoided by applying an (outer) polymer membrane coating (e.g., polysulfone or zwitterionic polymer coating) to the electrode probe structure of the microneedle electrodes of the microneedle sensor device 100.

[0043] FIG. 2 shows a block diagram illustrating an example embodiment of the electrochemical microneedle sensor device 100 of FIG. 1 in communication with an electronic device 200 according to the present technology.

[0044] It should be noted that for ease of explanation, the same reference numbers are used to identify some elements in different figures (e.g., electrodes 110, 120, and 130 in Figures 1 and 2), but it is understood that these designations do not necessarily mean that these elements are identical. In particular, it is contemplated that one or more characteristics (e.g., physical, chemical, electrochemical, material, dimensional, etc.) of an element may be modified based on the particular configuration of a microneedle sensor device according to the technology disclosed herein and / or based on its desired performance characteristics.

[0045] In various embodiments, for example, electronic device 200 may affect control, measurement, and monitoring functions, among other functions, of sensor device 100 related to the operation of sensor device 100. In some implementations, electronic device 200 may be included in an electronic unit of a wearable medical device that incorporates or otherwise interfaces with sensor device 100. For example, in some embodiments, electronic unit 200 is configured on the same substrate 105 as sensor device 100, enabling an all-in-one sensor patch implemented to accurately detect concentrations of target analytes (e.g., insulin and cortisol) using multiplexed (simultaneous (parallel) or sequential) detection of target analytes using different electrodes or different electrode groups for detection of each analyte.

[0046] For example, in some embodiments, the electrochemical microneedle sensor device 100 is configured on a patch that is attached and adapted to the skin of a user for mobile, remote monitoring applications. The patch may integrate the sensor device 100 and the electronic device 200 on a single substrate (e.g., substrate 105) or on a single convertible platform.

[0047] 3 shows a block diagram of an example embodiment of electronic device 200. For example, electronic device 200 can be configured to be electrically coupled to at least one electrode (or electrode probe structure) of sensor device 100. For example, electronic device 200 can be configured to provide a signal (e.g., voltage and / or current) across at least one electrode (or electrode probe structure) of device 100. Electronic device 200 can also be configured to receive and process an electrical signal (e.g., voltage, current) generated by at least one electrode (or electrode probe structure) of sensor device 100, for example. Electronic device 200 can be further configured to determine the concentration of one or more target analytes present in ISF and / or other fluids and / or body tissues in contact with at least one electrode (or electrode probe structure) of sensor device 100 based on a signal obtained by device 200 from one or more electrodes (or electrode probe structure) of device 100.

[0048] In some embodiments, electronic device 200 can address (e.g., apply signals across or acquire signals from, or both) electrodes of device 100 configured to detect a first target analyte (e.g., electrodes 120 configured to detect insulin) independently of electrodes of device 100 configured to detect a second target analyte different from the first target analyte (e.g., electrodes 130 configured to detect cortisol). For example, electronic device 200 can provide electrical connections to electrodes of device 100 configured to detect a first target analyte, and these connections can be independent of electrical connections provided by electronic device 200 to electrodes of device 100 configured to detect a second target analyte. For example, electronic device 200 can address electrodes of device 100 configured to detect different analytes (corresponding to different analytes) at the same time (simultaneously) but in a manner such that electrical signals applied to and / or acquired from the electrodes corresponding to the different analytes do not mix with each other. Alternatively, or in addition to the methods of addressing the electrodes of device 100 mentioned, electronic device 200 can acquire electronic signals from electrodes of device 100 corresponding to different analytes in a manner that provides a mixture of signals corresponding to the different analytes and then processes the mixture of signals to separate it into components corresponding to each individual analyte. In some embodiments, electronic device 200 can address electrodes of device 100 corresponding to different analytes sequentially, where electrodes corresponding to a first target analyte are addressed first (e.g., signals applied across or acquired from these electrodes, or both), followed by electrodes corresponding to a second target analyte (e.g., signals applied across or acquired from these electrodes, or both).

[0049] In some embodiments, electronic device 200 can use a single signal source of electronic device 200 to apply signals across electrodes of device 100 corresponding to different analytes. For example, a single signal source of electronic device 200 can be used to apply a signal (e.g., directly or via one or more passive or active electrical elements, such as, for example, a resistor, capacitor, transistor, or diode) across one or more electrodes of device 100 corresponding to a first target analyte (e.g., insulin), and a single signal source can also be used to apply the same or different signal across one or more electrodes of device 100 corresponding to a second target analyte (e.g., cortisol). In some implementations, a single signal source of electronic device 200 can generate signals (e.g., directly or via one or more passive or active electrical elements) simultaneously provided to one or more electrodes of device 100, including electrodes corresponding to different target analytes. As another example, a single signal source of electronic device 200 can first generate a signal corresponding to an electrode configured to detect a first target analyte, and then generate a signal corresponding to an electrode configured to detect a second target analyte. In other embodiments, the electronic device 200 can include multiple signal sources and can apply signals in a parallel (e.g., substantially simultaneously) or sequential manner across electrodes of the device 100 corresponding to different analytes using different signal sources.

[0050] Similarly, electronic device 200 can include a single data acquisition element (e.g., an analog-to-digital converter (ADC)) or multiple data acquisition elements that can be used to acquire electrical signals from electrodes of device 100. In some embodiments, electronic device 200 can include a first data acquisition element configured to acquire electrical signals from electrodes of device 100 corresponding to a first target analyte, and electronic device 200 can include a second data acquisition element configured to acquire electrical signals from electrodes of device 100 corresponding to a second target analyte. In some implementations, electronic device 200 can acquire signals from electrodes of device 100 corresponding to a first target analyte using the first data acquisition element simultaneously (or, in some implementations, substantially simultaneously) as it acquires signals from electrodes of device 100 corresponding to a second target analyte using the second data acquisition element. In other implementations, the electronic device 200 may use a data acquisition element to acquire a signal from an electrode of the device 100 corresponding to a first target analyte, followed by using the same data acquisition element to acquire a signal from an electrode of the device 100 corresponding to a second target analyte. In yet other implementations, the electronic device 200 may use a data acquisition element to acquire a signal from an electrode of the device 100 corresponding to a second target analyte, while simultaneously using the same data acquisition element to acquire a signal from an electrode of the device 100 corresponding to the first target analyte.

[0051] Although the above example includes electrodes of device 100 configured to detect two different target analytes, the techniques disclosed herein can be applied to the detection of any number of target analytes. Thus, device 100 can have any number of electrodes configured to detect any number of target analytes.

[0052] In various implementations, electronic device 200 is operable to store and execute software applications and algorithms to process signals obtained by electronic device 200 from sensor device 100 and to perform various controls of sensor device 100, such as applying various voltage waveforms across one or more electrodes of sensor device 100. In various embodiments, electronic device 200 can be implemented as a portable computing device, such as a mobile communications device, a wearable device such as a smartphone, tablet, or smartwatch, glasses, etc., and / or electronic device 200 can be implemented as a fixed computing device, such as a desktop computer.

[0053] In some embodiments, electronic device 200 includes a processor 220 configured to process data, a memory 210 in communication with processor 220 configured to store data, and an input / output (I / O) communication interface (or unit) 230 configured to interface processor 220 and / or memory 210 to other elements of electronic device 200, as well as various modules, units, or devices, including, for example, device 100 and / or external computing devices, data storage devices, or communication devices.

[0054] In some embodiments, I / O unit 230 electrically interfaces with at least one electrode of device 100. In some implementations, I / O 230 includes an analog-to-digital (ADC) converter that converts electrical signals (e.g., current or voltage) received by I / O 230 from the electrodes of device 100 into a digital format suitable for processing by processor 220 and / or storage within memory 210. In some implementations, I / O 230 includes a digital-to-analog (DAC) converter that converts digital waveforms obtained by processor 220 from memory 210 into voltage waveforms applied across the electrodes of device 100, for example, to perform one of the electrochemical measurement techniques described above or any other electrochemical measurement or detection technique using the electrodes. In some embodiments, I / O unit 230 can interface processor 220 and / or memory 210 to other modules, units, or devices, including other external computing devices.

[0055] For example, processor 220 may include a central processing unit (CPU) or a microcontroller unit (MCU) or a graphics processing unit (GPU). For example, memory 210 may include and store processor-executable code that, when executed by processor 220, configures electronic device 200 to perform various operations, such as receiving information, commands, and / or data, processing information, commands, and / or data, and transmitting or providing information, commands, and / or data to other elements of electronic device 200 and / or other devices external to electronic device 200.

[0056] In some implementations, the electronic device 200 may transmit raw data or processed data (e.g., a voltage profile captured by the electronic device 200 from one or more electrodes of the device 100) to a computer system or computer network that may include one or more remote computer processing devices (e.g., a server) and that may be accessible via a communications network, such as the Internet (such computer systems or networks are sometimes referred to as being located "in the cloud").

[0057] To support various functions of electronic device 200, memory 210 may store information and data, such as instructions, software, values, voltage or current profiles, and other data that is processed or referenced by processor 220. For example, various types of random access memory (RAM) devices, read-only memory (ROM) devices, flash memory devices, and other suitable storage media may be used to perform the storage functions of memory 210.

[0058] In some embodiments, I / O unit 230 includes a wireless communication interface 231, e.g., a wireless transmitter configured to transmit stored and / or processed data, or a wireless transceiver (Tx / Rx) configured to transmit and receive data. For example, in some embodiments, I / O unit 230 may also include a wireless communication interface 232 that can be used to exchange electrical signals, e.g., between elements of electronic device 200 and between electronic device 200 and device 100. I / O unit 230 can utilize various types of wired or wireless interfaces compatible with typical data communication standards, including, but not limited to, Bluetooth, Bluetooth Low Energy, Zigbee, IEEE 802.11, wireless local area networks (WLANs), wireless personal area networks (WPANs), wireless wide area networks (WWANs), WiMAX, IEEE 802.16 (Worldwide Interoperability for Microwave Access (WiMAX)), 3G / 4G / LTE / 5G cellular communication methods, NFC (Near Field Communication), and parallel interfaces, that can be used in communications with electronic device 200. In some embodiments, electronic device 200 uses I / O unit 230 for purposes of transferring data to and receiving data from another device (e.g., a voltage profile applied across one or more electrodes of sensor device 100 during its operation).

[0059] In some embodiments, electronic device 200 includes or is otherwise interfaced with display unit 240, which may include a visual display, e.g., a display screen, an audio display, e.g., a speaker, or any other type of display, or a combination thereof.

[0060] The I / O unit 230 of the electronic device 200 may interface with other external interfaces, data sources, data storage devices, and / or visual or audio display devices, etc., to receive and transfer data and information that may be processed by the processor 220, stored in the memory 210, or shown on an output unit (e.g., the display unit 240) of the electronic device 200 or an external device. For example, the display unit 240 may be configured to communicate data with the electronic device 200, e.g., via the I / O unit 230, to provide visual, audio, and / or other sensory displays that result in a software application user interface. In some examples, the display unit 240 may include various types of screen displays, speakers, or printing interfaces, including, but not limited to, a light-emitting diode (LED) or liquid crystal display (LCD) monitor or screen, a cathode ray tube (CRT) as a visual display, an audio signal transducer device as an audio display, and / or a toner, liquid inkjet, solid ink, dye-sublimation, inkless (e.g., thermal or UV) printing device, etc.

[0061] 4 shows a block diagram representing an example embodiment of an electrochemical microneedle sensor device 100 having at least two groups of microneedle-based working electrodes for sensing two different analytes according to the technology disclosed herein. As shown in FIG. 4, the electrochemical microneedle sensor device 100 can have a group 122 of working electrodes 120 ("WE-1" in FIG. 4), each electrode in the group 122 configured to measure the concentration of a first target analyte (e.g., insulin) in the ISF, for example, using an aptamer-assisted electrochemical detection method. Also shown in FIG. 4, the electrochemical microneedle sensor device 100 can have a group 132 of working electrodes 130 ("WE-2" in FIG. 4), each electrode in the group 132 configured to measure the concentration of a second target analyte (e.g., cortisol) different from the first target analyte in the ISF, for example, using an aptamer-assisted electrochemical detection method. 4, the electrochemical microneedle sensor device 100 can have a group 112 of electrodes 110, and each electrode in group 112 can be configured to be used as a reference electrode (RE) or as a counter electrode (CE) in cooperation with any electrode or electrodes in either group of electrodes 122 or 132. The electrode groups 112, 122, or 132 can have any number of corresponding electrodes, which can differ from the number of electrodes in those groups shown in FIG.

[0062] 5 shows a block diagram representing another example embodiment of an electrochemical microneedle sensor device 100 having at least three microneedle-based working electrodes for multiplexed sensing of different target analytes according to the present technology, where the three microneedle-based working electrodes are disposed on a substrate in proximity to microneedle-based reference and / or counter electrodes. As shown in the block diagram in FIG. 5, the device 100 can include a first microneedle electrode 110 configured as a reference electrode RE and / or a counter electrode CE for electrochemical sensing of one or more analytes, where the electrode 110 is disposed on a substrate 105. The device 100 can also include a second microneedle electrode 120 configured as a first working electrode WE-1, a third microneedle electrode 130 configured as a second working electrode WE-2, and a fourth microneedle electrode 140 configured as a third working electrode WE-3, each disposed on the substrate 105. Each of the microneedle electrodes 110, 120, 130, and 140 of the electrochemical microneedle sensor device 100 includes a microneedle structure and an electrode probe structure, as previously described. FIG. 5 illustrates the microneedle structures 113, 123, 133, and 143 of the microneedle electrodes 110, 120, 130, and 140, respectively. In various example embodiments, the electrode probe structure of any of the electrodes 110, 120, 130, or 140 may be disposed (e.g., at least partially) within the interior or opening or cavity of the microneedle structure of the respective electrode. In other embodiments, the electrode probe structure of any of the electrodes 110, 120, 130, or 140 may be attached to or otherwise integrated into the microneedle structure of the corresponding electrode that does not have an opening or cavity. FIG. 5 illustrates the electrode probe structures 117, 127, 137, and 147 of the microneedle electrodes 110, 120, 130, and 140, respectively.

[0063] In some embodiments, the second microneedle electrode 120, the third microneedle electrode 130, and the fourth microneedle electrode 140 may include one or more functionalizing materials, depending, for example, on the electrochemical sensing technique being applied at the particular working electrode. In Figure 5, the second microneedle electrode 120 includes a functionalizing material 125 disposed on or integrated with at least a portion of the electrode probe structure 127. Similarly, in some embodiments, the third microneedle electrode 130 includes a functionalizing material 135 disposed on or integrated with at least a portion of the electrode probe structure 137. Also, in some embodiments, the fourth microneedle electrode 140 includes a functionalizing material 145 disposed on or integrated with at least a portion of the electrode probe structure.

[0064] For example, in some implementations, functionalization material 125 can include an aptamer designed to have a conformation capable of receiving an analyte in a biological fluid, such as insulin in ISF, such that the aptamer-analyte binding complex induces a conformational change in functionalization material 125 that is detected at electrode 120 as an electrical signal. Functionalization material 125 can include, for example, a redox reporter molecule, such as methylene blue or anthraquinone. Processing of the detected electrical signal can provide information about the concentration or level of the analyte (e.g., insulin) in the biological fluid (e.g., ISF). Similarly, in some implementations, functionalization material 135 can include an aptamer designed to have a conformation capable of receiving another analyte in a biological fluid, such as cortisol in ISF, such that the aptamer-analyte binding complex induces a conformational change in functionalization material 135 that is detected at electrode 130 as an electrical signal. Functionalization material 135 can include, for example, the same type of redox reporter molecule (e.g., methylene blue or anthraquinone) as that included in functionalization material 125, or functionalization material 135 can include a different type of redox reporter molecule.

[0065] For example, in some implementations, the electrode probe structure 147 of the microneedle electrode 140 can be functionalized with a biocatalyst 145, such as an enzyme, e.g., glucose oxidase (GOx), or others, which can be attached to the electrode 140, e.g., via electropolymeric encapsulation. The enzyme (e.g., GOx) can be configured to be specific for a target analyte (e.g., glucose) present in a biological fluid (e.g., ISF or blood) or bodily tissue. In such embodiments, for example, enzymatic detection of a target analyte (e.g., glucose) can be performed using the functionalized microneedle electrode 140. In other implementations, non-enzymatic detection of a target analyte can be performed using an electrode 140, e.g., where the electrode probe structure 147 includes a metal (e.g., gold) wire at least partially covered by a graphene film functionalization material 145. In yet other embodiments, the functionalization material 145 of the electrode 140 can include a β-hydroxybutyrate dehydrogenase (HBD) enzyme, which enables the electrode 140 to detect ketone bodies. As another example, the functionalizing material 145 can include carbon (e.g., graphite) powder along with a binder (e.g., oil) and an enzyme specific for the target analyte (e.g., glucose or ketone bodies). As yet another example, the functionalizing material 145 can include a carbon paste-based functionalizing material having catalytic metal nanoparticles embedded therein to enhance the detection signal using non-enzymatic detection of the target analyte. The functionalizing materials 125, 135, and 145 of the microneedle sensor device 100 depicted in FIG. 5 can include any of the previously described functionalizing materials or any other functionalizing material suitable for detection of a target analyte at any of the functionalized electrodes 120, 130, and / or 140 using any of the electrochemical measurement techniques described above.

[0066] It should be noted that the target analytes referred to above may share common properties that are indicative of biomarkers associated with a particular disease, e.g., diabetes, or the state of one or more physiological systems and / or organs of the body.

[0067] 6 shows a block diagram representing another example embodiment of an electrochemical microneedle sensor device 100 having at least four microneedle-based working electrodes for multiplexed sensing of different target analytes according to the technology disclosed herein, where the four microneedle-based working electrodes are disposed on a substrate in proximity to microneedle-based reference and / or counter electrodes. As shown in the diagram of FIG. 6, the device 100 can include a first microneedle electrode 110 configured as a reference electrode RE and / or a counter electrode CE for electrochemical sensing of the target analytes, where the electrode 110 is disposed on a substrate 105. The device 100 can also include a second microneedle electrode 120 configured as a first working electrode WE-1, a third microneedle electrode 130 configured as a second working electrode WE-2, a fourth microneedle electrode 140 configured as a third working electrode WE-3, and a fifth microneedle electrode 150 configured as a fourth working electrode WE-4, each disposed on the substrate 105. Each of the microneedle electrodes 110, 120, 130, 140, and 150 of the electrochemical microneedle sensor device 100 can include a microneedle structure and an electrode probe structure, as previously described. The microneedle electrodes 110, 120, 130, 140, and 150 shown in FIG. 6 have microneedle structures 113, 123, 133, 143, and 153, respectively. In various example embodiments, the electrode probe structure can be disposed within the interior or an opening or cavity of the respective microneedle structure. The microneedle electrodes 110, 120, 130, 140, and 150 shown in FIG. 6 have electrode probe structures 117, 127, 137, 147, and 157, respectively.

[0068] In some embodiments, the first working electrode 120, the second working electrode 130, the third working electrode 140, and the fourth working electrode 150 can include one or more functionalization materials, depending, for example, on the electrochemical sensing technique applied at the particular working electrode. In FIG. 6 , the first working electrode 120 includes a functionalization material 125 disposed on or integrated with at least a portion of the electrode probe structure 127. Similarly, in some embodiments, the second working electrode 130 includes a functionalization material 135 disposed on or integrated with at least a portion of the electrode probe structure 137. Also, in some embodiments, the third working electrode 140 includes a functionalization material 145 disposed on or integrated with at least a portion of the electrode probe structure 147. Similarly, in some embodiments, the fourth working electrode 150 includes a functionalization material 155 disposed on or integrated with at least a portion of the electrode probe structure 157.

[0069] 6 can include any of the functionalization materials described above or any other functionalization material suitable for detection of a target analyte, which can provide information regarding assessing a particular bodily condition (e.g., diabetes) at any of the functionalized working electrodes 120, 130, 140, and / or 150 using any of the electrochemical measurement techniques mentioned above. For example, functionalization material 145 of electrode 140 can enable antibody-based detection of a target analyte (e.g., insulin), and functionalization material 155 of electrode 150 can provide aptamer-based detection of the same target analyte (insulin).

[0070] In some implementations, the functionalization material 145 is configured to facilitate electrochemical immunoassays (e.g., including label-free immunoassays) for detecting analyte concentrations in biological fluids. In some embodiments, an electrochemical microneedle sensor device 100 having at least three or four microneedle-based working electrodes for multiplexed sensing of one or more target analytes includes a microneedle electrode 140 in which a functionalized electrode probe structure 147 is disposed within a hollow interior formed as a channel spanning between two openings of a microneedle structure 143 of the electrode 140. Within the channel, the electrode probe structure 147 is functionalized with a functionalization material 145 comprising a capture antibody (e.g., an anti-analyte capture antibody and / or a detection antibody) covalently attached to a self-assembled monolayer (SAM) linked to the electrode probe structure 147. The channel allows fluid (e.g., ISF) to flow through the channel within the interior of the microneedle structure with the functionalized immunoassay electrodes present to facilitate a detectable reaction. In some implementations, the anti-analyte antibody functionalized material 145 can interact with the target analyte for detection at the working electrode (e.g., after covalent attachment of the analyte (e.g., insulin) to a capture antibody and / or subsequent binding of a detection antibody). The immunoassay reaction can give rise to an electrical signal that is detected at the electrode 140.

[0071] FIG. 7 illustrates an example embodiment of an electrochemical microneedle sensor device 100 having at least four microneedle-based working electrodes for multiplexed sensing of different target analytes according to the technology disclosed herein. The four microneedle-based working electrodes of device 100 shown in FIG. 7 are disposed on a substrate and are positioned proximate to microneedle-based reference and / or counter electrodes also disposed on the same substrate. As shown in FIG. 7, device 100 includes a first microneedle electrode 110 configured as a reference electrode RE and / or a counter electrode CE for electrochemical sensing of one or more target analytes, where electrode 110 is disposed on a substrate 105. Device 100 also includes a second microneedle electrode 120 configured as a first working electrode WE-1, a third microneedle electrode 130 configured as a second working electrode WE-2, a fourth microneedle electrode 140 configured as a third working electrode WE-3, and a fifth microneedle electrode 150 configured as a fourth working electrode WE-4. Each of the microneedle electrodes 120, 130, 140, and 150 is also disposed on the substrate 105. Each of the microneedle electrodes 110, 120, 130, 140, and 150 of the electrochemical microneedle sensor device 100 shown in Figure 7 includes a microneedle structure and an electrode probe structure. For example, either the microneedle structure and / or the electrode probe structure can be any of those previously described. The microneedle electrodes 110, 120, 130, 140, and 150 shown in Figure 7 have microneedle structures 113, 123, 133, 143, and 153, respectively. As shown in FIG. 7, the electrode probe structures 117, 127, 137, 147 and 157 of the microneedle electrodes 110, 120, 130, 140 and 150, respectively, are disposed within the interior of the microneedle structures or openings or cavities of the microneedle structures 113, 123, 133, 143 and 153, respectively.

[0072] In some embodiments, the first working electrode 120, the second working electrode 130, the third working electrode 140, and the fourth working electrode 150 can include one or more functionalizing materials, depending, for example, on the electrochemical sensing technique applied at the particular working electrode. In FIG. 6 , the first working electrode 120 includes a functionalizing material 125 disposed on or integrated with at least a portion of the electrode probe structure 127 and / or at least partially disposed within the opening or cavity of the microneedle structure 123. Similarly, in some embodiments, the second working electrode 130 includes a functionalizing material 135 disposed on or integrated with at least a portion of the electrode probe structure 137 and / or at least partially disposed within the opening or cavity of the microneedle structure 133. Also, in some embodiments, the third working electrode 140 includes a functionalizing material 145 disposed on or integrated with at least a portion of the electrode probe structure 147 and / or at least partially disposed within the opening or cavity of the microneedle structure 143. Similarly, in some embodiments, the fourth working electrode 150 includes a functionalizing material 155 disposed on or integrated with at least a portion of the electrode probe structure 157 and / or at least partially disposed within an opening or cavity of the microneedle structure 153.

[0073] The functionalization materials 125, 135, 145, and 155 of the microneedle sensor device 100 depicted in FIG. 7 can include any of the functionalization materials described above or any other functionalization material suitable for detecting a target analyte, which can provide information regarding a particular condition of the body (e.g., diabetes) at any of the functionalized working electrodes 120, 130, 140, and / or 150 using any of the electrochemical measurement techniques mentioned above.

[0074] The embodiment of the microneedle sensor device 100 shown in Figure 7 is configured for real-time measurement of cortisol, insulin, glucose, and ketone bodies in a single microneedle array patch, all integrated into a substrate 105. The concentrations of the first two analytes (cortisol and insulin) are tracked through the reversible binding of conformation-dependent aptamers, while the concentrations of the remaining two analytes, glucose and ketone bodies, are monitored using the specific biocatalytic redox reactions of these molecules in the presence of the enzymes glucose oxidase (GOx) and β-hydroxybutyrate dehydrogenase (HBD), respectively. Such multiplexed simultaneous monitoring of four important diabetes-related markers can provide a more comprehensive understanding of a patient's health status compared to monitoring a single marker, leading to enhanced glycemic control. Other embodiments of device 100 can provide measurements of other disease biomarkers, including proteins such as interleukin-6 (IL-6), tumor necrosis factor (TNF), C-reactive protein (CRP), etc., in combination with metabolites such as alcohol, glutamate, lactate, etc., as well as in combination with potentiometric-based detection of various electrolytes such as sodium, potassium, lithium, etc.

[0075] The microneedle sensor device 100 shown in FIG. 7 includes four hollow microneedle-based working electrodes 120, 130, 140, and 150. The two Au wire-integrated microneedle electrodes 120 and 130 on the right are functionalized with a cortisol-specific aptamer and an insulin-specific aptamer, respectively, while the two hollow microneedle electrodes 140 and 150 on the left are filled with a carbon paste matrix and act as working electrodes for ketone bodies and glucose, respectively. The Ag / AgCl wire-integrated microneedle-based electrode 110 is a common reference electrode. Electrode 110 is shown centered between the working electrodes. Other relative arrangements of the electrodes in the sensor device are possible as well.

[0076] A carbon paste-filled microneedle electrode 140 for monitoring ketone body concentrations, shown in FIG. 7, can be constructed by incorporating a suitable mediator into the carbon paste, followed by droplet casting of an outer polymer membrane that allows for stable entrapment of HBD / NAD+ enzymes / cofactors and continuous monitoring of ketone bodies.

[0077] Similarly, the carbon paste-filled microneedle electrode 150 shown in Figure 7 and used for monitoring glucose concentrations relies on immobilized glucose oxidase (GOx) enzyme and the reduction of hydrogen peroxide (HO) generated on a graphite / mineral oil carbon paste modified with Prussian blue (PB). The GOx enzyme and PB can be easily incorporated into the carbon paste without leaching issues during prolonged use in a fluid matrix.

[0078] 8 illustrates an example embodiment of a method 200 for fabricating an electrochemical sensing device. The method 200 includes providing a substrate 210. The method 200 further includes coupling at least two microneedles to the substrate 220.

[0079] One aspect of the disclosed technology relates to a device comprising a substrate and at least two microneedles coupled to the substrate, each microneedle of the at least two microneedles comprising a protruding needle structure and an electrode probe structure, the protruding needle structure comprising an outer wall extending outward from a surface of the substrate, the outer wall surrounding an interior volume of the protruding needle structure and forming an apex at the end of the outer wall, the electrode probe structure configured to generate a signal in response to one or more chemical or biological substances in a biological fluid contacting the electrode probe structure, the electrode probe structure of a first microneedle of the at least two microneedles comprising an aptamer sequence specific to a first analyte, the electrode probe structure of the first microneedle operable as a working electrode for detection of the first analyte using a first electrochemical detection technique, and the electrode probe structure of a second microneedle of the at least two microneedles operable as an electrochemical counter electrode or an electrochemical reference electrode.

[0080] In some exemplary embodiments, the electrode probe structure is incorporated into or attached to the protruding needle structure. In certain exemplary embodiments, the aptamer sequence is tethered to the electrode probe structure of the first microneedle via a 5'-thiol. In other exemplary embodiments, the aptamer sequence is functionalized with a redox reporter molecule. In certain exemplary embodiments, the redox reporter molecule is methylene blue. In other exemplary embodiments, the redox reporter molecule is anthraquinone. In exemplary embodiments, the functionalization is at the 3' end of the aptamer sequence. In another exemplary embodiment, the functionalization is at the 5' end of the aptamer sequence.

[0081] In some exemplary embodiments, the device includes at least two electrically conducting channels, each electrically connected to an electrode probe structure of a microneedle in at least two microneedles for transmitting a signal from the electrode probe structure or applying a control signal to the electrode probe structure. In other exemplary embodiments, the electrode probe structure of a third microneedle in the at least two microneedles includes an aptamer sequence specific to a second analyte, and the electrode probe structure of the third microneedle is operable as a working electrode for detection of the second analyte using a second electrochemical detection technique. In yet another exemplary embodiment, the second analyte is different from the first analyte. According to exemplary embodiments, the second electrochemical detection technique is different from the first electrochemical detection technique.

[0082] In certain exemplary embodiments, for at least one microneedle of the at least two microneedles, the internal volume of the protruding needle structure of the microneedle comprises a hollow interior defined by an inner wall, the outer wall of the protruding needle structure of the microneedle comprises an opening to the hollow interior, and the electrode probe structure of the microneedle is at least partially disposed within the hollow interior. In yet another exemplary embodiment, the electrode probe structure of the microneedle of the at least two microneedles comprises a metal film. In some exemplary embodiments, the electrode probe structure of the microneedle of the at least two microneedles comprises a metal wire. According to certain exemplary embodiments, the metal is gold. In other exemplary embodiments, the electrode probe structure of a second microneedle of the at least two microneedles comprises silver / silver chloride (Ag / AgCl).

[0083] In yet other exemplary embodiments, the electrode probe structure of a fourth microneedle of the at least two microneedles comprises a coating on a surface of the electrode probe structure, the coating functionalized with a first enzyme, the coating configured to interact with a third analyte, and the electrode probe structure of the fourth microneedle is operable as a working electrode for detection of the third analyte using a third electrochemical detection technique. According to some exemplary embodiments, the electrode probe structure of a fifth microneedle of the at least two microneedles comprises a coating on a surface of the electrode probe structure, the coating functionalized with a second enzyme, the coating configured to interact with a fourth analyte, and the electrode probe structure of the fifth microneedle is operable as a working electrode for detection of the fourth analyte using a fourth electrochemical detection technique. In certain exemplary embodiments, the second enzyme is different from the first enzyme.

[0084] In other exemplary embodiments, the electrode probe structure of a sixth microneedle of the at least two microneedles includes a coating on a surface of the electrode probe structure, the coating being functionalized with an ionophore receptor, the coating being configured to interact with a fifth analyte, and the electrode probe structure of the sixth microneedle is operable as a working electrode for detection of the fifth analyte using a fifth electrochemical detection technique, the fifth analyte being an electrolyte. In some exemplary embodiments, any electrochemical detection technique from the first, second, third, and fourth electrochemical detection techniques is one of a cyclic voltammetry technique, a fast-scan cyclic voltammetry technique, a square-wave voltammetry technique, a potentiometry technique, or a chronoamperometry technique. In certain exemplary embodiments, the fifth electrochemical detection technique is one of a cyclic voltammetry technique, a fast-scan cyclic voltammetry technique, a square-wave voltammetry technique, a potentiometry technique, or a chronoamperometry technique.

[0085] In other exemplary embodiments, the electrode probe structure of at least one macroneedle of the at least two microneedles includes a conformal coating. According to some exemplary embodiments, the conformal coating includes an electrically insulating polymer or a dielectric material. In some exemplary embodiments, the conformal coating includes at least one of a poly(p-xylene) polymer, a polyethyleneimine polymer, or SiO2. In other exemplary embodiments, the first analyte is a protein. According to some exemplary embodiments, the protein is a cytokine. In yet other exemplary embodiments, the protein is one of insulin, interleukin-6 protein, tumor necrosis factor protein, or C-reactive protein. In some exemplary embodiments, the third analyte is a small molecule compound. In other exemplary embodiments, the fourth analyte is a small molecule compound. According to certain exemplary embodiments, the small molecule compound is one of lactose, lactic acid, alcohol, glucose, glutamic acid, or ketone bodies. In other exemplary embodiments, the first analyte is a drug. In some exemplary embodiments, the first analyte is cortisol and the second analyte is insulin. In other exemplary embodiments, the first enzyme is glucose oxidase and the third analyte is glucose. In yet other exemplary embodiments, the second enzyme is β-hydroxybutyrate dehydrogenase and the fourth analyte is ketone bodies. According to certain exemplary embodiments, the electrolyte is one of sodium, potassium, or lithium. According to other exemplary embodiments, the third analyte is different from the first analyte, and the third analyte is different from the second analyte. In some exemplary embodiments, the fourth analyte is different from the first analyte, and the fourth analyte is different from the second analyte, and the fourth analyte is different from the third analyte. In other exemplary embodiments, the device is configured to be placed on human skin.

[0086] Another aspect of the disclosed technology relates to a method of manufacturing an electrochemical sensing device, the method comprising the steps of providing a substrate and coupling at least two microneedles to the substrate, wherein each microneedle of the at least two microneedles comprises a protruding needle structure and an electrode probe structure, the protruding needle structure comprising an outer wall extending outwardly from a surface of the substrate, the outer wall surrounding an interior volume of the protruding needle structure and forming an apex at a terminal end of the outer wall, the electrode probe structure configured to generate a signal in response to one or more chemical or biological substances in a biological fluid contacting the electrode probe structure, the electrode probe structure of a first microneedle of the at least two microneedles comprising an aptamer sequence specific to a first analyte, the electrode probe structure of the first microneedle operable as a working electrode for detection of the first analyte using a first electrochemical detection technique, and the electrode probe structure of a second microneedle of the at least two microneedles operable as an electrochemical counter electrode or an electrochemical reference electrode.

[0087] Yet another aspect of the disclosed technology relates to a method of electrochemical-based sensing, comprising the steps of: providing a device according to the technology disclosed herein, the device comprising at least two electrically conductive channels, each channel in the at least two electrically conductive channels electrically coupled to an electrode probe structure of a microneedle in at least two microneedles of the device, for transmitting a signal from the electrode probe structure (e.g., a sensing signal, e.g., a signal generated in response to a compound in a biological fluid or tissue) or applying a control signal to the electrode probe structure, the method further comprising transmitting a signal from the electrode probe structure of a first microneedle in the at least two microneedles using a first electrically conductive channel in the at least two electrically conductive channels.

[0088] An aspect of the disclosed technology relates to a method of electrochemical-based sensing, the method comprising: preparing a device according to the technology disclosed herein, the device including at least two electrically conductive channels, each of which is electrically coupled to an electrode probe structure of at least two microneedles of the device, for transmitting a signal (e.g., a sensing signal, e.g., a signal generated in response to a compound in a biological fluid or tissue) from the electrode probe structure or applying a control signal to the electrode probe structure. The method further comprises applying a first control signal to the electrode probe structure of a first microneedle of the at least two microneedles using a first electrically conductive channel of the at least two electrically conductive channels. The method also includes transmitting a signal from the electrode probe structure of a second microneedle of the at least two microneedles using a second electrically conductive channel of the at least two electrically conductive channels. In some implementations, the method of electrochemical-based sensing further comprises determining a concentration of an analyte using the signal transmitted from the electrode probe structure of the second microneedle.

[0089] In some exemplary embodiments of the electrochemical-based sensing method, the first microneedle and the second microneedle refer to the same microneedle in the at least two microneedles. In other exemplary embodiments of the method, the first microneedle and the second microneedle refer to different microneedles in the at least two microneedles. In yet other exemplary embodiments of the method, the first electrically conductive channel and the second electrically conductive channel refer to the same electrically conductive channel in the at least two electrically conductive channels. In certain exemplary embodiments of the method, the first electrically conductive channel and the second electrically conductive channel refer to different electrically conductive channels in the at least two electrically conductive channels. According to some exemplary embodiments of the method, the electrically conductive channel in the at least two electrically conductive channels is a wire made of a conductive material or a trace of a conductive material on a circuit board.

[0090] The specification, together with the drawings, are intended to be considered exemplary only, where exemplary means example. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Additionally, the use of "or" is intended to include "and / or" unless the context clearly dictates otherwise.

[0091] While this application contains many details, these should not be construed as limitations on the scope of any invention or what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of a particular invention. Certain features described in this application in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment can also be implemented separately in multiple embodiments or in any suitable subcombination. Also, while features may be described above as working in a particular combination and even initially claimed as such, one or more features from a claimed combination may in some cases be deleted from the combination, and the claimed combination may be subject to subcombinations or variations of the subcombination.

[0092] Similarly, although operations are depicted in a particular order in the figures, this should not be understood as requiring that such operations be performed in the particular shown order or sequential order, or that all of the depicted operations be performed, to achieve desired results. Additionally, the separation of various system components in the embodiments described herein should not be understood as requiring such separation in all embodiments.

[0093] It is understood that the various disclosed embodiments can be implemented individually or collectively in devices comprised of various optical, electronic, and / or software modules and components. For example, these devices may include a processor, a memory unit, and an interface communicatively connecting them to one another, and can range from desktop and / or laptop computers to mobile devices, etc. The processor and / or controller can perform the various disclosed operations based on the execution of program code stored on a storage medium. The processor and / or controller can communicate directly or indirectly with at least one memory and at least one communication unit that enables the exchange of data and information, e.g., through communication links with other entities, devices, and networks. The communication unit can provide wired and / or wireless communication capabilities via one or more communication protocols, and thus can include suitable transmitter / receiver antennas, electrical circuits and ports, and encoding / decoding capabilities that may be necessary for the proper transmission and / or reception of data and other information. For example, the processor can be configured to receive electrical signals or information from the described sensors (e.g., CMOS sensors) and process the received information to generate the desired image or other information.

[0094] Various information and data processing operations described herein may, in one embodiment, be performed by a computer program product embodied in a computer-readable medium containing computer-executable instructions, e.g., program code, executed by computers in a networked environment. Computer-readable media may include removable and non-removable storage devices, including, but not limited to, read-only memory (ROM), random access memory (RAM), compact discs (CDs), digital versatile discs (DVDs), and the like. Accordingly, computer-readable media as described herein includes non-transitory storage media. Generally, program modules may include routines, programs, objects, components, data structures, and the like that perform particular tasks or implement particular abstract data types. Computer-executable instructions, associated data structures, and program modules represent examples of program code for executing steps of the methods disclosed herein. A particular arrangement of such executable instructions or associated data structures represents examples of corresponding acts for performing the functions described in such steps or processes.

[0095] Only a few implementations and examples are described; other implementations, enhancements and variations can be made based on what is described and illustrated herein.

Claims

1. a substrate, and At least two microneedles connected to a substrate A device comprising: Each microneedle in the at least two microneedles a protruding needle structure; and Electrode Probe Structure Including, the protruding needle structure includes an outer wall extending outwardly from a surface of the substrate, the outer wall enclosing an interior volume of the protruding needle structure and forming an apex at the end of the outer wall; the electrode probe structure is configured to generate a signal in response to one or more chemical or biological substances in a biological fluid in contact with the electrode probe structure; the electrode probe structure of a first microneedle of the at least two microneedles comprises an aptamer sequence specific to a first analyte, the electrode probe structure of the first microneedle being operable as a working electrode for detection of the first analyte using a first electrochemical detection technique; the electrode probe structure of a second microneedle of the at least two microneedles is operable as an electrochemical counter electrode or an electrochemical reference electrode; device.

2. The device of claim 1 , wherein the electrode probe structure is incorporated into or attached to a protruding needle structure.

3. The device of claim 1 , wherein the aptamer sequence is tethered to the electrode probe structure of the first microneedle via a 5′-thiol.

4. The device of claim 1 , wherein the aptamer sequence is functionalized with a redox reporter molecule.

5. The device of claim 4 , wherein the redox reporter molecule is methylene blue.

6. The device of claim 4 , wherein the redox reporter molecule is an anthraquinone.

7. The device of claim 4, wherein the functionalization is at the 3' end of the aptamer sequence.

8. The device of claim 4, wherein the functionalization is at the 5' end of the aptamer sequence.

9. 10. The device of claim 1, comprising at least two electrically conductive channels, each of which is electrically coupled to an electrode probe structure of a microneedle of at least two microneedles for transmitting a signal from the electrode probe structure or applying a control signal to the electrode probe structure.

10. 10. The device of claim 1, wherein the electrode probe structure of a third microneedle of the at least two microneedles comprises an aptamer sequence specific to a second analyte, and the electrode probe structure of the third microneedle is operable as a working electrode for detection of the second analyte using a second electrochemical detection technique.

11. The device of claim 10 , wherein the second analyte is different from the first analyte.

12. The device of claim 10 , wherein the second electrochemical detection technique is different from the first electrochemical detection technique.

13. 10. The device of claim 1, wherein for at least one microneedle in the at least two microneedles, the internal volume of the protruding needle structure of the microneedle includes a hollow interior defined by an inner wall, the outer wall of the protruding needle structure of the microneedle includes an opening to the hollow interior, and the electrode probe structure of the microneedle is at least partially disposed within the hollow interior.

14. 10. The device of claim 1, wherein the electrode probe structure of the microneedles in at least two of the microneedles comprises a metal film.

15. 14. The device of claim 13, wherein the electrode probe structure of the microneedle comprises a metal wire.

16. 16. A device according to claim 14 or claim 15, wherein the metal is gold.

17. 10. The device of claim 1, wherein the electrode probe structure of a second microneedle of the at least two microneedles comprises silver / silver chloride (Ag / AgCl).

18. 11. The device of claim 10, wherein the electrode probe structure of a fourth microneedle of the at least two microneedles comprises a coating on a surface of the electrode probe structure, the coating functionalized with a first enzyme, the coating configured to interact with a third analyte, and the electrode probe structure of the fourth microneedle operable as a working electrode for detection of the third analyte using a third electrochemical detection technique.

19. 20. The device of claim 18, wherein the electrode probe structure of a fifth microneedle of the at least two microneedles comprises a coating on a surface of the electrode probe structure, the coating functionalized with a second enzyme, the coating configured to interact with a fourth analyte, and the electrode probe structure of the fifth microneedle operable as a working electrode for detection of the fourth analyte using a fourth electrochemical detection technique.

20. 20. The device of claim 19, wherein the second enzyme is different from the first enzyme.

21. 20. The device of claim 18 or claim 19, wherein the electrode probe structure of a sixth microneedle of the at least two microneedles comprises a coating on a surface of the electrode probe structure, the coating being functionalized with an ionophore receptor, the coating being configured to interact with a fifth analyte, and the electrode probe structure of the sixth microneedle is operable as a working electrode for detection of the fifth analyte using a fifth electrochemical detection technique, and the fifth analyte is an electrolyte.

22. 20. The device of claim 19, wherein any electrochemical detection technique from the first, second, third and fourth electrochemical detection techniques is one of a cyclic voltammetry technique, a fast scan cyclic voltammetry technique, a square wave voltammetry technique, a potentiometry technique or a chronoamperometry technique.

23. 22. The device of claim 21, wherein the fifth electrochemical detection technique is one of a cyclic voltammetry technique, a fast scan cyclic voltammetry technique, a square wave voltammetry technique, a potentiometry technique, or a chronoamperometry technique.

24. 10. The device of claim 1, wherein the electrode probe structure of at least one microneedle of the at least two microneedles comprises a conformal coating.

25. 25. The device of claim 24, wherein the conformal coating comprises an electrically insulating polymer or a dielectric material.

26. The conformal coating is a poly(p-xylylene) polymer, a polyethyleneimine polymer, or SiO 2 25. The device of claim 24, comprising at least one of:

27. The device of claim 1 , wherein the first analyte is a protein.

28. 28. The device of claim 27, wherein the protein is a cytokine.

29. 28. The device of claim 27, wherein the protein is one of insulin, interleukin-6 protein, tumor necrosis factor protein, or C-reactive protein.

30. 20. The device of claim 18, wherein the third analyte is a small molecule compound.

31. 20. The device of claim 19, wherein the fourth analyte is a small molecule compound.

32. 32. The device of claim 30 or claim 31, wherein the small molecule compound is one of lactose, lactic acid, alcohol, glucose, glutamic acid, or ketone bodies.

33. The device of claim 1 , wherein the first analyte is a drug.

34. 11. The device of claim 10, wherein the first analyte is cortisol and the second analyte is insulin.

35. 20. The device of claim 18, wherein the first enzyme is glucose oxidase and the third analyte is glucose.

36. 20. The device of claim 19, wherein the second enzyme is beta-hydroxybutyrate dehydrogenase and the fourth analyte is a ketone body.

37. 22. The device of claim 21, wherein the electrolyte is one of sodium, potassium, or lithium.

38. 20. The device of claim 18, wherein the third analyte is different from the first analyte and the third analyte is different from the second analyte.

39. 20. The device of claim 19, wherein the fourth analyte is different from the first analyte, the fourth analyte is different from the second analyte, and the fourth analyte is different from the third analyte.

40. The device of claim 1 , wherein the device is configured to be placed on human skin.

41. 1. A method of manufacturing an electrochemical sensing device, comprising: providing a substrate; and Connecting at least two microneedles to a substrate Including, Each microneedle in the at least two microneedles a protruding needle structure; and Electrode Probe Structure Including, the protruding needle structure includes an outer wall extending outwardly from a surface of the substrate, the outer wall enclosing an interior volume of the protruding needle structure and forming an apex at the end of the outer wall; the electrode probe structure is configured to generate a signal in response to one or more chemical or biological substances in a biological fluid in contact with the electrode probe structure; the electrode probe structure of a first microneedle of the at least two microneedles comprises an aptamer sequence specific to a first analyte, the electrode probe structure of the first microneedle being operable as a working electrode for detection of the first analyte using a first electrochemical detection technique; the electrode probe structure of a second microneedle of the at least two microneedles is operable as an electrochemical counter electrode or an electrochemical reference electrode; method.

42. 1. A method of electrochemical-based sensing comprising: Preparing a device according to any one of claims 1 to 8 or 10 to 40, wherein the device comprises at least two electrically conducting channels, each channel in the at least two electrically conducting channels being electrically coupled to an electrode probe structure of a microneedle in at least two microneedles for transmitting a signal from the electrode probe structure or applying a control signal to the electrode probe structure; and transmitting a signal from the electrode probe structure of a first microneedle of the at least two microneedles using a first electrically conductive channel of the at least two electrically conductive channels; A method comprising:

43. 1. A method of electrochemical-based sensing comprising: providing a device according to any one of claims 1 to 8 or 10 to 40, wherein the device comprises at least two electrically conducting channels, each channel in the at least two electrically conducting channels being electrically coupled to an electrode probe structure of a microneedle in at least two microneedles for transmitting a signal from the electrode probe structure or applying a control signal to the electrode probe structure; applying a first control signal to an electrode probe structure of a first microneedle of the at least two microneedles using a first electrically conductive channel of the at least two electrically conductive channels; and transmitting a signal from the electrode probe structure of a second microneedle of the at least two microneedles using a second electrically conductive channel of the at least two electrically conductive channels; A method comprising:

44. Determining the concentration of the analyte using the signal transmitted from the electrode probe structure of the second microneedle.

44. The method of claim 43, comprising:

45. 45. The method of claim 43 or claim 44, wherein the first microneedle and the second microneedle refer to the same microneedle in the at least two microneedles.

46. 45. The method of claim 43 or claim 44, wherein the first microneedle and the second microneedle refer to different microneedles in the at least two microneedles.

47. 45. The method of claim 43 or claim 44, wherein the first electrically conducting channel and the second electrically conducting channel refer to the same electrically conducting channel in the at least two electrically conducting channels.

48. 45. The method of claim 43 or claim 44, wherein the first electrically conducting channel and the second electrically conducting channel refer to different electrically conducting channels in the at least two electrically conducting channels.

49. 49. The method of any one of claims 42 to 48, wherein the electrically conductive channels in the at least two electrically conductive channels are wires made of electrically conductive material or traces of electrically conductive material on a circuit board.

Images