Biosensors for placement at non-predetermined skin depths - Patent Application 20070122997

Abstract

A wearable device for continuous monitoring of at least one analyte is provided. The device includes a plurality of aptamer sensors for a particular analyte and means for determining at least one position measurement. The sensors are positionable at distinct locations with respect to depth within a user's skin. The position measurement has distinct measurement responses between at least two skin tissues, such as the epidermis, dermis, and subcutaneous tissue. At least one of the sensors is positionable at a distinct tissue location.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 448,511, filed February 27, 2023, which is incorporated herein by reference in its entirety.

[0002] The present invention generally relates to the placement of biosensors in the skin at multiple penetration depths achievable with a single device. [Background technology]

[0003] This section is intended to introduce the reader to various aspects of the art that may be related to various aspects of the present invention, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of the various aspects of the present invention. As such, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0004] Historically, continuous glucose monitoring has been nearly the only successful example of implanting a wearable sensor into the skin to continuously measure a target analyte (e.g., glucose in interstitial fluid), with the ultimate goal of replacing repeated fingersticks or blood sampling procedures. Continuous glucose meters have undergone considerable development effort, and the selection of the penetration depth into the skin has included factors such as reliability during ambulatory use, representativeness of analyte status in other parts of the body, inflammatory response, and the ideal analyte flux or concentration for accurate sensor operation. Commercially available continuous glucose meters today employ a single needle inserted into the skin, with options for insertion depth including the epidermis, dermis, and subcutaneous tissue (also referred to as the subcutaneous layer). An insertion depth of more than 5 mm into the subcutaneous tissue is chosen for glucose monitors because it is thick enough to allow glucose sensors to be placed on a variety of skin types and users of various ages (which have varying skin roughness), and because glucose sensors almost always reach the subcutaneous tissue despite expected variability in penetration depth. Furthermore, even if the wire depth increases or decreases slightly as the patient moves (e.g., walking), the sensor remains within the subcutaneous tissue, resulting in more reliable glucose readings. More recently, microneedles have begun to appear in commercial development, promising a "painless" procedure despite the fact that the majority of users of existing continuous glucose meters experience little or no pain when inserting the sensor into the skin. To avoid deeper penetration (greater than 1 mm) and any pain sensation, as well as providing reliable access to interstitial fluid, microneedles employ two needles in the same horizontal plane to ensure that at least one of the needles is properly positioned within the dermis. Having multiple needles has inherent drawbacks, including multiple skin penetrations and increased skin inflammatory responses.

[0005] In general, each tissue layer, such as the epidermis, dermis, and subcutaneous tissue, has its own advantages and disadvantages. The epidermis is metabolically more active due to the cellular content that supports skin growth. The dermis is largely acellular and therefore, for some analytes, is considered closest to blood in terms of both lag time and concentration. Subcutaneous tissue can present analytes at deeper tissue levels due to its fat cell content and has the advantage of being sufficiently thick, making repeated sensor insertion within a single tissue type easier to achieve and maintain. However, subcutaneous tissue may have a longer lag time and a lower analyte concentration, which can be a disadvantage. For dilute analytes (e.g., insulin or BNP) for which a high density of receptors may exist in tissue, an additional consideration is that cellular uptake may distort the measurable concentration, making insulin or BNP measurement in subcutaneous tissue inferior in one or more aspects. For example, as demonstrated in dogs in Yang YJ, Hope ID, Ader M, Bergman RN. Insulin transport across capillaries is rate-limiting for insulin action in dogs. J Clin Invest. 1989 Nov;84(5):1620-8. doi: 10.1172 / JCI114339. PMID: 2681272, insulin (hormone) measurements in the subcutaneous tissue or dermis may be inferior to inulin (carbohydrate) measurements in terms of concentration and lag time when compared with blood measurements due to cellular uptake of insulin at cellular insulin receptors in the skin. Thus, receptor-rich subcutaneous tissue may have different insulin concentrations than receptor-sparse dermis, thereby affecting the accuracy of the intended measurement. Depending on the application, the accuracy of the intended measurement can be compared to blood concentrations (representing an environment with a low receptor density) or tissue concentrations (representing a high receptor density).For example, blood concentrations may be desirable for determining how much of a drug or hormone is present in the circulation, while tissue concentrations may be desirable for determining how much of the drug or hormone has reached its target tissue. For example, in the case of a blood infection, circulating blood concentrations may be most important for measuring a drug to treat the infection, while in the case of a tissue infection, tissue concentrations may be a more desirable and representative measure of the effectiveness of drug delivery. Similar considerations may extend to the smallest analytes, such as glucose, or very large analytes, such as C-reactive protein (120 kDa), which have size-dependent transport from blood to tissue. Similar considerations may also extend to analytes, such as cytokines, which are produced locally in tissues and therefore may not be equivalent to blood concentrations. In summary, it would be valuable to identify or increase opportunities for placing sensors for analytes in the bodily environment that most represents the ideal measure for the analyte.

[0006] There remains a need for devices and methods for targeting specific depths and / or multiple depths and locations within the skin, and for doing so in a repeatable, reliable, and / or verifiable manner, which would enable greater accuracy, precision, time lag, or predictability of biosensor measurements. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 10973443B2 [Patent Document 2] PCT Application No. 21 / 51972 [Patent Document 3] US Patent No. 7,381,184 B2 [Non-patent literature]

[0008] [Non-Patent Document 1] Yang YJ, Hope ID, Ader M, Bergman RN. Insulin transport across capillaries is rate limiting for insulin action in dogs. J Clin Invest. 1989 Nov;84(5):1620~8. doi: 10.1172 / JCI114339. PMID: 2681272 [Non-patent document 2] Friedel M, Werbovetz B, Drexelius A, Watkins Z, Bali A, Plaxco KW, Heikenfeld J. Continuous molecular monitoring of human dermal interstitial fluid with microneedle-enabled electrochemical aptamer sensors. Lab Chip. 2023 Jul 12;23(14):3289~3299 pages. doi: 10.1039 / d3lc00210a. PMID: 37395135 Summary of the Invention [Problem to be solved by the invention]

[0009] Certain illustrative aspects of the present invention are described below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms that the invention may take, and that these aspects are not intended to limit the scope of the invention. Indeed, the present invention may encompass a variety of aspects that may not be explicitly set forth below.

[0010] Many of the above-mentioned drawbacks and limitations can be resolved by creating new and advanced interactions of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs that bring sensing technologies into proximity with biological fluids and analytes affordably, effectively, conveniently, intelligently, or reliably. [Means for solving the problem]

[0011] One aspect of the present invention relates to a wearable device for continuous monitoring of at least one analyte. The device includes a plurality of aptamer sensors for a particular analyte. The aptamer sensors are carried by a support. At least a subset of the plurality of sensors are each positionable at a distinct location with respect to depth within a user's skin. The device also includes means for determining at least one position measurement. The position measurement has a distinct measurement response value between at least two skin tissues selected from the group consisting of the epidermis, the dermis, and the subcutaneous tissue. Additionally, at least one of the sensors is positionable at a distinct location selected from the group consisting of the epidermis, the dermis, and the subcutaneous tissue.

[0012] In one embodiment, the at least one location measurement is selected from the group consisting of electrical impedance, a measured rate of change of concentration of at least one analyte, and the concentration of at least one analyte. In another embodiment, the at least one location measurement is an electrical impedance measurement. In one embodiment, the sensor is positionable at multiple discrete locations, and the same type of electrical impedance measurement is measured at the multiple discrete locations.

[0013] In another embodiment, the electrical impedance measurements have a threshold between the plurality of distinct locations, and when the distinct locations are in the dermis, the threshold has an electrical impedance that is equal to or less than one-half. In one embodiment, the monitoring of at least one analyte is an electrical measurement from a first electrical waveform, and the electrical impedance measurements are an electrical measurement from a second electrical waveform. In another embodiment, the first electrical waveform and the second electrical waveform are the same waveform. In one embodiment, the first electrical waveform and the second electrical waveform are square wave voltammetric waveforms.

[0014] In another embodiment, the location measurements are electrical impedance measurements with a pre-associated measure of cell density at the individual locations. In one embodiment, the location measurements are electrical impedance measurements with a pre-associated correction factor for monitoring at least one analyte. In one embodiment, the subset of the plurality of sensors is further included within a smaller subset of sensors, the smaller subset of sensors may all be disposed within a skin tissue layer selected from the group consisting of the epidermis, dermis, and subcutaneous tissue, and the monitoring of the analyte measurements is predominantly associated with the smaller subset of sensors. In another embodiment, the at least one analyte has a molecular weight greater than 1 kDa. In one embodiment, the individual locations further include a separation distance between the individual locations, the separation distance being a value between about 0.05 mm and about 5 mm. In another embodiment, the support is a single support.

[0015] In one embodiment, the support is a plurality of individual supports, and at least a portion of the plurality of sensors is distributed across the plurality of individual supports. In another embodiment, the individual locations further comprise blood vessels selected from the group consisting of veins, arteries, and capillaries. In one embodiment, the device also includes at least one component used to position a horizontal position of the vein, artery, or capillary below the user's skin.

[0016] Another aspect of the present invention relates to a wearable device for continuous monitoring of at least one analyte. The device includes at least one aptamer sensor for the analyte, at least one aptamer sensor depth adjustment component, and at least one discrete location measurement for the at least one sensor carried by a support. The discrete location is selected from the group consisting of the epidermis, dermis, subcutaneous tissue, veins, arteries, and capillaries.

[0017] In one embodiment, the position measurement is an electrical impedance measurement. In another embodiment, the position measurement is an analyte concentration measurement. In one embodiment, the electrical impedance measurement has a pre-associated correction factor for monitoring at least one analyte. In another embodiment, the at least one analyte has a molecular weight greater than 1 kDa. In one embodiment, the device also includes at least one component used to horizontally position a vein, artery, or capillary.

[0018] Another aspect of the present invention relates to a method for continuously monitoring at least one analyte at a discrete location within a subject's skin using a wearable device. The method includes placing at least one aptamer sensor for the analyte at a discrete location within the subject's skin, the at least one aptamer sensor being in communication with the wearable device. Then, performing at least one location measurement for the at least one aptamer sensor. Next, performing an action. Here, the action can be a first action of adjusting the sensor location within the skin, thus adjusting the discrete location to be the epidermis, dermis, or subcutaneous tissue, or blood in a vein or venous capillary. Alternatively, the action can be an action of monitoring at least one analyte measurement from multiple sensors for the same analyte, where the measurement is dominated by a subset of the multiple sensors, which can be as small as one sensor. A combination of both actions can also be performed.

[0019] In one embodiment, the method also includes performing at least one location measurement to determine a discrete location of the at least one aptamer sensor. In another embodiment, the at least one location measurement is selected from the group consisting of electrical impedance, a measured rate of change of concentration of at least one analyte, and the concentration of at least one analyte. In one embodiment, the at least one location measurement is performed via the same type of electrical impedance measurement at multiple discrete locations.

[0020] In another embodiment, the electrical impedance measurements have a threshold between the plurality of discrete locations, and if the location is within the dermis, the threshold has an electrical impedance that is equal to or less than one-half. In one embodiment, the at least one analyte measurement is an electrical measurement from a first electrical waveform and the electrical impedance measurement is an electrical measurement from a second electrical waveform. In another embodiment, the first electrical waveform and the second electrical waveform are the same waveform.

[0021] In one embodiment, the first electrical waveform and the second electrical waveform are square wave voltammetric waveforms. In another embodiment, the position measurements are electrical impedance measurements with pre-associated measurements of cell density at the location. In one embodiment, the position measurements are electrical impedance measurements with pre-associated correction factors for monitoring at least one analyte measurement.

[0022] In another embodiment, for multiple sensors measuring the same analyte, there is a subset of the multiple sensors, with a smaller subset of the sensors all located within a skin tissue layer selected from the group consisting of the epidermis, dermis, and subcutaneous tissue, and at least one analyte measurement is associated with only the smaller subset of sensors. In one embodiment, at least one analyte has a molecular weight greater than 1 kDa. In another embodiment, the discrete locations further include a separation distance between the discrete locations, the separation distance being a value between about 0.05 mm and about 5 mm. In one embodiment, the multiple sensors are carried by a single support. In another embodiment, at least a portion of the multiple sensors are distributed across multiple discrete supports. In one embodiment, the method also includes at least one component used to horizontally position the vein, artery, or capillary.

[0023] The objects and advantages of the disclosed invention will be better understood in light of the following detailed description and drawings. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows a schematic diagram of a conventional prior art sensor device based on microneedles. [Figure 2] FIG. 1 shows a schematic diagram of a conventional prior art sensor device based on a single needle. [Figure 3] FIG. 1 shows a schematic of an embodiment of the present invention implemented with multiple needle insertion depths. [Figure 4] FIG. 1 shows a schematic of an embodiment of the invention with single needle insertion using multiple redundant working electrodes at multiple depths. [Figure 5] FIG. 1 shows a schematic diagram of an embodiment of the present invention with an adjustable single needle insertion. [Figure 6] FIG. 1 shows an exemplary diagram of an electrochemical aptamer sensor. [Figure 7] FIG. 1 shows exemplary data for a subcutaneous electrochemical aptamer sensor. [Figure 8] FIG. 1 illustrates an exemplary environment for connecting a monitoring device to one or more additional processing devices. [Figure 9] 1A-1C illustrate system and component level diagrams of an embodiment of a monitoring device. [Figure 10] FIG. 5 illustrates the exemplary embodiment of FIG. 4 shown in more detail. [Figure 11] FIG. 5 illustrates the exemplary embodiment of FIG. 4 shown in more detail. [Figure 12] FIG. 1 illustrates an exemplary embodiment of the present invention including continuous blood access. [Figure 13] FIG. 13 shows a plan view of the device of FIG. 12 placed on the skin. [Figure 14] FIG. 1 shows a representative plot of skin reflectance (620 nm light wavelength) versus distance for vein location. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition As used herein, "analyte sensor," or "continuous sensing" using a "continuous sensor" or "continuous analyte sensor," or "continuous monitor or monitoring" refers to a sensor that provides measurements responsive to changes in the concentration of at least one analyte in a solution, and includes the ability of a device to provide multiple measurements of the analyte over time. Such sensors may include electrochemical sensors (such as sensors that use aptamers for affinity-based sensing) or other suitable means, and may include mechanical or optical sensors (such as sensors based on biolayer interferometry), or other sensing mechanisms in support of one or more embodiments of the present invention.

[0026] As used herein, the term "analyte" means any solute in a solution or fluid that can be measured using a sensor. An analyte can be a small molecule, protein, peptide, electrolyte, acid, base, antibody, small molecule-bound molecule, DNA, RNA, drug, chemical, pollutant, or other solute in a solution or fluid.

[0027] As used herein, the term "working electrode" refers to an electrode that performs sensing (e.g., an electrode that carries a sensing chemical such as an aptamer). A counter electrode and a reference electrode, or simply a counter electrode, are further required to operate in a three-electrode or two-electrode electrochemical measurement system, respectively. The working electrode may also be referred to as "a sensor" or "the sensor."

[0028] As used herein, "support" or "sensor support" refers to at least one material that allows the sensor to be placed within the skin and that can maintain the position of the sensor within the skin. The support may be an insulating material (e.g., plastic or ceramic, etc.) or may be, for example, an electrode (e.g., gold, tantalum, tungsten, or other wire type, etc.). In some cases, the support may also be an electrode required for the operation of the sensor.

[0029] As used herein, the term "about," when referring to a numerical value or an amount of mass, weight, time, volume, pH, size, concentration, or percentage, is meant to encompass variations of, in some embodiments, ±20%, in some embodiments, ±10%, in some embodiments, ±5%, in some embodiments, ±1%, in some embodiments, ±0.5%, and in some embodiments, ±0.1% from the specified amount, since such variations are suitable for performing the disclosed methods.

[0030] As used herein, the term "electrode" refers to any electrically conductive material (e.g., gold, platinum, nickel, silicon, a conductive liquid infusion material (e.g., an ionic liquid), PEDOT:PSS, a conductive oxide, carbon, boron-doped diamond, a nanotube or nanowire mesh, or other suitable electrically conductive material).

[0031] As used herein, the term "protective layer" refers to a uniform or non-uniform layer of material or one or more types of molecules on an electrode that can reduce electrochemical background current and / or current due to electrochemical interference and also facilitate the appropriate freedom of movement for the aptamer needed to produce a measurable response to analyte concentration.

[0032] As used herein, the term "anti-fouling layer" refers to a homogeneous or heterogeneous layer of a material or one or more types of molecules on a surface that reduces fouling on the surface compared to when such an anti-fouling layer is not utilized. A protective layer can also act as an anti-fouling layer. A selectively permeable membrane such as polybetaine can also be an anti-fouling layer.

[0033] As used herein, the term "aptamer" refers to a molecule that undergoes a conformational or binding change upon binding of an analyte to the molecule, and satisfies the general operating principle of the sensing method described herein. Such molecules include, for example, natural or modified DNA, RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers, and affimers. Modifications may include substituting non-natural nucleobases for natural bases in the aptamer sequence, substituting non-natural sequences for natural sequences, or other suitable modifications that improve sensor function but behave similarly to conventional aptamers. Two or more aptamers bound together may also be referred to as aptamers (i.e., do not separate in solution). Aptamers may have a molecular weight of at least 1 kDa, 10 kDa, or 100 kDa.

[0034] As used herein, the term "redox tag" or "redox molecule" refers to any species (e.g., a small molecule or a large molecule) having a redox-active moiety that can reversibly donate or accept at least one electron to or from an electrode when introduced adjacent to the electrode. Examples of redox tags or molecules include methylene blue, ferrocene, quinones, or other suitable species that meet the definition of a redox tag or molecule. In some cases, redox tags or molecules are referred to as redox mediators. Redox tags or molecules may also exchange electrons or change behavior when introduced adjacent to other redox tags or molecules. Exogenous redox molecules are those that are added to the device, e.g., they are not endogenous but are provided by the sample fluid being tested.

[0035] As used herein, the term "change in electron transfer" refers to a redox molecule whose electron transfer to or from an electrode has been altered in a measurable manner. This change in electron transfer may be due, for example, to the availability of electron transfer, distance from the electrode, diffusion rate to or from the electrode, a shift or increase / decrease in the electrochemical activity of the redox molecule, or any other embodiment that results in a measurable change in electron transfer between the redox molecule and the electrode. Numerous redox tags or redox molecules are possible, and numerous structural switching mechanisms exist for how an aptamer can change the electron transfer properties of a redox tag or molecule when it binds to or releases the target analyte; such alternative switching mechanisms are included herein even if not specifically mentioned.

[0036] As used herein, the term "sensing monolayer" refers to at least a plurality of aptamers on a working electrode, and may also include a plurality of molecules or mixtures of molecules that form a protective and / or antifouling layer.

[0037] Detailed Description of the Invention One or more specific embodiments of the present invention are described below. While an effort has been made to describe these embodiments concisely, not all features of an actual implementation are described herein. It should be understood that the development of any such actual implementation, as with any engineering or design project, will involve numerous implementation-specific decisions to achieve the developer's specific goals (e.g., adhering to system- and business-related constraints that may vary from implementation to implementation). It should further be understood that such a development effort might be complex and time-consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill in the art having the benefit of this disclosure.

[0038] Certain embodiments of the disclosed invention present sensors as individual, simple elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional supporting technologies or features not captured in the description herein. Sensors may be dual, triple, or more to provide improved data and readings. Sensors may provide continuous or discrete data and / or readings. Certain embodiments of the disclosed invention reveal subcomponents of sensing devices with many more subcomponents (e.g., reference or counter electrodes, batteries, antennas, adhesives) that are required and known for using the device in various applications. However, for the sake of simplicity and focus on inventive aspects, such components may not be explicitly shown in the diagrams or described in the embodiments of the disclosed invention. All ranges of parameters disclosed herein include the endpoints of the ranges.

[0039] Prior Art Devices Referring to FIG. 1 , a conventional prior art sensor device 100 is shown initially placed in a sample fluid (e.g., dermal interstitial fluid) on the skin 12. The device includes a microneedle support 110, which may be made of, for example, a metal, semiconductor, or plastic, and at least one working electrode 120 (e.g., gold, carbon, platinum, or other suitable electrode material). Counter and reference electrodes are not shown and may be included in feature 110. The device may also include electronics for reading the sensor 120 and communicating the data to a user or a smartphone (not shown). The sensor 120 may also be an aptamer sensor including at least one blocking layer of multiple molecules (e.g., mercaptohexanol) thiol-linked to the electrode and at least one aptamer responsive to binding to an analyte and containing a redox tag (e.g., methylene blue). Additional aptamer examples are provided in subsequent examples. The working electrodes 120 generally target the same analyte (e.g., cortisol) and are implanted through the epidermis 12a into the dermis 12b and, although less likely, into the subcutaneous tissue 12c. For microneedle arrays such as those shown in FIG. 1, the penetration depth of the device 100 into the skin 12 is typically several hundred microns (often 500-700 microns). The epidermis 12a may range from approximately 0.05 mm to 1.5 mm in thickness, depending on the body location. The dermis 12b may range from approximately 0.5 mm to 4 mm in thickness, depending on the body location. The subcutaneous tissue 12c is typically much thicker, ranging from 1 mm to several centimeters, depending on the body location. As with any biosensor (e.g., continuous glucose monitor, etc.), the user is typically advised on proper placement to minimize excessive variability in the thickness of these lower layers of the skin 12. Microneedle devices such as device 100 generally target the dermis and utilize the redundancy of multiple working electrodes 120 to ensure that at least one microneedle working electrode is properly positioned within the dermis. Accordingly, the prior art has at least one approach in which the penetration depth of the working electrode into the tissue is predetermined.

[0040] With further reference to the prior art and Figure 1, device 100 may also use hollow microneedles and sensors (not shown) that are either internal to the hollow microneedles or external to the body, as taught in Friedel M, Werbovetz B, Drexelius A, Watkins Z, Bali A, Plaxco KW, Heikenfeld J. Continuous molecular monitoring of human dermal interstitial fluid with microneedle-enabled electrochemical aptamer sensors. Lab Chip. 2023 Jul 12;23(14):3289-3299. doi: 10.1039 / d3lc00210a. PMID: 37395135. In this hollow microneedle example, the sensor is ex vivo, but effectively this is also an example of at least one approach in which the penetration depth of the working electrode into the tissue is predetermined. This is because the access point (depth) to the interstitial fluid is predetermined, and therefore the sensor only comes into contact with interstitial fluid originating from the predetermined depth, regardless of its location (outside the body, inside the hollow microneedle, etc.).

[0041] Referring to FIG. 2 , where like numerals refer to like features, a conventional prior art sensor device 200 includes a single needle element with a working electrode 220 implanted in the subcutaneous tissue 12c. This configuration is commonly employed in continuous glucose monitors. For most users of the device 200, the working electrode 220 is implanted deep enough, maintaining an appropriate penetration depth (e.g., approximately 5 mm), so that the working electrode 220 remains securely fixed in the subcutaneous tissue 12c throughout the device's life. Thus, the prior art has at least one approach in which the penetration depth of the working electrode into the tissue is predetermined. Even if the insertion depth is set pre-insertion for multiple possible depths, such a configuration also falls under the predetermined depth because a trial depth of insertion is set before the needle element is placed in the skin. Single needle element type sensors that are several millimeters or longer in length generally require an additional mechanical inserter, as taught, for example, in U.S. Pat. No. 10,973,443 B2 (Sensor inserter assembly - Jeffery V. Funderburk, Duane O. Yamaski, Brian VanHiel, Stephen J. Flynn).

[0042] General Description of the Invention 3 , where like numbers refer to like features, in one embodiment of the present invention, for a working electrode used to continuously monitor an analyte, the working electrode depth into tissue is not predetermined. Also, where like numbers refer to like features, device 300, including housing 310, includes multiple working electrodes that are independently physically supported in skin 12 at multiple, permanently fixed distances by physical supports 360, 362, 364. Physical supports 360, 362, 364 may be an insulated metal, plastic, glass, ceramic, semiconductor material, or other suitable material, and are typically less than a few millimeters in diameter or width, preferably less than 1 mm in diameter or width, and more preferably less than 500 μm in diameter or width, to minimize the size of a puncture hole created in skin 12. The term "distance" is defined as the vertical distance from the skin surface (exposed surface of the stratum corneum) to the average penetration depth of the exposed area of ​​the working electrode. For example, for working electrodes with surface areas ranging from 0.5 mm to 0.8 mm, the average penetration depth is 0.65 mm. For example, when targeting working electrodes within each layer of tissue (12a, 12b, 12c), the distance may be 0.1 mm for 320, 1 mm for 322, and 10 mm for 324. For example, when simply aiming for at least one working electrode to be located within the dermis with a high degree of certainty, the distance may be 0.5 mm for 320, 1.5 mm for 322, and 2.5 mm for 324. For example, the separation distance between electrodes may be greater than 0.05 mm and less than 5 mm. Multiple working electrodes 320, 322, and 324 are used to measure the same analyte (e.g., cortisol, BNP, NT-proBNP, insulin, albumin, C-reactive protein, or other suitable target analyte). Analytes above 1 kDa (such as vasopressin, vancomycin, or even larger analytes) are likely to benefit most from the present invention due to their greater restriction in transport from blood to tissues.For example, in embodiments of the present invention, at least one working electrode 320, 322, 324 may be within an ideal tissue layer (e.g., if the dermis is the ideal tissue layer, then working electrode 322 is located within dermis 12b) to achieve maximum blood correlation, or maximum tissue correlation, or other performance parameters.

[0043] With further reference to embodiments of the present invention, the present invention may therefore include a device for sensing at least one analyte in a biological fluid of a subject, the device comprising at least one sensor connected to at least one structure capable of penetrating the skin of the subject, wherein the at least one sensor can be positioned in the skin of the subject at a non-predetermined depth, and further wherein the sensor can be positioned in at least one layer of the skin of the subject, said layer being selected from the group consisting of the epidermis, the dermis, and the subcutaneous tissue.

[0044] The discrete locations may further include a separation distance between the discrete locations, the separation distance being at least greater than 0.05 mm and at least less than 5 mm.

[0045] The support may further comprise a plurality of individual supports 360, 362, 364, wherein at least a portion of said plurality of sensors 320, 322, 324 are distributed across the plurality of individual supports. The analyte may have a molecular weight greater than 1 kDa.

[0046] With further reference to the present invention, at least one of the plurality of sensors may be permanently implanted in one of the epidermis, dermis, or subcutaneous tissue throughout a human population, or may be implanted at least 90% of the time the device is applied to human skin. At least one of the plurality of sensors is a first sensor and may be permanently implanted in one of the epidermis, dermis, or subcutaneous tissue, and a second sensor of the plurality of sensors may be permanently implanted in one of the epidermis, dermis, or subcutaneous tissue not occupied by the first sensor.

[0047] 4, where like numbers refer to like features, and in an alternative embodiment of the present invention, where like numbers refer to like features, a device 400 having a housing 410 includes multiple working electrodes 420, 422, 424 having a common physical support 460, the working electrodes 420, 422, 424 positioned at multiple permanently fixed distances in the skin 12. The multiple working electrodes 420, 422, 424 are used to measure the same analyte (e.g., vancomycin, cortisol, NT-proBNP, insulin, or other suitable target analyte). As a result, at least one working electrode 420, 422, 424 is in a target tissue layer, such as working electrode 422 in the dermis 12b, or two electrodes are individually in two tissue layers, such as electrode 422 in the dermal tissue 12b and electrode 424 in the subcutaneous tissue 12c. Thus, Figure 4 teaches another case in which the depth or tissue location of the working electrode is not predetermined with respect to the tissue layer targeted for continuous analyte monitoring. In Figure 4, physical support 460 is perpendicular to the skin surface, but the physical support may be angled, curved, or of other geometric shape as long as it meets the requirements of the present invention. In Figure 4, the device measures only a first analyte, but additional sets of working electrodes at multiple discrete depths, on the same support 460 or on a second support, could similarly be included, thereby enabling the device to measure a second analyte while retaining the benefits of the present invention across both the first and second analytes.

[0048] With further reference to embodiments of the present invention, the present invention may therefore include multiple aptamer sensors for the same analyte carried by a support, although the support is a single support.

[0049] With further reference to Figures 3 and 4, in one embodiment of the present invention, multiple working electrodes for the same analyte can be directly connected to an electronic measurement circuit (described in more detail below). For example, working electrodes can be patterned using photolithography on Kapton or PET film, similar to the film used in continuous glucose meters, with one metal electrode used per working electrode. Each electrode can be connected to its own dedicated measurement circuit, or wires can be collectively connected to a multiplexer, which in turn connects to a single measurement circuit. Measurements can include electrochemical scans (e.g., voltammetry, amperometry, potentiometry, or other suitable methods for detecting target analytes). One of the more preferred measurement methods for aptamer sensors is square-wave voltammetry, which is a form of linear potential sweep voltammetry that uses a combined square wave and step potential applied to the working electrode.

[0050] 3 and 4, in one embodiment of the present invention, multiple working electrodes for the same analyte may also include at least one measurement to determine the depth or surrounding tissue location of one or more working electrodes. Without the ability to determine tissue location, the present invention may lose the advantages of prior art techniques having predetermined depths and tissue locations in some applications. For example, if at least one measurement is a measurement of the electrical impedance between a working electrode and a counter electrode (where the counter electrode is, for example, a cardiac gel pad (shown in a later figure) placed on the skin surface or a counter electrode located near each working electrode (not shown) within the body), or between multiple working electrodes where the other working electrode acts as a counter electrode, within the dermis, working electrodes 322 and 422 may have the lowest electrical impedance (especially at low electrical measurement frequencies) because they are not surrounded by significant cellular content that could increase the electrical impedance. For example, cell-dense tissues may have high impedances on the order of 0.01 S / m, while acellular matrices (e.g., interstitial fluid-filled collagen) may have low impedances on the order of only 0.1 S / m. For example, electrodes 320, 322, 324, 420, 422, 424 may be measured at the same negative or positive potential of tens to hundreds of millivolts, at the same frequency, or at frequencies scanned with AC frequencies ranging from 0.1 Hz to 100 kHz. Because the dermis is an acellular mixture of collagen compared to the adipocyte-rich subcutaneous tissue and cell-dense epidermis, the electrical conductivity of the dermis may be 10 to 20 times higher than the epidermis and subcutaneous tissue, and thus may be distinguishable between electrodes. Therefore, by using electrical impedance, the present invention can identify which electrodes are accurately inserted at which tissue depth or in which tissue environment. The present invention has the advantage that by placing multiple electrodes within the body, it allows for a ratiometric comparison of impedance levels between electrodes instead of relying on absolute quantitative impedance measurements, which may be more error prone.For example, if eight electrodes are spaced 200 μm apart on a single support (similar to FIG. 4 ) and the relative positions of the electrodes are known at the time the device is constructed (which is the case in most cases), then when comparing any working electrode with the other working electrodes, a high impedance at the electrode closest to the skin surface suggests that it is within the epidermis; an impedance that is near infinity or is infinity suggests that the electrode closest to the skin surface is outside the skin; a low impedance at intermediate electrodes (not located near the skin surface and not near the distal end of the support) suggests that these electrodes are within the dermis; and if the electrodes closest to the distal end of the support have a higher electrical impedance than the electrodes in the dermis, then they suggest that they are in the subcutaneous tissue.

[0051] In one embodiment, the present invention further includes at least one location measurement, wherein the location measurement has a distinct measurement response value between at least two tissues, including the epidermis, dermis, or subcutaneous tissue. The at least one location measurement may be an electrical impedance measurement, and may be a homogenous electrical impedance measurement at a plurality of distinct locations. The at least one location measurement may be an electrical impedance measurement having a threshold value between the plurality of distinct locations, the threshold value having an electrical impedance that is half or less when the distinct location is the dermis.

[0052] With further reference to Figures 3 and 4, in one embodiment of the present invention, multiple working electrodes for the same analyte can also be measured using square-wave voltammetry, chronoamperometry, or other suitable electrochemical techniques that simultaneously provide at least one measure of analyte concentration and at least one measure of electrical impedance. For example, square-wave voltammetry or continuous square-wave voltammetry is a preferred measurement technique because the electrochemical current from the redox tag on the aptamer can be sampled after 0.1 to 1 milliseconds, at which point the majority of the capacitive current has dissipated. This capacitive current can be directly exploited for impedance measurements by measuring the current magnitude or change in current with time during the first 0.1 to 1 millisecond (e.g., 0.05 to 0.1 milliseconds, or 0.5 milliseconds, or other examples) of each square-wave measurement pulse. At the beginning of the square-wave measurement pulse, the current is dominated by the resistance-capacitance change time, which is directly dependent on the electrical impedance of the surrounding fluid or tissue. This measurement can be used not only to determine the depth of the working electrode or the tissue environment around it, but also to determine the cell density around the electrode, which, for example, in the case of BNP or insulin, can affect concentration measurements by two-fold or more due to cellular receptor uptake. If cell density needs to be measured near the working electrode, the working electrode can be factory calibrated for its impedance in artificial interstitial fluid or serum (0% or minimum cell density) and for its impedance when placed in, for example, adipose tissue (100% or maximum cell density). Comparative measurements then taken in the body during human use provide a quantitative measure of the tissue or cell density near the working electrode. For example, it is known that high cell density reduces local insulin concentrations by half compared to blood insulin concentrations, and if the electrical impedance of the working electrode confirms during use that it is surrounded by a high density of cells, the insulin concentration measured by the working electrode can be multiplied by 2 for the device to report a stronger correlation with blood insulin levels.

[0053] Thus, in one embodiment, the present invention further includes a position measurement that is an electrical impedance measurement, where the monitoring of at least one analyte is an electrical measurement from a first electrical waveform and the electrical impedance measurement is an electrical measurement from a second electrical waveform. The first electrical waveform and the second electrical waveform can be the same waveform. The first electrical waveform and the second electrical waveform can be square wave voltammetric waveforms.

[0054] 3 and 4, in one embodiment of the present invention, multiple working electrodes for the same analyte may also include at least one measurement to determine the depth or surrounding tissue location of one or more working electrodes. For example, the at least one measurement may be a measure of rise time in concentration or a concentration measured locally about the working electrode. Higher tissue or cell density may increase measurement lag time (e.g., albumin in the subcutaneous tissue may increase in concentration more slowly than albumin in the dermis due to increased diffusion resistance toward the working electrode) or may decrease the local concentration of an analyte (e.g., insulin, whose concentration in adipose tissue in the subcutaneous tissue is half that of the dermis, which is a largely acellular collagen matrix). Thus, the present invention may further include at least one measurement for determining the tissue location surrounding one or more sensors, where the at least one measurement is electrical impedance, and the location is used to correct the measured response, for example, to improve correlation with blood levels, or dermal levels, or subcutaneous levels, or lymph levels. For example, if the measurement location is subcutaneous tissue, the analyte is insulin, and the goal of the measurement is to correlate with blood levels of insulin, the measurement may be doubled to correct for the reduced insulin in subcutaneous tissue compared to blood insulin. If the device (not shown) uses optical measurements, electrical impedance measurements may still be utilized, such as by patterning an optical waveguide on one side of a support (such as a PET or Kapton film) and impedance-measuring electrodes on the other side of the support.

[0055] In one embodiment, the invention further includes a location measurement that is an electrical impedance measurement, the location measurement having a pre-associated measure of cell density at the location. Thus, the invention may further include a location measurement, the location measurement being an electrical impedance measurement having a pre-associated correction factor for monitoring at least one analyte. The at least one location measurement may be selected from at least one of electrical impedance; a measured rate of change of the concentration of the at least one analyte; or the concentration of the at least one analyte.

[0056] 3 and 4, in one embodiment of the present invention, any single working electrode of the present invention may include at least one impedance measurement of the healing progress of local tissue adjacent to the working electrode. When the working electrode is inserted into the skin, it may initially cause inflammation and local tissue damage, causing a decrease in electrical impedance, but over a period of hours to days the insertion site may heal and the electrical impedance may increase.

[0057] 3 and 4, in one embodiment of the present invention, multiple working electrodes for the same analyte may include at least one working electrode whose measurement is determined to have greater accuracy. For example, when sensing BNP or insulin, if the desired measurement correlates well with blood concentration, the measurement preference may be in the dermis 12b, and electrodes 322, 422 may be preferred. Alternatively, if the target measurement is tissue delivery, the epidermis or subcutaneous tissue may be preferred. For example, if the target measurement is tissue delivery of the antibiotic vancomycin, a working electrode with an aptamer sensor for vancomycin may represent the tissue level if implanted in subcutaneous tissue. If multiple sensors are utilized for a target analyte, and only one sensor or subset of sensors is located in the preferred tissue(s) (and thus there may be multiple sensors), only one sensor or subset of sensors, or only one of them, may be used to collect and / or report data to the user. With further reference to embodiments of the present invention, as the body and device move, are shocked, or are otherwise mechanically agitated, the working electrode position may change, resulting in a shallower or deeper depth within the tissue, and the sensors may be recalibrated to determine which sensors are in the desired tissue(s), and therefore which sensors to use to collect and / or report data to the user. The present invention may include only one sensor or a subset of multiple sensors, and may be used to report sensor data.

[0058] Thus, the subset of sensors according to the present invention may further be included as part of a smaller subset of sensors, but the smaller subset of sensors may all be located within a skin tissue layer selected from the group consisting of the epidermis, dermis, and subcutaneous tissue, and the monitoring of analyte measurements is predominantly associated with the smaller subset of sensors.

[0059] 3 and 4, in one embodiment of the present invention, other measures can be used to determine the depth of a working electrode or electrodes. For example, in addition to the electrodes illustrated in the figures (e.g., electrical, optical, mechanical, thermal, acoustic, or other sensing modalities), additional electrodes or sensors can be used to determine the depth or local tissue or cell density for a particular working electrode.

[0060] 5, where like numbers refer to like features, in one embodiment of the present invention, at least one working electrode 520 is positioned by device 500 to target a preferred tissue of epidermis 12a, dermis 12b, or subcutaneous tissue 12c. This can be accomplished, for example, by initially inserting electrode 520 into subcutaneous tissue 12c to a maximum depth of, for example, 6 mm, and then retracting electrode 520 to a depth of, for example, 2 mm with one or more components 570, 580, 590, thereby positioning electrode 520 within dermis 12b at a depth of 2 mm determined using one or more methods (e.g., measuring differences in electrical impedance between tissue layers) as taught herein. For example, element 590 can be epoxy that securely holds wire 560 to housing 510, element 580 can be a heater, and element 570 can be a heater and a volume of solder or wax. The heater 570 and solder or wax can be meltable, and the element 580 can heat the wire 560 (e.g., a shape-memory alloy such as Nitinol) to cause a shape change in the wire, retracting the wire 560 until it reaches the reduced impedance expected from being within the dermis 12b for the electrode 520; the heater 570 is then first cooled, allowing the solder or wax to solidify and hold the wire 560 in place; and the heater 580 is then cooled. This example is instructive because all components can be simple, low-cost components mounted on a printed circuit board that is already part of the device construction. In an alternative embodiment, the element 570 can be a motor or micro-linear actuator, for which there are many commercially available industrial examples, and the wire 560 can be adjusted in both directions (deeper or shallower) to place the electrode 520 in the desired tissue. Positioning of the sensor can be done after the device 500 is applied to the skin or at a later time if the sensor location moves out of the desired location due to walking or other movements by the user or mechanical agitation.The positioning of the sensor can be shallower only, deeper only, or both shallower and deeper (bidirectional depth control). While numerous linear actuation methods are suitable for the operation of FIG. 5, the present invention does not rely on the actuation method, but rather relies on depth determination and selection into various tissues as taught herein. Thus, FIG. 5 teaches another example in which the electrode depth or tissue location is not predetermined. Thus, the present invention can include at least one component for adjusting the penetration depth of at least one sensor in the skin. At least one component can be unidirectionally adjustable for penetration depth into the skin. At least one component can be bidirectionally adjustable for penetration depth into the skin. At least one component can be only retractably adjustable for penetration depth into the skin. At least one component can be thermally activated. At least one component can be a linear actuator. At least one component can be active when the device is initially applied to the skin. At least one component can be active when the position of the at least one sensor needs to be maintained at a depth. At least one component can be actuated in two directions (deeper or shallower on the skin) until a concentration maximum is found or a concentration minimum is found in at least one sensor indicating the optimal site for sensing.

[0061] With further reference to embodiments of the present invention, two or more working electrodes in different tissue layers can together provide superior measurements compared to a single electrode alone. For example, a working electrode in subcutaneous tissue may report the steady-state concentration of an analyte, such as a drug that must penetrate adipose tissue or brain parenchyma, while a working electrode in the dermis may more closely reflect blood concentrations. An example of an analyte may be vancomycin, which is used to treat both blood-borne infections and early infections originating from tissues (the antibiotic must penetrate both infection sites without reaching toxic levels). Thus, the present invention may include embodiments in which a sensor is placed at a predetermined depth in at least two of the epidermis, dermis, or subcutaneous tissue. Like other potential analytes (e.g., peptides), vancomycin has a molecular weight of less than 1 kDa, so the concentration or time profile of concentration change may differ between the dermis and subcutaneous tissue; therefore, the present invention may, in some specific embodiments, be limited to analytes with a molecular weight greater than 1 kDa. Preparation and operation of the working electrode

[0062] 6 and 7, where like numerals refer to like features, additional non-limiting examples and details regarding the construction and operation of a working electrode are provided in embodiments of the present invention. The working electrode 620 is comprised of an electrode material 658 (e.g., gold, etc.). The gold is then incubated with an aptamer 650 via thiol binding to the gold electrode, where the aptamer includes a redox tag (e.g., methylene blue 652, etc.). Between the aptamers, the gold is further incubated with a protective monolayer 656 (e.g., mercaptohexanol, mercaptooctanol, or other suitable chemicals, etc.). Although not shown, a protective film (e.g., polybetaine or other suitable material, etc.) can be added to prevent fouling of the monolayer surface. The working electrode 620 can be stored in a preservative (e.g., trehalose, etc.) to allow for dry storage. In a specific, non-limiting example, binding of aptamer 650 to target 654 causes a shape change that brings redox tag 652 into closer proximity with electrode 658, resulting in increased electron transfer (increased current). As the concentration of target 654 increases, more binding of target 654 to aptamer 650 occurs, resulting in more electron transfer (generating more measurable current). Conversely, as the concentration of target 654 decreases, the current decreases. Numerous other potential ways exist for an analyte to bind to an aptamer and cause a change in electron transfer with the electrode; therefore, embodiments of the present invention are not limited to the specific, simple example illustrated and discussed in FIG. 6 . The devices taught herein allow a support bearing a working electrode to be inserted into the skin using one or more methods, such as those commonly employed to insert glucose sensor needles for continuous glucose monitoring (e.g., slotted insertion guides or other methods). An example of device fabrication and testing is as follows:

[0063] material Sulfuric acid (96%, PA), sodium hydroxide (98%, pellet), powdered phosphate-buffered saline (PBS, pH 7.4), Tris-EDTA solution (TE buffer, pH 8), bovine serum, tris(2-carboxyethyl)phosphine hydrochloride (TCEP; 98%), sodium azide (99.5%), 1,6-d6-mercapto-1-hexanol (MCH; 98%), and 8-mercapto-1-octanol (MCO; 97%) were obtained from Sigma-Aldrich (USA). 2-Hydroxy-2-methylpropiophenone (photoinitiator, purity: >96%) was purchased from TCI Chemicals. [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (monomer, purity: >95%) was obtained from Chem-Impex Inc. (USA). Absolute ethanol (100%; anhydrous) was purchased from Fisher Scientific (USA). Vancomycin hydrochloride (94.6%) and ethylene glycol-dimethacrylate (crosslinker, purity: >98%) were obtained from Alfa Aesar. 3'-methylene blue and 5'-thiol modified oligonucleotide sequences were synthesized by Integrated DNA Technologies (IDT, USA).

[0064] Examples of aptamer sequences are as follows:

[0065] [Table 1]

[0066] 1. Preparation of Sensors The gold electrode with a titanium adhesion layer is laminated onto a PET or Kapton strip, providing a support for the electrode (e.g., support 460 in Figure 4). The gold can be patterned via photolithography or chemical etching, and a photosensitive or screen-printable electrical insulator can also be applied, as described below in Figure 10. The gold can be further electroplated with additional gold and electrochemically roughened or cleaned prior to incubation with the aptamer 650 and protective layer 656. Electrochemical cleaning can be performed in a standard three-electrode electrochemical cell consisting of a gold working electrode, a platinum counter electrode, and an Ag / AgCl reference electrode by performing 700 cycles of cyclic voltammetry scans in 0.5 M NaOH from -1 V to -1.6 V at a scan rate of 1 V / s, followed by 150 scans in 0.5 M H2SO4 from 0 V to 1.6 V at 1 V / s. After electrochemical cleaning is complete, the electrode is thoroughly rinsed with DI water and dried in a stream of nitrogen (99.999% purity) for subsequent incubation. The lyophilized pellet of modified aptamer can be diluted to a 100 μM stock solution using TE buffer and stored at -20 °C until use. The aptamer working solution can be prepared by first mixing a 100 μM aptamer stock solution with an equal volume of 0.5 M TCEP dissolved in Milli-Q water. The mixture can then be left for 1 hour to ensure complete reduction of any disulfide aptamer molecules. The resulting solution can then be diluted to an intermediate concentration of approximately 4 μM with 1x PBS / 2 mM MgCl2 buffer, and the concentration can be confirmed via absorbance measured at 260 nm using a Nanodrop UV / Vis spectrophotometer. This solution can then be diluted to 500 nM in 1x PBS / 2 mM MgCl2 buffer to allow the aptamer to incubate for 1 hour. The aptamer-functionalized electrodes can then be rinsed with DI water and incubated overnight at room temperature in 5 mM MCH (mercaptohexanol) or MCO (mercaptooctanol) prepared in 1x PBS.The functionalized sensors can then be rinsed with DI water before being coated with trehalose for storage and ultimately used for measurements. If electrochemical roughening and hydrogel protection are desired, prior to incubation with the aptamer and MCH or MCO, the electrodes can be immersed in a 5 M NaOH solution and each electrode can be subjected to alternating potential steps of -5 V and +0.8 V (vs. Hg / HgSO, saturated NaSO) for 20 ms (6000 s total) in an electrochemical cell consisting of a Kapton®-carbon counter electrode and a saturated Hg / HgSO reference electrode. Once roughening is complete, the electrodes can be rinsed with copious amounts of DI water and then incubated with the aptamer and MCH or MCO as described above. The sensor was modified with an antibiofouling zwitterionic polybetaine-based hydrogel by drop-casting 1 μl of an aqueous mixture consisting of monomer / crosslinker / photoinitiator (2.8 g / 1.8 μl / 36 μl dissolved in 1 ml of DI water, respectively) onto the sensor and exposing it to UV light (λ: 280–450 nm, Bluewave LEDPrime UVA, Dynamax, USA) for 45 min.

[0067] Electrochemical measurements Electrochemical measurements can be performed using a miniaturized potentiostat (detailed in Figure 9) or a benchtop CHI 620E potentiostat (Austin, TX) connected to a 64-channel multiplexer in a standard three-electrode system with the aptamer / alkylthiolate-functionalized electrode serving as the working electrode. Counter and reference electrodes can be inserted into the skin using a platinum counter electrode and an Ag / AgCl reference electrode, or they can be large gel electrode pads on the skin surface, as taught in PCT application U.S. Patent No. 21 / 51972, entitled "Aptamer Sensors with Reference and Counter Voltage Control." Cyclic voltammograms can be recorded within a window of -0.1 V to -0.5 V at a scan rate of 100 mV / s. Square-wave voltammetry can be performed at the measurement frequency optimal for each aptamer, with an amplitude of 25 mV, within a potential window of -0.1 V to -0.5 V. Dynamic differential, dual-frequency, or continuous square-wave voltammetry can be utilized to improve calibration-free sensor operation. An example of in vivo data collected on a cortisol sensor inserted subcutaneously in a rat is shown in Figure 7, where cortisol injections were administered at 5 or 10 mg / kg.

[0068] Additional Targets The present invention applies generally to aptamer sensors and other types of affinity biosensors and is therefore not limited to the specific examples taught herein. Available electrochemical sensors for analytes (e.g., cortisol, vancomycin, phenylalanine, insulin, BNP, NT-proBNP, IL-6, C-peptide, C-reactive protein, etc.), as well as sensors for other analyte targets, can be incorporated into the present invention without limitation. Analytes can be endogenous or exogenous to the body, without limitation.

[0069] System Overview With a basic understanding of the above sensors in mind, reference is now made to Figure 8, which illustrates a general diagram of a computer system 800 in accordance with various aspects of the present disclosure. Like numerals within Figure 8 do not necessarily refer to like features in other figures. The computer system 800 includes multiple hardware processing devices (collectively referred to as 802) linked together by one or more networks (collectively referred to as 804).

[0070] The network 804 may be supported by networking components 806 (including, for example, routers, hubs, firewalls, network interfaces, wired or wireless communication links and corresponding interconnections, cellular stations and corresponding cellular conversion technology (e.g., to convert between cellular and TCP / IP, etc.)) that provide communications links between and interconnect the various processing devices 802. Additionally, the network 804 may include connections using one or more intranets, extranets, local area networks (LANs), wide area networks (WANs), wireless networks (Wi-Fi), the Internet, including the World Wide Web, cellular, and / or other configurations to enable communications between the processing devices 802 in real time or other manners (e.g., via time shifting, batch processing, etc.).

[0071] The processing device 802 may be any device capable of communicating with another processing device 802 in the network 804 or a combination thereof, for example, via Bluetooth, ultra-wideband, near field communication (NFC), via one or more radio frequencies (RF), or via any other form of wired or wireless communication.

[0072] Some examples of processing devices 802 include cellular devices (including cellular mobile phones (i.e., smartphones)), tablet computers, netbook computers, notebook computers, personal computers, servers, cloud devices, edge devices, and the like.

[0073] It is also intended that in certain contexts and roles, processing device 802 is a wearable monitoring device. Examples of wearable monitoring devices include purpose-driven appliances, Internet of Things (IoT) devices, special-purpose devices, etc. Processing device 802 implemented as a wearable monitoring device is illustrated schematically in FIG. 8 solely for convenience of illustration as a wearable device attached to a patient's arm. In practical applications, the wearable monitoring device may be attached to other parts of the patient's body.

[0074] In some embodiments, the wearable monitoring device can communicate locally (e.g., with a smartphone) via Bluetooth, ultra-wideband, via one or more radio frequencies (RF), or via any other form of wired or wireless communication. In other embodiments, the wearable monitoring device can communicate across a network, e.g., via Wi-Fi, and / or can communicate locally with another processing device 802.

[0075] The exemplary computer system 800 also includes a processing device implemented as a server 812 (e.g., a web server, a file server, and / or other processing device) that supports an analytics engine 814 and corresponding data sources (collectively identified as data sources 816). The analytics engine 814 and data sources 816 provide resources for implementing and storing data related to the collection and aggregation of data from wearable monitoring devices, captured events, combinations thereof, etc., as described in more detail herein.

[0076] In an exemplary implementation, data sources 816 are implemented by a collection of databases storing various types of information. By way of example only, data sources 816 may include device data 818, e.g., data related to the wearable monitoring device, including configuration data, version data, software versioning and control, data generated from wearing the wearable monitoring device, etc. Data sources 816 may also include medical data 820, e.g., medical research used to calibrate, adjust, design, modify, etc. the wearable monitoring device. Data sources 816 may also optionally include user data, e.g., data related to patients wearing the wearable monitoring device (if such data is collected). As yet another example, data sources 816 may include platform data 824, e.g., data used by analysis engine 814, e.g., computer drivers, GUI information, algorithms for processing physiological conditions, etc. As another further example, data sources 816 may optionally include other data 826, such as any data required by analytics engine 814 and not otherwise considered above.

[0077] Considering FIG. 8 as an environment used by a wearable monitoring device, in some embodiments, processing of physiological data of a corresponding patient wearing the wearable monitoring device (such as biochemical sensing using additional sensing modalities to enhance the patient's care or health and well-being) may be performed entirely on processing device 802 (e.g., on the wearable monitoring device itself), on processing device 802 (e.g., a smartphone), by analysis engine 814, or via a combination thereof (e.g., by distributing processing tasks between two or more processing devices).

[0078] With particular attention to processing device 802 implemented as a wearable monitoring device (see processing device 802 generally attached to the patient's arm), it may be desirable to perform all processing on the wearable monitoring device itself. In this regard, a compatible device (e.g., a smartphone, etc.) may optionally provide a graphical user interface for displaying dashboard measurements, but all processing is performed on the wearable monitoring device itself.

[0079] In other embodiments, the smartphone may perform some processing, such as to execute algorithms, rules, or other processing to compare the computed data to dashboard thresholds, as described more fully herein.

[0080] In still other embodiments, the analytics engine 814 may collect data from each wearable monitoring device, for example, to trend patient data, monitor device health (e.g., to detect malfunctions in the wearable device itself), monitor battery charge levels, and for version control (e.g., to perform software updates, etc.).

[0081] In some embodiments, the analysis engine 814 is controlled by a third party, for example, the manufacturer of the wearable monitoring device that is deployed in the environment.

[0082] In some embodiments, the analytics engine 814 generally provides integration into an electronic health record system, for example, to connect a patient with their physician and enable the physician to access electronic data generated by a corresponding wearable monitoring device.

[0083] Exemplary Monitoring Devices 9, which schematically illustrates an example of a wearable monitoring device 900 according to aspects of the present disclosure. Like numerals in FIG. 9 do not necessarily refer to like features as in other figures. The wearable monitoring device 900 may represent an exemplary embodiment of the processing device 802 (FIG. 8) as previously described, e.g., a wearable monitoring device.

[0084] The wearable monitoring device 900 includes a housing 910 that attaches to the patient. The housing can be attached to the patient via adhesive 904, straps, or other fasteners.

[0085] Wearable monitoring device 900 also includes at least a first working electrode 920, and may include a second working electrode 922, and may further include a third working electrode 924, or more working electrodes. In some embodiments, one or more electrodes include an analyte-sensing material, such as an aptamer, so that continuous sensing can be performed. Electrode 950 may be a gel electrode pad, as previously described, and may also serve as a reference and counter electrode.

[0086] In practical applications, the housing 910 is connectable to the electrodes 920, 922, 924. As used herein, unless specifically noted otherwise, "connectable" shall be interpreted broadly to mean any one of permanently connected, removably connected, adjustable, temporarily connected, user-attachable, user-detachable, user-attachable and removable, factory-attachable, factory-detachable, user-attachable, factory-attachable and removable, or any combination thereof.

[0087] As illustrated, the housing 910 contains a potentiostat 991 communicatively coupled to the electrodes 920, 922, 924 (or a combination thereof) using an optional multiplexer 990, or each of the electrodes 920, 922, 924 may be directly and dedicatedly connected to the potentiostat 991. In practical applications, the term "potentiostat" is intended to be broadly construed and is not limited to a particular number of sensors. For example, a potentiostat may be described as a bipotentiostat, polypotentiostat, etc., depending on the sensor configuration provided by the wearable monitoring device 900.

[0088] Additionally, the wearable monitoring device 900 includes a controller 993 that is communicatively coupled to the memory 992. The controller 993 is also communicatively coupled to a communication interface 994.

[0089] The controller 993 includes the necessary electronics that enable the controller 993 to perform the intended functions of the wearable monitoring device. For example, the controller 993 may include a processor, bus interface, ports, registers, memory, etc. that enable the wearable monitoring device 900 to perform the functions more fully described herein.

[0090] Also, as illustrated, the controller 993 is communicatively coupled to one or more of an optional multiplexer 990, a potentiostat 991, a memory 992, a transceiver 994, optional other sensors 995, an optional display / output 996, combinations thereof, and the like.

[0091] The communication interface 994 may include at least one transceiver that communicates via, for example, Bluetooth, Wi-Fi, ultra-wideband, near field communication, combinations thereof, and the like.

[0092] Optional display / output 996 may include a display screen, a dimensionally limited display screen, a touch screen, a haptic output, an optical output, a speaker / alarm, or a combination thereof.

[0093] The controller 993 collects measurements from the electrodes 920a, 920b, 920c using a potentiostat 991 and stores the collected measurements in a memory 992. The controller 993 may further provide filtering, analysis, control, authorization, authentication, and other controller-specific functions. The communication interface 994 facilitates coupling of the wearable monitoring device 900 with an external computing device (e.g., a smartphone, a cloud computer, etc.). In this regard, the communication interface 994 may include one or more modalities (each with different data and / or authorizations). For example, as illustrated in FIG. 10 , a patient may access data from the wearable monitoring device 900 on a smartphone 1006, while a physician may be able to access more detailed information from a cloud server and / or through an electronic health record (see FIG. 9 ). In this regard, multiple communication modalities may be utilized with the wearable monitoring device 900.

[0094] In some embodiments, the adhesive 904 of the wearable cardiac monitoring device 900 is or includes a gel electrode 950 that is connected to at least one of the potentiostat 991, the controller 993, or the sensor 995. For example, the gel electrode 950 can be a counter or reference electrode to the electrodes 920, 922, 924.

[0095] Working electrode configuration Referring to FIG. 10, where like numerals refer to like features in FIGS. 1-6, a more detailed description of an example physical layout of multiple working electrodes is illustrated. FIG. 10 shows that side-by-side electrodes can be used to implement the principles of the present invention. The support 1060 can be constructed, for example, from tens to hundreds of microns of PET or Kapton, or an electrically insulated metal foil. An exemplary support 1060 can have dimensions similar to a glucose monitor strip (e.g., 400 microns wide and 5 mm long). The support carries a working electrode film (e.g., 1020 and 1022, etc.) tens to hundreds to thousands of nanometers thick, which can be, for example, a layer of titanium or a chromium adhesion-promoting layer followed by a gold coating. These layers are then further coated with an electrical insulator 1055 (e.g., a screen-printable insulating paste used to fabricate glucose sensors, a photosensitive insulator (e.g., Microchem SU-8, HD Microsystems PI2611, and Fujifilm Durimide 7020, etc.), other photosensitive polymer coatings, adhesive tape, patterned parylene coatings, or other suitable film-based insulators), which can range in thickness from a few nanometers to over 100 μm. The remaining areas of the exposed working electrodes 1020, 1022 can then be incubated with sensing chemistry as taught herein and coated with other materials as needed. FIG. 10 shows an example of two depth-excursion working electrodes 1020, 1022, where electrode 1020 penetrates the skin the deepest. This example, which teaches case 10, may use alternative patterning methods, alternative electrode shapes, and alternative numbers of multiple working electrodes, and is therefore a non-limiting example of all possible embodiments of the present invention. For example, a PET or Kapton film can also carry an optical waveguide for optically sensing molecular beacon aptamers bound to the waveguide surface, in which case, instead of using an electrical insulator as shown in Figure 10, an optical cladding on the waveguide serves a similar purpose and function to limit the exposed area of ​​the optical-based sensor to a specific depth in the skin.

[0096] Referring to FIG. 11, where like numerals refer to like features in FIGS. 1-6 and 10, a more detailed description of one example physical layout of multiple working electrodes is illustrated. FIG. 11 shows that the principles of the present invention can also be realized by layering electrodes. FIG. 11 simply represents a side cross-sectional view of layers of solid support 1160, working electrode 1120, insulator 1555, working electrode 1122, and insulator 1155. Overall, FIGS. 10 and 11 illustrate that multiple methods for creating multiple working electrodes are possible, including methods not specifically illustrated herein (e.g., electrode vias, coaxial wires, and conductors, etc.), as well as other techniques known in the fields of microelectronics, printed circuit boards, and other similar techniques.

[0097] Access of the working electrode to blood at an undetermined depth With reference to further embodiments of the present invention, the present invention may also be applied to continuous blood measurements. Continuous blood measurements have not been actively pursued due to the challenges of placing sensors in veins or arteries compared to simpler subcutaneous placement. Continuous blood measurements may be advantageous, for example, for measuring vancomycin during blood-borne infections, for drugs with narrow therapeutic windows whose pharmacokinetics are clearly defined by blood and / or tissue concentrations, or for large analytes (some antibodies, some proteins) that are so large that they have difficulty distributing from blood to interstitial fluid. Further motivations for performing blood measurements include cases where measurements are more diagnostically accurate (e.g., in skin inflammation, inflammatory markers may be produced locally and biased, potentially not representative of blood concentrations). Methods exist for chronic intravenous line access (e.g., Port-a-Cath, peripherally inserted central catheters, or central venous lines), but all are more invasive and typically require trained medical personnel for placement. When applied to continuous blood monitoring, an important aspect of the present invention is that the user can reliably insert the device into the bloodstream without the need for trained medical personnel. Referring to Figures 12, 13, and 14, where like numbers refer to similar procedures, the present invention, like previous embodiments, allows for sensor placement in arteries and veins at undetermined depths, since arterioles and venules, even close to the skin, are only 30 μm in diameter, making reliable sensor placement extremely difficult, if not impossible. In contrast, more methods (e.g., manual visual inspection, ultrasound, portable near-infrared vein finder, etc.) allow for identification and targeting of a superficial artery or vein (e.g., cephalic, cubital, basilic, or other vein or artery, etc.). One of multiple working electrodes (e.g., 1220, 1222, 1224, etc.) can then enter and reside within vein 13 and / or artery, enabling precise blood access.While the present invention allows for reliable insertion of a working electrode into a vein or artery, perhaps a greater challenge remains in locating the vein or artery and aligning a working electrode inserter (such as those used in continuous glucose meters) with the target vein or artery. In a more detailed example, FIG. 13 shows a wearable sensor 1300 with an opening for sensor insertion into the skin 12, similar to that used with Abbott's Libre2 or Libre3 glucose monitors. This same opening can be used as a simple viewport for a camera or red light source (such as an LED or laser), and as the device 1300 is moved horizontally across the skin 12, the red light reflectance measured by a photodiode or spectrometer decreases, indicating the presence of a vein due to deoxygenated hemoglobin, as shown in FIG. 14 . This provides a more automated example of horizontal positioning over a vein, after which the sensor can be inserted into the skin using one or more insertion methods, such as those used for inserting continuous glucose monitor sensors (e.g., Funderburk et al., U.S. Patent No. 7,381,184 B2). The method of positioning the working electrode on an artery or vein is not a limiting factor for embodiments of the present invention used in continuous blood monitoring, although the ability to determine which tissue or fluid the sensor is in is crucial to the embodiments taught herein. The ability to have multiple redundant working electrodes for the same analyte and / or the ability to adjust the insertion depth of the working electrode after applying the device to the skin is also crucial. In addition to the previously described methods for determining which tissue location the working electrode is in, blood measurements can distinguish between working electrodes in blood and tissue using, by non-limiting example, comparison of maximum concentration between blood and tissue, comparison of the rate of initial concentration increase between blood and tissue, pulsatile pressure effects (such as turbulence at red blood cells and the exposed working electrode surface), or other suitable measures.

[0098] Accordingly, embodiments of the present invention may be applicable to direct sensing in blood, and thus the present invention may include at least one of the plurality of sensors being a first sensor and at least one of the plurality of sensors being a second sensor, wherein the at least one sensor is disposed in the blood at a depth that is not predetermined.

[0099] Thus, the present invention may further include at least one component used to horizontally locate the position of a vein, artery, or capillary.

[0100] Other steps not described in detail herein but readily apparent from or incorporated with the disclosed embodiments are intended to be included as part of the present invention. The embodiments described herein provide specific examples to illustrate elements of the invention, but do not necessarily encompass all possible embodiments known to those skilled in the art. [Explanation of symbols]

[0101] 12 Skin 12a Epidermis 12b Dermis 12c subcutaneous tissue 13 Veins 100 Sensor Devices, Devices 110 Microneedle support, feature 120 Working electrode, sensor 200 Sensor Devices, Devices 220 Working electrode 300 devices 310 Housing 320 Working electrodes, sensors, electrodes 322 Working electrodes, sensors, and electrodes 324 Working electrodes, sensors, and electrodes 360 Physical Support, Individual Support 362 Physical Support, Individual Support 364 Physical Support, Individual Support 400 devices 410 Housing 420 Working electrode, electrode 422 Working electrode, electrode 424 Working electrode, electrode 460 physical support, support 500 devices 510 Housing 520 Working electrode, electrode 560 Wire 570 Components, Heaters, Elements 580 Components, Heaters, Elements 590 Components, Elements 620 working electrode 650 Aptamers 652 Methylene Blue, Redox Tag 654 Target 656 Protective monolayer, protective layer 658 Electrode materials, electrodes 800 Computer Systems 802 Processing Device 804 Network 806 Networking Components 812 Server 814 Analysis Engine 816 Data Sources 818 Device Data 820 Medical Data 824 Platform Data 826 Other Data 900 Wearable monitoring devices, wearable cardiac monitoring devices 904 Adhesive 910 Housing 920 first working electrode, electrode 920a electrode 920b electrode 920c electrode 922 Second working electrode, electrode 924 Third working electrode, electrode 950 electrodes, gel electrodes 990 Multiplexer 991 Potentiostat 992 Memory 993 Controller 994 Communication Interface, Transceiver 995 Other Sensors 996 Optional Display / Output 1006 Smartphone 1020 Working electrode film, working electrode, depth-deflection type working electrode 1022 Working electrode film, working electrode, depth-deflection type working electrode 1055 Electrical insulators 1060 Support 1120 Working electrode 1122 Working electrode 1155 Insulator 1160 Solid Support 1220 Working electrode 1222 Working electrode 1224 Working electrode 1300 Wearable Sensors and Devices 1555 Insulator

Claims

1. 1. A wearable device for continuous monitoring of at least one analyte, comprising: a. a plurality of aptamer sensors for a particular analyte, the aptamer sensors being carried by a support, and at least a subset of the plurality of sensors being each positionable at a distinct location with respect to depth within a user's skin; b. means for determining at least one position measurement, said position measurement having a distinct measurement response between at least two skin tissues selected from the group consisting of the epidermis, the dermis, and the subcutaneous tissue; and wherein at least one of the sensors is capable of being positioned at a discrete location selected from one of the group consisting of the epidermis, the dermis, and the subcutaneous tissue.

2. 10. The device of claim 1, wherein the at least one position measurement is selected from the group consisting of electrical impedance, a measured rate of change of concentration of the at least one analyte, and a concentration of the at least one analyte.

3. The device of claim 1 , wherein the at least one position measurement is an electrical impedance measurement.

4. 4. The device of claim 3, wherein the sensor is positionable at multiple discrete locations and the same type of electrical impedance measurement is taken at the multiple discrete locations.

5. 4. The device of claim 3, wherein the electrical impedance measurements have a threshold between a plurality of distinct locations, and when the distinct locations are in the dermis, the threshold has an electrical impedance that is at least half or less.

6. 4. The device of claim 3, wherein the monitoring of at least one analyte is an electrical measurement from a first electrical waveform and the electrical impedance measurement is an electrical measurement from a second electrical waveform.

7. 7. The device of claim 6, wherein the first electrical waveform and the second electrical waveform are the same waveform.

8. 7. The device of claim 6, wherein the first electrical waveform and the second electrical waveform are square wave voltammetric waveforms.

9. The device of claim 1 , wherein the location measurements are electrical impedance measurements having a pre-associated measure of cell density at a discrete location.

10. 10. The device of claim 1, wherein the position measurements are electrical impedance measurements having pre-associated correction factors for monitoring at least one analyte.

11. 10. The device of claim 1, wherein a subset of the plurality of sensors is further comprised within a smaller subset of sensors, the smaller subset of sensors being capable of being all disposed within a skin tissue layer selected from the group consisting of the epidermis, the dermis, and the subcutaneous tissue, and wherein monitoring of analyte measurements is predominantly associated with the smaller subset of sensors.

12. The device of claim 1 , wherein at least one analyte has a molecular weight greater than 1 kDa.

13. 10. The device of claim 1, wherein the discrete locations further comprise a separation distance between the discrete locations, the separation distance being a value between about 0.05 mm and about 5 mm.

14. The device of claim 1 , wherein the support is a single support.

15. The device of claim 1 , wherein the support is a plurality of individual supports and at least some of the plurality of sensors are distributed across the plurality of individual supports.

16. The device of claim 1 , wherein the discrete location further comprises a blood vessel selected from the group consisting of a vein, an artery, and a capillary.

17. 17. The device of claim 16, further comprising at least one component used to position a vein, artery, or capillary horizontally below the user's skin.

18. 1. A wearable device for continuous monitoring of at least one analyte, comprising: At least one aptamer sensor for an analyte carried by a support; at least one aptamer sensor depth adjustment component; at least one individual position measurement for at least one sensor; wherein the discrete location is selected from the group consisting of the epidermis, the dermis, the subcutaneous tissue, a vein, an artery, and a capillary.

19. 20. The device of claim 18, wherein the position measurements are electrical impedance measurements.

20. 20. The device of claim 18, wherein the position measurement is a concentration measurement of the analyte.

21. 20. The device of claim 18, wherein the electrical impedance measurements have a pre-associated correction factor for monitoring at least one analyte.

22. 19. The device of claim 18, wherein at least one analyte has a molecular weight greater than 1 kDa.

23. 20. The device of claim 18, further comprising at least one component used to position a vein, artery, or capillary horizontally.

24. 1. A method for continuously monitoring at least one analyte at discrete locations within a subject's skin using a wearable device, comprising: placing at least one aptamer sensor for an analyte at a discrete location within the skin of a subject, the at least one aptamer sensor being in communication with a wearable device; performing at least one location measurement for at least one aptamer sensor; a first action of adjusting the sensor location within the skin, thus adjusting the individual location so that it is the epidermis, dermis, or subcutaneous tissue, or blood in a vein or venous capillary; monitoring at least one analyte measurement from a plurality of sensors for the same analyte, the measurement being dominated by a subset of the plurality of sensors, which may be as small as one sensor; and a combination of both actions performing an action selected from the group consisting of: A method comprising:

25. 25. The method of claim 24, further comprising performing at least one position measurement to determine the individual positions of at least one aptamer sensor.

26. 26. The method of claim 25, wherein the at least one location measurement is selected from the group consisting of electrical impedance, a measured rate of change of concentration of the at least one analyte, and the concentration of the at least one analyte.

27. 26. The method of claim 25, wherein the at least one location measurement is performed via electrical impedance measurements of the same type at multiple discrete locations.

28. 28. The method of claim 27, wherein the electrical impedance measurements have a threshold between a plurality of distinct locations, the threshold having an electrical impedance at least two times lower when the location is within the dermis.

29. 27. The method of claim 26, wherein the at least one analyte measurement is an electrical measurement from a first electrical waveform and the electrical impedance measurement is an electrical measurement from a second electrical waveform.

30. 30. The method of claim 29, wherein the first electrical waveform and the second electrical waveform are the same waveform.

31. 31. The method of claim 30, wherein the first electrical waveform and the second electrical waveform are square wave voltammetric waveforms.

32. 26. The method of claim 25, wherein the location measurements are electrical impedance measurements having a pre-associated measure of cell density at the location.

33. 26. The method of claim 25, wherein the position measurements are electrical impedance measurements having pre-associated correction factors for monitoring at least one analyte measurement.

34. 26. The method of claim 25, wherein for the plurality of sensors to measure the same analyte, there is a subset of the plurality of sensors, the smaller subset of sensors all disposed within a skin tissue layer selected from the group consisting of the epidermis, dermis, and subcutaneous tissue, and at least one analyte measurement is associated with only the smaller subset of sensors.

35. 10. The method of claim 1, wherein at least one analyte has a molecular weight greater than 1 kDa.

36. 25. The method of claim 24, wherein the discrete locations further comprise a separation distance between the discrete locations, the separation distance being a value between about 0.05 mm and about 5 mm.

37. The method of claim 24 , wherein the plurality of sensors are carried by a single support.

38. 25. The method of claim 24, wherein at least a portion of the plurality of sensors are distributed across a plurality of separate supports.

39. 25. The method of claim 24, further comprising at least one component used to horizontally position the vein, artery, or capillary.

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