EP4673049A1 - Biosensor for placement at skin depths that are not predetermined - Google Patents

Abstract

A wearable device for continuous monitoring of at least one analyte is provided. The device includes a plurality of aptamer sensors for a specific analyte and a means to determine at least one location measurement. The sensors are capable of being placed in a distinct location with respect to depth into a user's skin. The location measurement has a distinct measurement response between at least two skin tissues such as epidermis, dermis, and hypodermis. Also, at least one of the sensors is capable of being placed at a distinct tissue location.

Description

BIOSENSOR FOR PLACEMENT AT SKIN DEPTHS THAT ARE NOT PREDETERMINED

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/448,511, filed February 27, 2023, which application is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to placement of biosensors into the skin with multiple depths of penetration achievable by a single device.

BACKGROUND OF THE INVENTION

[0003] This section is intended to introduce the reader to various aspects of 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 various aspects of the present invention. Accordingly, 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 stands as a nearly singular success in terms of embedding a wearable sensor into the skin to continuously measure a target analyte such as glucose in interstitial fluid with the goal of replacing repeated finger prick or blood draw measures. Considerable development efforts went into continuous glucose meters, and choice of depth of penetration into the skin included factors such as ambulatory reliability, representation of analyte status in other parts of the body, inflammation response, and an analyte flux or concentration which is ideal for accurate sensor operation. For continuous glucose meters on the market today, which employ a single needle inserted into the skin, the options for depth insertion including epidermis, dermis, and hypodermis (also called subcutaneous layer). A >5 mm insertion depth, which is into the hypodermis, has been chosen for glucose monitors because it is adequately thick that a glucose sensor could be placed on a variety of skin types and for a variety of ages of users (variable skin roughness), and despite expected variations in depth of penetration the glucose sensor will nearly always end up in the hypodermis. Furthermore, as the patient moves (ambulatory), even if the wire depth increases or decreases slightly, the sensor remains in the hypodermis. As a result, a more reliable glucose reading is obtained. More recently, microneedles have started to emerge in commercial development and are promising ‘pain free’ operation despite the fact that the vast majority of users of existing continuous glucose meters feel little or no pain upon sensor insertion into the skin. To avoid deeper penetration (>1 mm) and any pain-sensation but also provide reliable access to interstitial fluid, microneedles therefore employ duplicate needles in the same horizontal plane to ensure at least one of the needles has proper placement in the dermis. Having multiple needles has its own drawbacks including multiple skin perforations and increased skin inflammation response.

[0005] Generally, each layer of tissue, epidermis, dermis, and hypodermis brings its own advantages and disadvantages. The epidermis is metabolically more active due to cellular content to support skin growth. The dermis is largely acellular and may therefore be closest to blood for some analytes in terms of both lag times and concentrations. The hypodermis can represent deeper tissue levels of analytes due to its fat cell content and has an advantage of being thick enough that it is easier to repeatedly achieve and maintain sensor insertion in a single tissue type. However, the hypodermis can also have greater lag times and lower concentration of analytes which can be a disadvantage. A further consideration is for dilute analytes such as insulin or BNP for which a high density of receptors can exist in tissue, cellular uptake can skew measurable concentrations and could make insulin or BNP measurement in the hypodermis 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 (a hormone) measurement in the hypodermis or the dermis can be inferior to inulin (a carbohydrate) measurement in concentration and lag time compared to blood, because of cellular update of insulin at insulin receptors for cells in the skin. Therefore, the receptor rich hypodermis may have different insulin concentrations than the receptor dilute dermis, therefore effecting the accuracy of the intended measure. Depending on application, the accuracy of the intended measure could be compared to blood concentrations (representing an environment with a low-density of receptors), or the accuracy of the intended measure could be compared to tissue concentrations (representing a high density of receptors). For example, blood concentrations may be preferred for knowing how much of a drug or hormone is in circulation, whereas tissue concentrations may be preferred for knowing how much of a drug or hormone is reaching its targeted tissue. For example, for a blood infection, circulating blood concentrations may be the most important to measure for a drug to treat the infection, whereases for a tissue infection tissue concentrations would be the more preferred and representative measure of efficacy of delivery of the drug. Similar considerations can extend to the smallest of analytes such as glucose or very large analytes such as C-reactive protein (120 kDa), which will have a size dependency on their transport from blood into tissue. Similar considerations can extend to analytes like cytokines that can be generated locally in tissue, and therefore being not equal to blood concentrations. In summary, it can be valuable to be able to confirm or to be able to increase the chances of placing the sensor for an analyte in an environment in the body that is most representative of the ideal measure for the analyte.

[0006] A need still exists for devices and methods to target specific and/or multiple depths and locations into skin, and do so in a repeatable, reliable, and or confirmable manner. If such devices and methods can be achieved, greater accuracy, precision, time lags, or predictive value of biosensor measurements can be achieved.

SUMMARY OF THE INVENTION

[0007] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0008] Many of the drawbacks and limitations stated above can be resolved by creating novel and advanced interplays of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs, in a manner that affordably, effectively, conveniently, intelligently, or reliably brings sensing technology into proximity with biofluid and analytes.

[0009] One aspect of the present invention is directed to a wearable device for continuous monitoring of at least one analyte. The device includes a plurality of aptamer sensors for a specific analyte. The aptamer sensors are carried by a support. At least a subset of the plurality of sensors are each capable of being placed in a distinct location with respect to depth into a user’s skin. The device also includes a means to determine at least one location measurement. The location measurement has a distinct measurement response between at least two skin tissues selected from the group consisting of epidermis, dermis, and hypodermis. Also, at least one of the sensors is capable of being placed at a distinct location that is selected from the group consisting of epidermis, dermis, and hypodermis tissue.

[0010] In one embodiment, at least one location measurement is selected from the group consisting of electrical impedance, rate of measured change of concentration of the at least one analyte, and concentration of the at least one analyte. In another embodiment, the at least one location measurement is an electrical impedance measurement. In one embodiment, sensors are capable of being placed in a plurality of distinct locations and the same type of electrical impedance measurement is measured at a plurality of the distinct locations.

[0011] In another embodiment, the electrical impedance measurement has a threshold between a plurality of distinct locations and the threshold has at least 2X lower electrical impedance when the distinct location is the dermis. In one embodiment, monitoring of the at least one analyte is an electrical measurement from a first electrical waveform, and wherein 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. In one embodiment, the first electrical waveform and the second electrical waveform are a square wave voltammetry waveform.

[0012] In another embodiment, the location measurement is an electrical impedance measurement that has a pre-associated measure of cellular density at the distinct location. In one embodiment, the location measurement is an electrical impedance measurement that has a pre-associated correction factor for monitoring of the at least one analyte. In one embodiment, the subset of the plurality of sensors is further comprised in part of a smaller subset of sensors, and the smaller subset of sensors are capable of all being placed within a skin tissue layer selected from the group consisting of epidermis, dermis, and hypodermis, and the monitoring of the analyte measurement is dominantly associated with the smaller subset of sensors. In another embodiment, the at least one analyte is greater than 1 kDa in molecular weight. In one embodiment, the distinct locations further include a separation distance between the distinct locations and the separation distance is a value from about 0.05 mm to about 5 mm. In another embodiment, the support is a single support.

[0013] In one embodiment, the support is plurality of distinct supports and at least a portion of the plurality of sensors are distributed across a plurality of distinct supports. In another embodiment, the distinct location further comprises a blood vessel selected from the group consisting of a vein, artery, and capillary. In one embodiment, the device also includes at least one component used to locate beneath the user’s skin a horizontal position of a vein, artery, or capillary.

[0014] Another aspect of the present invention is directed to a wearable device for continuous monitoring of at least one analyte. The device includes at least one aptamer sensor for the analyte carried by a support, at least one aptamer sensor depth adjustment component, and at least one distinct location measurement for the at least one sensor. The distinct location is selected from the group consisting of epidermis, dermis, hypodermis, a vein, an artery and a capillary.

[0015] In one embodiment, the location measurement is an electrical impedance measurement. In another embodiment, the location measurement is a concentration measurement of the analyte. In one embodiment, the electrical impedance measurement has a pre-associated correction factor for the monitoring of the at least one analyte. In another embodiment, the at least one analyte is greater than 1 kDa in molecular weight. In one embodiment, the device also includes at least one component used to horizontally locate a vein, artery, or capillary.

[0016] Another aspect of the present invention is directed to a method for using a wearable device to continuously monitor at least one analyte in a distinct location in a subject’s ski. The method involves placing at least one aptamer sensor for the analyte in a distinct location in a subject’s skin, wherein the at least one aptamer sensor is in communication with the wearable device. Then, performing at least one location measurement for the at least one aptamer sensor. Next, performing an action, where the action may be a first action of adjusting sensor position into skin and therefore the distinct location such that it is the epidermis, dermis, or hypodermis, or blood in a vein or venous capillary. Alternatively, the action may be, from a plurality of sensors for the same analyte, monitoring of the at least one analyte measurement being dominated by a subset of the plurality of sensors that may be as a small as one sensor. It is also possible to perform a combination of both actions.

[0017] In one embodiment, the method also involves performing at least one location measurement to determine the distinct location of at least one aptamer sensor. In another embodiment, the at least one location measurement is selected from the group consisting of electrical impedance, rate of measured change of concentration of the at least one analyte, and concentration of the at least one analyte. In one embodiment, the at least one location measurement is performed via the same type of electrical impedance measurement at a plurality of the distinct locations.

[0018] In another embodiment, the electrical impedance measurement has a threshold between a plurality of the distinct locations and the threshold has at least 2X lower electrical impedance when the location is in the dermis. In one embodiment, the at least one analyte measurement is an electrical measurement from a first electrical waveform, and wherein 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. [0019] In one embodiment, the first electrical waveform and the second electrical waveform are a square wave voltammetry waveform. In another embodiment, the location measurement is an electrical impedance measurement that has a pre-associated measure of cellular density at the location. In one embodiment, the location measurement is an electrical impedance measurement that has a pre-associated correction factor for monitoring of the at least one analyte measurement.

[0020] In another embodiment, for the plurality of sensors for measuring the same analyte there is a subset of the plurality of sensors, and the smaller subset of sensors are all placed in a skin tissue layer selected from the group consisting of the epidermis, dermis, and hypodermis, and the at least one analyte measurement is only associated with the smaller subset of sensors. In one embodiment, the at least one analyte is greater than 1 kDa in molecular weight. In another embodiment, the distinct locations further comprise a separation distance between the distinct locations and the separation distance is a value from about 0.05 mm to about 5 mm. In one embodiment, the plurality of sensors is carried by a single support. In another embodiment, at least a portion of the plurality of sensors are distributed across a plurality of distinct supports. In one embodiment, the method also includes at least one component used to horizontally locate a vein, artery, or capillary.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0011] FIG. 1 is a schematic of a conventional prior art sensor device based on microneedles.

[0012] FIG. 2 is a schematic of a conventional prior art sensor device based on a single needle.

[0013] FIG. 3 is a schematic of an embodiment of the present invention implemented with multiple needle insertion depths.

[0014] FIG. 4 is a schematic of an embodiment of the present invention with a single needle insertion with a plurality of redundant working electrodes at multiple depths.

[0015] FIG. 5 is a schematic of an embodiment of the present invention with a single needle insertion that is adjustable.

[0016] FIG. 6 is an example diagram of an electrochemical aptamer sensor.

[0017] FIG. 7 is example data for a subcutaneous electrochemical aptamer sensor. [0018] FIG. 8 is an example environment for connecting a monitoring device to one or more additional processing devices.

[0019] FIG. 9 is a system level and component level diagram of an embodiment of a monitoring device.

[0020] FIG. 10 is an example embodiment of FIG. 4 shown in greater detail.

[0021] FIG. 11 is an example embodiment of FIG. 4 shown in greater detail.

[0022] FIG. 12 is an example embodiment of the present invention including continuous blood access.

[0023] FIG. 13 is a top view of the device of FIG. 12 placed on skin.

[0024] FIG. 14 is a representative plot of skin reflectance at 620 nm wavelength of light vs. distance in order to identify the location of a vein.

DEFINITIONS

[0025] As used herein, “analyte sensor” or “continuous sensing” with a “continuous sensor” or “continuous analyte sensor” or “continuous monitor or monitoring” means a sensor that provides a measurement that responds to changing concentration of at least one analyte in a solution, and which includes the capability of a device to provide multiple measurements of an analyte over time. Such sensors can include electrochemical sensors such as those using aptamers for affinity based sensing or other suitable measures, and may include mechanical or optical sensors, such as those based on biolayer interferometry, or other sensing mechanisms that support one or more embodiments of the present invention.

[0026] As used herein, the term “analyte” means any solute in a solution or fluid which can be measured using a sensor. Analytes can be small molecules, proteins, peptides, electrolytes, acids, bases, antibodies, molecules with small molecules bound to them, DNA, RNA, drugs, chemicals, pollutants, or other solutes in a solution or fluid.

[0027] As used herein, the term “working electrode” means the electrode that is performing the sensing, such as the electrode that carries sensing chemistry such as aptamers. Counter and reference electrodes or just counter electrodes are further required for operation in a 3 or 2 electrode electrochemical measurement system, respectively. A working electrode may also be referred to as “a sensor” or “the sensor”.

[0028] As used herein, “support” or “sensor support” means at least one material that allows placement of the sensor into skin and which is able to maintain position of the sensor in skin. The support may be an insulating material such as a plastic or ceramic, or for example may be an electrode such as a gold, tantalum, tungsten, or other type of wire. In some cases, the support may also be an electrode required for operation of the sensor.

[0029] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the specified amount, as such variations are appropriate to perform the disclosed method.

[0030] As used herein, the term “electrode” means any material that is electrically conductive such as gold, platinum, nickel, silicon, conductive liquid infused materials such as ionic liquids, PEDOT:PSS, conductive oxides, carbon, boron-doped diamond, nanotubes or nanowire meshes, or other suitable electrically conducting materials.

[0031 ] As used herein, the term “protective layer” means a homogeneous or heterogeneous layer of material or of one or more types of molecules on an electrode which reduce electrochemical background current and/or current due to electrochemical interference, and which may promote proper freedom of movement for the aptamer which is required for creating a measurable response to analyte concentration.

[0032] As used herein, the term “antifouling layer” means a homogeneous or heterogeneous layer of material or of one or more types of molecules on a surface which reduces fouling on a surface compared to if such an antifouling layer was not utilized. A protective layer may also act as an antifouling layer. A permi-selective membrane such as polybetaine may also be an anti-fouling layer.

[0033] As used herein, the term “aptamer” means a molecule that undergoes a conformation or binding change as an analyte binds to the molecule, and which satisfies the general operating principles of the sensing method as described herein. Such molecules are, e.g., natural or modified DNA, RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers, and affimers. Modifications may include substituting unnatural nucleic acid bases for natural bases within the aptamer sequence, replacing natural sequences with unnatural sequences, or other suitable modifications that improve sensor function, but which behave analogous to traditional aptamers. Two or more aptamers bound together can also be referred to as an aptamer (i.e., not separated in solution). Aptamers can have molecular weights of at least 1 kDa, 10 kDa, or 100 kDa.

[0034] As used herein, the term “redox tag” or “redox molecule” means any species such as small or large molecules with a redox active portion that when brought adjacent to an electrode can reversibly transfer at least one electron with the electrode. Redox tag or molecule examples include methylene blue, ferrocene, quinones, or other suitable species that satisfy the definition of a redox tag or molecule. In some cases, a redox tag or molecule is referred to as a redox mediator. Redox tags or molecules may also exchange electrons or change in behavior when brought into proximity with other redox tags or molecules. Exogenous redox molecules are those added to a device, e.g. they are not endogenous and provided by the sample fluid to be tested.

[0035] As used herein, the term “change in electron transfer” means a redox molecule whose electron transfer with an electrode has changed in a measurable manner. This change in electron transfer can, for example, originate from availability for electron transfer, distance from an electrode, diffusion rate to or from an electrode, a shift or increase or decrease in 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. There are numerous redox tags or redox molecules that are possible, and there are numerous structural switching mechanisms for how aptamers can change electron transfer properties for the redox tag or molecule when the aptamer binds or releases the target analyte, and such alternative switching mechanisms are herein included even if not specifically mentioned.

[0036] As used herein, the term “sensing monolayer” means at least a plurality of aptamers on a working electrode, which may also include a plurality of molecules or mixtures of molecules that form a protective layer and/or an anti-fouling layer.

DETAILED DESCRIPTION OF THE INVENTION

[0037] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated 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 having the benefit of this disclosure.

[0038] Certain embodiments of the disclosed invention show sensors as simple individual elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional supporting technology or features which are not captured in the description herein. Sensors can be in duplicate, triplicate, or more, to provide improved data and readings. Sensors may provide continuous or discrete data and/or readings. Certain embodiments of the disclosed invention show sub-components of what would be sensing devices with more sub-components needed for use of the device in various applications, which are known (e.g., a reference or counter electrode, a battery, antenna, adhesive), and for purposes of brevity 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.

Prior Art Devices

[0039] With reference to FIG. 1, a conventional prior art sensor device 100 as placed initially in a sample fluid such as dermal interstitial fluid of skin 12 is shown, comprising: a microneedle support 110 that can be made of metal, semiconductor, or plastic for example, and at least one working electrode 120 such as gold, carbon, platinum, or other suitable electrode material. Counter and references electrodes are not shown and may be included on feature 110. The device may also comprise electronics for reading the sensor 120 and communicating data to a user or smart phone (not shown). Sensor 120 may also be an aptamer sensor comprising at least one blocking layer of a plurality of molecules such as mercaptohexanol that are thiol bonded to the electrode, and at least one aptamer that is responsive to binding to an analyte and which contains a redox tag such as methylene blue. Further aptamer examples will be taught in later examples. The working electrode(s) 120 are typically for the same analyte, such as cortisol for example, and are embedded through the epidermis 12a and in the dermis 12b, and potentially but unlikely into the hypodermis 12c. The depth of penetration into skin 12 by the device 100 is typically 100’s of pm (often 500-700 pm) for microneedle arrays as shown in Fig. 1. The epidermis 12a can range from roughly 0.05 mm to 1.5 mm thick depending on body location depending on body location. The dermis 12b can range from roughly 0.5 mm to 4 mm thick depending on body location. The hypodermis 12c is typically much thicker ranging from 1mm to even several centimeters depending on body location. Like with any biosensor such as a continuous glucose monitor users are typically advised on proper locations for placement to minimize too much variation in thickness of these sublayers of skin 12. Microneedle devices like device 100 typically target the dermis and leverage the redundancy of multiple working electrodes 120 to ensure at least one microneedle working electrode is properly placed in the dermis. Hence, the prior art has at least one approach where the working electrode depth of penetration into tissue is predetermined. [0040] With further reference to the prior art and FIG. 1, the device 100 may also use hollow microneedles and a sensor which is inside the hollow microneedles or which is outside the body (not shown), as taught by, 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. While in this hollowmicroneedle example the sensor is ex-vivo, effectively this is also an example least one approach where the working electrode depth of penetration into tissue is predetermined, because the access point (depth) for interstitial fluid is predetermined and therefore the sensor regardless of its location (ex-vivo, inside a hollow microneedle, etc.) is only coupled to interstitial fluid originating at predetermined depth.

[0041] With reference 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 embedded in the hypodermis 12c. This arrangement is commonly employed in continuous glucose monitors. The working electrode 220 is embedded deeply enough with adequate penetrating depth (e.g. ~5 mm) such that for most users of the device 200 the working electrode 220 will always be securely in the hypodermis 12c during use of the device 200. Hence, the prior art has at least one approach where the working electrode depth of penetration into tissue is predetermined. Even if the insertion depth can be configured for multiple possible depths before insertion, such a configuration still represents a predetermined depth because the attempted depth of insertion is configured before the needle element is placed into skin. Single needle element type sensors that are several mm or more in length typically require an additional mechanical inserter, as taught for example in US10973443B2 - Sensor inserter assembly - Jeffery V. Funderburk, Duane O. Yamaski, Brian VanHiel, Stephen J. Flynn.

General Description of the Present Invention

[0042] With reference to FIG. 3, where like numerals refer to like features, in an embodiment of the present invention where working electrode depth into tissue is not predetermined in terms of the working electrode that is to be used for continuous monitoring of an analyte, and where like numerals refer to like features, a device 300 with housing 310 includes a plurality of working electrodes independently physically supported at multiple permanent fixed distances into skin 12 by physical supports 360, 362, 364. Physical supports 360, 362, 364 can be insulated metals, plastics, glasses, ceramics, semiconductor materials, or other suitable materials and typically are less than several mm in diameter or width and preferably less than 1 mm in diameter or width and more preferably <500 pm in diameter or width to minimize the size of puncture hole(s) 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 distance of penetration for the exposed area of the working electrode, which for example with a working electrode that had a surface area that spans 0.5 and 0.8 mm would be the average of 0.65 mm penetration depth. For example, if targeting a working electrode in each layer of tissue (12a, 12b, 12c) distances could be 0.1 mm for 320, 1 mm for 322 and 10 mm for 324. For example if the goal is simply for at least one working electrode to be in the dermis with a high degree of certainty then distances could be 0.5 mm for 320, 1.5 mm for 322 and 2.5 mm for 324. For example, the separation distances between electrodes can be >0.05 mm but less than 5 mm. The plurality of working electrodes 320, 322, 324 are used to measure the same analyte, such as cortisol, BNP, NT -proBNP, insulin, albumin, C-reactive protein, or other suitable target analyte. Analytes >1 kDa (such as vasopressin, vancomycin, or even larger analytes) may benefit most from the present invention due to greater restriction of their transport from blood into tissues. For example, with embodiments of the present invention, at least one working electrode 320, 322, 324 can be inside the ideal tissue layer for maximum blood correlation or for maximum tissue correlation or other performance parameter, such as having the dermis be the ideal tissue layer and therefore working electrode 322 is placed in the dermis 12b.

[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 biofluid of a subject comprising at least one sensor connected to at least one structure that is capable of penetrating the subject’s skin, wherein the at least one sensor is capable of being placed in the subject’s skin at a depth that is not predetermined and further, wherein the sensor is capable of being placed into at least one layer of the subject’s skin, said layer being selected from the group consisting of epidermis, dermis, and hypodermis..

[0044] The distinct locations may further include a separation distance between the distinct locations and the separation distance is at least >0.05 mm and at least < 5 mm.

[0045] The support may further include a plurality of distinct supports 360, 362, 364, and at least a portion of said plurality of the sensors 320, 322, 324, are distributed across a plurality of distinct supports. The analyte may be greater than 1 kDa in molecular weight.

[0046] With further reference to the present invention, at least one of the plurality of sensors may always be embedded into one of the epidermis, dermis, or hypodermis, or at least >90% of the time that the device is applied to human skin across the population of humans. At least one of the plurality of sensors is a first sensor and may always embedded into one of the epidermis, dermis, or hypodermis, and a second sensor of the plurality of sensors may always embedded into one of the epidermis, dermis, or hypodermis not occupied by first sensor.

[0047] With reference to FIG. 4, where like numerals refer to like features, in an alternate embodiment of the present invention, where like numerals refer to like features, a device 400 with housing 410 includes a plurality of working electrodes 420, 422, 424 with a shared physical support 460, with the working electrodes 420, 422, 424 positioned at multiple permanent fixed distances into skin 12. The plurality of working electrodes 420, 422, 424 are used to measure the same analyte, such as vancomycin, cortisol, NT -proBNP, insulin, or other suitable target analyte. As a result, at least one working electrode 420, 422, 424 is in the targeted tissue layer, such as working electrode 422 in the dermis 12b, or alternately there are two electrodes distinctly in two layers of tissue such as electrode 422 in dermal tissue 12b and electrode 424 in hypodermal tissue 12c. Therefore FIG. 4 teaches another example where the working electrode depth or tissue location is not predetermined in terms of which tissue layer is targeted for continuous monitoring of the analyte. While in FIG. 4 the physical support 460 is perpendicular to the skin surface, the physical support may be angled, curved, or other geometries as long as they satisfy the requirements of the present invention. While in FIG. 4 the device is measuring just one first analyte, and additional set of working electrodes may be included, similarly at multiple distinct depths, on the same support 460 or on a second support, allowing the device to measure a second analyte while retaining the benefits of the present invention across both the first and the second analyte.

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

[0049] With further reference to FIGS. 3 and 4, in an embodiment of the present invention the multiple working electrodes for the same analyte can be directly connected to electronic measurement circuit (shown in greater detail later). For example, working electrodes can be patterned using photolithography on Kapton or PET films like that used in continuous glucose meters, and one metal electrode be used per working electrode. Each electrode can connect to its own measurement circuit or the wires can collectively connect to multiplexer which then connects to a single measurement circuit. Measurements may include electrochemical scans such as voltammetry or amperometry or potentiometry or other suitable methods for detection of the target analyte. 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 staircase potential applied to the working electrode.

[0050] With further reference to FIGS. 3 and 4, in an embodiment of the present invention the multiple working electrodes for the same analyte may also include at least one measurement to determine the depth or surrounding tissue position of one or more working electrodes. Without being able to determine the tissue position, the present invention in some applications may lack an advantage of the prior art which has a pre-determined depth and tissue position. For example, if the at least one measurement was that of electrical impedance between a working electrode and a counter electrode, where the counter electrode for example may be a cardiac gel pad placed on the skin surface (shown in a later figure) or a counter electrode placed inside the body and near each working electrode (not shown) or the electrical impedance between multiple working electrodes where other working electrodes act as the counter electrode, then working electrodes 322 and 422 may have the lowest electrical impedance (especially at lower electrical measurement frequencies) since in the dermis electrodes 322 and 422 are not surrounded by significant cellular content that can increase electrical impedance. For example, tissue that is packed with cells may have a high impedance of 0.01’s of S/m whereas an acellular matrix such as collagen filled with interstitial fluid may have a low impedance of only 0.1’ s of S/m. For example, electrodes 320, 322, 324, 420, 422, 424 could be measured at the same negative or positive potentials of 10’s to 100’s of mV at the same frequencies or frequencies scanned from AC frequencies spanning 0.1 Hz to 100 kHz. Because the dermis is an acellular mix of collagen compared to the adipocyte rich hypodermis and packed cells of the epidermis, dermal electrical conductance can be 10-20X higher than the epidermis and the hypodermis and therefore distinguishable across the electrodes. Therefore, with electrical impedance the present invention can identify which electrodes are reliably inserted at what tissue depths or into what tissue environments. The present invention, by having multiple electrodes placed into the body, has an advantage by allow ratiometric comparisons of impedance levels between electrodes instead of having to rely on absolute quantitative impedance measurements which could be more error prone. For example, if 8 electrodes were spaced at 200 pm spacing on a single support (similar to FIG. 4) and the relative position of the electrodes is known at the time of building the device (which is most always the case), then for any working electrode relative to the other working electrodes, high impedance for the electrodes closest to the skin surface would imply they are in the epidermis, near infinite or infinite impedance could imply electrodes closest to the skin surface are outside of skin, low impedance for electrodes in the middle (neither near skin surface nor near the distal end of the support) would imply those electrodes are in the dermis, and electrodes closest to the distal end of the support if having higher electrical impedance than the electrodes in the dermis would imply they are in the hypodermis.

[0051] In one embodiment, the present invention further includes at least one location measurement, where said location measurement has a distinct measurement response between at least two tissues including the epidermis, dermis, or hypodermis. The at least one location measurement may be an electrical impedance measurement and may be the same type of electrical impedance measurement at a plurality of the distinct locations. The at least one location measurement may be an electrical impedance measurement that has a threshold between a plurality of the distinct locations and said threshold has at least 2X lower electrical impedance when the distinct location is the dermis.

[0052] With further reference to FIGS. 3 and 4, in an embodiment of the present invention the multiple working electrodes for the same analyte may 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 tags on the aptamers can be sampled after 0.1’ s to l’s of ms which is a time point at which much of the electrical capacitive current has dissipated. This electrical capacitive current can be directly leveraged for impedance measurement, by measuring the current magnitude, or change in current vs. time during the first 0.1 ’ s to 1 ’ s of ms for each square wave measurement pulse (e.g from 0.05 to 0.1 ms, or at 0.5 ms, or other examples). At the beginning of the square wave measurement pulse the current is dominated by resistance-capacitance changing times which are directly dependent on the electrical impedance of surrounding fluid or tissue. This measurement can be used not only to determine depth of a working electrode or its surrounding tissue environment, but also the cellular density around the electrode which for example for BNP or insulin can affect concentration measures by 2X or even more due to cellular receptor uptake. If a measure of cellular density is required near a working electrode, then the working electrode(s) can be calibrated at the factory for their impedance in artificial interstitial fluid or serum (0% or minimum cellular density) and for impedance when placed for example into adipose tissue at the factory (100% or maximum cellular density), and then a comparative measure in body during use on a human will provide a quantified measure of the tissue or cellular density adjacent to the working electrode. For example, if it is known that a high cellular density decreases local insulin concentration by 2X compared to blood concentrations of insulin, and electrical impedance of the working electrode confirms during use that the working electrode is surrounded by a high cellular density, than the measured insulin concentration by the working electrode can be multiplied by 2X for the device to report a stronger correlation to blood insulin levels.

[0053] Therefore, in one embodiment the present invention further includes a location measurement that is an electrical impedance measurement, and wherein monitoring of the least one analyte is a current measurement from a first electrical waveform, and wherein the electrical impedance measurement is an electrical measurement from a second electrical waveform. The first electrical waveform and the second electrical waveform may be the same waveform. The first electrical waveform and the second electrical waveform may be a square wave voltammetry waveform.

[0054] With further reference to FIGS. 3 and 4, in an embodiment of the present invention the multiple working electrodes for the same analyte may also include at least one measurement to determine the depth or surrounding tissue position of one or more working electrodes. For example, if the at least one measurement can be a measure of rise time in concentration or concentration measured locally for a working electrode. A high tissue or cellular density can increase measurement lag times (for example, albumin in the hypodermis may rise more slowly in concentration than in the dermis due to increased diffusion resistance to the working electrode) or a high tissue or cellular density can decrease concentration locally for an analyte such as insulin being 2X lower in concentration in adipose tissue in the hypodermis than in the largely acellular collagen matrix of the dermis. The present invention may therefore further comprise at least one measurement to determine the surrounding tissue position of one or more sensors, and wherein the at least one measurement is electrical impedance, and the position location used to correct the measured response for improved correlation for example with blood concentrations, or correlation with dermis concentrations, or correlation with hypodermis concentrations, or correlations with lymph concentrations. For example, if the measurement location was the hypodermis and the analyte was insulin, and the measurement goal is to correlate to blood levels of insulin, then the measurement could be multiplied by 2X to correct for the decrease of insulin in the hypodermis compared to blood. If a device (not shown) were to use optical measurements, an electrical impedance measurement could still be utilized such as pattering an optical waveguide on one side of a support such as a PET or Kapton film, and an impedance measuring electrode on the other side of the support.

[0055] In one embodiment, the present invention further includes a location measurement that is an electrical impedance measurement and that has a pre-associated measure of cellular density at the location. Therefore, the present invention may further include a location measurement, and the location measurement is an electrical impedance measurement that has a pre-associated correction factor for monitoring of the at least one analyte. The at least one location measurement may be selected from at least one of: electrical impedance; rate of measured change of concentration of said at least one analyte; concentration of said at least one analyte.

[0056] With further reference to FIGS. 3 and 4, in an embodiment of the present invention any single working electrode of the present invention may include at least one impedance measurement to the healing evolution of the local tissue adjacent to the working electrode. When a working electrode is inserted into skin, it initially causes inflammation and local tissue damage that can decrease electrical impedance, and over time of hours to days the insertion site heals and electrical impedance may increase.

[0057] With further reference to FIGS. 3 and 4, in an embodiment of the present invention the multiple working electrodes for the same analyte may include at least one working electrode whose measurement is determined to have superior accuracy. For example, if sensing BNP or insulin the measurement preference may be in the dermis 12b and electrodes 322, 422 preferred if the desired measurement is accurate correlation to blood concentrations. Alternately the epidermis or hypodermis may be preferred if the target measurement is delivery to tissue. For example, if the target measurement is delivery of the antibiotic vancomycin to tissue, then a working electrode with an aptamer sensor for vancomycin may represent tissue levels if the working electrode is embedded in the hypodermis. If a plurality of sensors are utilized for the targeted analyte and only one or a subset of sensors is in the preferred tissue or tissues and therefore the sensor(s) then only one or only a subset or one of the sensors can be used to collect and/or report data to the user. With further reference to embodiments of the present invention, the working electrode positions may change and become less or more deep in tissue as the body and device are moved, bumped, or otherwise mechanically disturbed, and the sensors remeasured to determine which sensor is in the preferred tissue or tissues and therefore determine which of the sensor(s) are used to collect and/or report data to the user. The present invention may include only one or a subset of the plurality of sensors that are used to report sensor data.

[0058] The subset of the sensors for the present invention may therefore be further comprised in part of a smaller subset of sensors, and the smaller subset of sensors are capable of all being placed within a skin tissue layer selected from the group consisting of epidermis, dermis, and hypodermis, and the monitoring of the analyte measurement is dominantly associated with the smaller subset of sensors.

[0059] With further reference to FIGS. 3 and 4, in an embodiment of the present invention, other measures may be utilized to determine depth of a working electrode or multiple working electrodes. For example, additional electrodes or sensors beyond electrodes illustrated in the drawings, such as electrical, optical, mechanical, thermal, acoustic, or other sensing modalities may be used to determine depth or local tissue or cellular density for a specific working electrode.

[0060] With further reference to FIGS. 5, where like numerals refer to like features, in an embodiment of the present invention at least one working electrode 520 is adjusted in position by the device 500 such that the working electrode 520 targets the preferred tissue of epidermis 12a, dermis 12b, or hypodermis 12c. This can be achieved for example by first inserting electrode 520 at a maximum depth into hypodermis 12c of for example 6 mm and retracting the electrode 520 by one or more components 570, 580, 590 to a depth of 2 mm for example which would provide placement in the dermis 12b with the 2 mm depth being determined using one or more methods as taught herein such as measurement of electrical impedance differences between tissue layers. For example, element 590 could be epoxy rigidly holding wire 560 to housing 510, element 580 a heater, and element 570 a heater and volume of metal solder or wax. Heater 570 and solder or wax could be melted and element 580 could heat a wire 560 such as shape-memory allow nitinol causing a shape change in the wire to retract wire 560 until reaching a decreased impedance for electrode 520 expected from being in the dermis 12b, and then heater 570 would first cooled to solidify metal solder or wax to hold wire 560 at position after which heater 580 would be cooled. This example is taught because all the components can be simple and low-cost components mounted onto a printed circuit board that is already part of the device construction. In an alternate embodiment, element 570 could be a motor or micro-linear actuator for which there are many industrial examples commercially available, and wire 560 adjusted in either direction (deeper or less deep) to target electrode 520 in the preferred tissue. Sensor positioning can be done after application of the device 500 to skin or later if sensor positioning moves out of preferred position due to ambulation or other action or mechanical disturbance by the user. Sensor positioning may be only less deep, or only more deep, or may be both less or more deep (two way depth control). There are numerous methods of linear actuation suitable for operation in FIG. 5 and the present invention is not dependent on the method of actuation but more importantly on depth determination and selection into various tissues as taught herein. Therefore FIG. 5 teaches another example where the electrode depth or tissue location is not predetermined. The present invention may therefore include at least one component to adjust penetration depth of at least one sensor in the skin. The least one component may be capable of unidirectional adjustment of penetration depth into the skin. The least one component may be capable of bidirectional adjustment of penetration depth into the skin. The least one component may be only capable of retractable adjustment of penetration depth into the skin. The least one component may be thermally activated. The least one component may be a linear actuator. The least one component may be active when the device is first applied to the skin. The least one component may be active when necessary to maintain position of at least one sensor at depth. The at least one component can be actuated bidirectionally (deeper or shallower into skin) until a concentration maximum is found for at least one sensor, indicating an optimal site for sensing, or alternately until a minimum concentration is found.

[0061] With further reference to embodiments of the present invention, two or more working electrodes in separate tissue layers can together provide superior measurements compared to a single electrode alone. For example, a working electrode in the hypodermis could report steady state concentrations of an analyte that is a drug that must penetrate fatty tissue or into brain matter, while a working electrode in the dermis could more closely reflect blood concentrations. An analyte example could be vancomycin which is used to treat both blood infection and to treat the original infection originating from tissue (the antibiotic must penetrate both sites of infection without reaching toxic levels). Therefore, the present invention may include embodiments where the sensor is placed at the non-predetermined depth into at least two of the epidermis, dermis, or hypodermis. Like other potential analytes such as peptides, Vancomycin is than 1 kDa in molecular weight such that the concentrations or time profiles of changing concentrations may differ between the dermis and hypodermis, and therefore the present invention may in some specific embodiments be limited to analytes >1 kDa in molecular weight.

Working Electrode Fabrication and Operation

[0062] With reference to FIG. 6 and FIG. 7 where like numerals refer to like features, for embodiments of the present invention additional non-limiting examples and details are provided on working electrode construction and operation. The working electrode 620 is comprised of an electrode material 658 such as gold. The gold is then incubated with aptamers 650 via thiol attachment to the gold electrode, and the aptamer includes a redox tag such as methylene blue 652. In between the aptamers the gold is further incubated with a protective monolayer 656 such as mercaptohexanol, mercaptoocotanol, or other suitable chemistry. Not shown, a protective membrane such as polybetaine or other suitable material may be added to prevent fouling of the monolayer surface. The working electrode 620 may be preserved in a preservative such as trehalose to enable dry storage. In a non-limiting but specific example, binding of aptamer 650 to target 654 causes a shape confirmation change which brings the redox tag 652 closer to the electrode 658 resulting in increased electron transfer (increased electrical current). As concentration of target 654 increases, more binding of target 654 to aptamer 650 occurs, and more electron transfer occurs (more measurable electrical current). As concentration of target 654 decreases, conversely electrical current decreases. There are numerous other potential methods of analyte binding to aptamers and resulting changes in electron transfer with an electrode and the embodiments of the present invention are therefore not limited to the specific simple example illustrated and discussed for FIG. 6. Devices as taught herein can insert the supports carrying the working electrodes into skin using one or methods such as those commonly deployed for the insertion of glucose sensors needles for continuous glucose monitors (such as a slotted insertion guide or other methods). An example of device fabrication and testing is as follows:

Materials

[0063] Sulfuric acid (96%, p.a.), Sodium hydroxide (98%, pellets), Pulverized phosphate buffered saline (PBS, pH 7.4), Tris-EDTA solution (TE buffer; pH: 8), Bovine Serum, tris(2- carboxy ethyl) phosphine hydrochloride (TCEP; 98%), sodium azide (99.5%), l,6-d6- mercapto-1 -hexanol (MCH; 98%) and 8-mercapto-l -octanol (MCO; 97%) were obtained from Sigma Aldrich (USA). 2-hydroxy-2methylpropiophenone (photo-initiator, 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 (cross-linker, purity: >98%) were obtained from Alfa Aesar. The 3 ’-methylene blue and 5 ’-thiol modified oligonucleotide sequences were synthetized by Integrated DNA Technologies (IDT, USA).

[0064] Example aptamer sequences are as follows:

Sensor preparation

[0065] Gold electrodes with a titanium adhesion layer are deposited on PET or Kapton strips that provide a support for the electrodes such as support 460 in Fig. 4. The gold can be patterned via photolithography and chemical etching, and electrical insulators may also be applied that are photo-defineable or screen-printable, as will be detailed later in FIG. 10. The gold can be further electroplated with additional gold and electrochemically roughened or cleaned prior to aptamer 650 and protective layer 656 incubation. Electrochemical cleaning can be performed in a standard three-electrode electrochemical cell consisting of a gold working electrode, platinum counter electrode, and Ag/AgCl reference electrode by running 700 cyclic voltammetry scans in 0.5 M NaOH from -1 V to -1.6 V at a scan rate of 1 V/s and subsequently 150 scans in 0.5 M H2SO4 solution from 0V to 1.6 V at 1 V/s. Once the electrochemical cleaning is complete, electrodes can be thoroughly rinsed with DI water, dried in a nitrogen stream (99.999% purity), and used for subsequent incubation. A lyophilized pellet of modified aptamer can be diluted down to a 100 pM stock solution using TE buffer and kept at -20 °C until use. Preparation of aptamer working solution can be performed by first mixing an aliquot of the 100 pM aptamer stock solution with equal volume of 0.5 M TCEP dissolved in Milli-Q water. The mixture can then be set aside for Ihr to ensure complete reduction of any disulfide aptamer molecules. The obtained solution can then be diluted to an intermediary concentration of ~4 uM with lx PBS/2 mM MgCh buffer and the concentration confirmed via the absorbance measured at 260 nm using a Nanodrop UV/Vis Spectrophotometer. This solution can then be subsequently diluted to 500 nM with lx PBS/2 mM MgCh buffer for incubation of aptamer onto for one 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 lx PBS. The functionalized sensors can then be rinsed with DI water prior to coating with trehalose for storage and ultimately then used for measurement. If electrochemical roughening and hydrogel protection is desired, prior to aptamer and MCH or MCO incubation, electrodes can be immersed in 5 M NaOH solution and subject to 20 ms long alternating potential steps of -5 V and +0.8 V (vs. Hg/Hg2SO4, sat. Na2SO4) respectively, for a total duration of 6000s in an electrochemical cell consisting of a Kapton®-carbon counter and saturated Hg/Hg2SO4 reference electrode. Once the roughening is completed, the electrodes can be rinsed with copious amounts of DI water and aptamer and MCH or MCO then incubated as described above. Modification of the sensors with an antibiofouling zwitterionic polybetaine-based hydrogel can be performed by drop-casting 1 pL of the aqueous mixture consisting of monomer/cross-linker/photo-initiator (2.8g/1.8pl/36pl respectively dissolved in 1 ml of DI water) over the sensor and exposing it to UV light (X: 280- 450 nm, Bluewave LEDPrime UVA, Dynamax, USA) for 45 min.

Electrochemical measurements

[0066] Electrochemical measurements can be performed with a miniaturized potentiostat (details in FIG. 9) or performed by a benchtop CHI 620E potentiostat (Austin, Texas) connected to a 64-channel multiplexer in a standard three-electrode system with aptamer/alkylthiolate functionalized electrodes serving as working electrodes. The counter and reference electrodes can be inserted into the skin using a platinum counter electrode, and a Ag/AgCl reference electrode, or alternately the counter and reference electrodes can be a large gel-electrode-pad electrode on the surface of the skin as taught in PCT/US21/51972 - ‘APTAMER SENSORS WITH REFERENCE AND COUNTER VOLTAGE CONTROL’. Cyclic voltammograms can be recorded in a window from -0.1 V to -0.5 V at a scan rate of 100 mV/s. Square-wave voltammetry can be performed in a potential window from -0.1 V to -0.5 V at 25 mV amplitude at the optimal frequency of measurement for each aptamer. Kinetic differential measurements, two frequency measurement, or continuous square wave voltammetry may be utilized to improve calibration-free operation of the sensors. Example in- vivo data collected for a cortisol sensor inserted subcutaneously in a rat is shown in FIG. 7, where injections of cortisol are performed at 5 orlO mg/kg.

Additional Targets

[0067] The present invention applies generally to aptamer sensors and other types of affinity biosensors and is therefore not limited to specific examples as taught herein. Available electrochemical sensors for analytes such as cortisol, vancomycin, phenylalanine, insulin, BNP, NT -proBNP, IL-6, C-peptide, C-Reactive protein, and sensors for other analyte targets may be incorporated in the present invention without limitation. Analytes may be endogeneous or exogenous to the body, without limitation.

System Overview

[0068] With a basic understanding of the above-described sensors in place, reference is now drawn to FIG. 8, which illustrates a general diagram of a computer system 800 according to various aspects of the present disclosure. Like numerals in FIG. 8 do not necessarily refer to like features like that in the other figures. The computer system 800 comprises a plurality of hardware processing devices (designated generally by the reference 802) that are linked together by one or more network(s) (designated generally by the reference 804).

[0069] The network(s) 804 provides communications links between the various processing devices 802 and may be supported by networking components 806 that interconnect the processing devices 802, including for example, routers, hubs, firewalls, network interfaces, wired or wireless communications links and corresponding interconnections, cellular stations and corresponding cellular conversion technologies (e.g., to convert between cellular and TCP/IP, etc.). Moreover, the network(s) 804 may comprise connections using one or more intranets, extranets, local area networks (LAN), wide area networks (WAN), wireless networks (Wi-Fi), the Internet, including the world wide web, cellular and/or other arrangements for enabling communication between the processing devices 802, in either real time or otherwise (e.g., via time shifting, batch processing, etc.).

[0070] A processing device 802 can be any device capable of communicating with another processing device 802, e.g., via Bluetooth, Ultrawide band, near field communication (NFC), via one or more radio frequencies (RF) or via any other form of wired or wireless communication, over the network 804, or combinations thereof.

[0071] Some examples of processing devices 802 are cellular devices (including cellular mobile telephones (i.e., smartphones)), tablet computers, netbook computers, notebook computers, personal computers, servers, cloud devices, edge devices, etc.

[0072] Also, in certain contexts and roles, a processing device 802 is intended to be a wearable monitoring device. Examples of a wearable monitoring device include a purpose- driven appliance, Internet of Things (loT) device, special purpose device, etc. A processing device 802 implemented as a wearable monitoring device is schematically illustrated in FIG. 8 as a wearable device mounted to a patient’s arm solely for convenience of illustration. In practical applications, the wearable monitoring device can attach to other parts of a patient’s body.

[0073] In some embodiments, the wearable monitoring device can communicate locally (e.g., to a smart phone) via Bluetooth, ultrawide band, 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 communicate locally to another processing device 802.

[0074] The illustrative computer system 800 also includes a processing device implemented as a server 812 (e.g., a web server, file server, and/or other processing device) that supports an analysis engine 814 and corresponding data sources (collectively identified as data sources 816). The analysis engine 814 and data sources 816 provide the resources to implement and store data related to collecting and aggregating data from wearable monitoring devices, captured events, combinations thereof, etc., as described in greater detail herein.

[0075] In an exemplary implementation, the data sources 816 are implemented by a collection of databases that store various types of information. Solely by way of example, the data sources 816 can include device data 818, e.g., data related to wearable monitoring devices, including configuration data, version data, software versioning and control, data generated from wearing a wearable monitoring device, etc. The data sources 816 can also include medical data 820, e.g., medical research, etc., used to calibrate, tune, design, modify, etc., wearable monitoring devices. The data sources 816 can also optionally include user data, e.g., data regarding the patients that are wearing the wearable monitoring devices, where such data is collected. As yet further examples, the data sources 816 can include platform data 824, e.g., data used by the analysis engine 814, e.g., computer drivers, GUI information, algorithms for processing physiological conditions, etc. As yet a further example, the data sources 816 can optionally include miscellaneous data 826, e.g., any data needed by the analysis engine 814 that is not otherwise accounted for above.

[0076] Considering FIG. 8 as an environment used by wearable monitoring devices, in some embodiments, the processing of physiological data of a corresponding patient wearing the wearable monitoring device (e.g., biochemical sensing with additional sensing modalities that enhance patient care or health and wellness) can be carried out entirely on a processing device 802 (such as a wearable monitoring device itself); on a processing device 802 such as a smartphone, by the analysis engine 814, or via combinations thereof (e.g., by distributing processing tasks among two or more processing devices). [0077] With specific regard to a processing device 802 implemented as a wearable monitoring device (see processing device 802 schematically attached to a patient’s arm), it may be desirable to carry out all of the processing on the wearable monitoring device itself. In this regard, a corresponding device such as a smartphone can optionally provide a graphical user interface for displaying dashboard measurement results, but all processing is carried out on the wearable monitoring device itself.

[0078] In other embodiments, the smart phone can carry out some processing, e.g., to compare computed data to dashboard thresholds, to carry out algorithms, rules, or other processing, as described more fully herein.

[0079] In still other embodiments, the analysis engine 814 can collect data from each wearable monitoring device, e.g., for trend analysis of patient data, for device state of health monitoring (e.g., to detect faults in the wearable devices themselves), for battery charge level monitoring, for versioning (such as to carry out software updates), etc.

[0080] In some embodiments, the analysis engine 814 is controlled by a third party, e.g., the manufacturer of the wearable monitoring devices that are implemented in the environment. [0081] In some embodiments, the analysis engine 814 schematically represents integration into an electronic health record system, e.g., to connect a patient to the patient’s doctor so that the doctor can access the electronic data generated by a corresponding wearable monitoring device.

Example Monitoring Device

[0082] Referring now to FIG. 9, an example wearable monitoring device 900 is schematically illustrated, according to aspects of the present disclosure. Like numerals in FIG. 9 not necessarily refer to like features like that in the other figures. The wearable monitoring device 900 can represent an example embodiment of a processing device 802 (FIG. 8), e.g., a wearable monitoring device as previously described.

[0083] The wearable monitoring device 900 includes a housing 910 that attaches to a patient. The housing can attach to the patient via an adhesive 904, a strap, or other securement. [0084] The wearable monitoring device 900 also includes at least a first working electrode 920 and may include a second working electrode 922 and further may include a third working electrode 924 or even more working electrodes. In some embodiments, one or more electrodes include an analyte detecting material, e.g., aptamers, such that continuous sensing can be carried out. Electrode 950, as previously described may be a gel electrode pad and serve the roles of a reference and counter electrode. [0085] In practical applications, the housing 910 is couplable to the electrodes 920, 922, 924. As used herein, "couplable" is to be construed broadly to mean any one of permanently coupled, detachably coupled, adjustable, temporarily coupled, user attachable, user detachable, user attachable and detachable, factory attachable, factory detachable, user attachable, factory attachable and detachable, or any combination thereof, unless specifically noted otherwise.

[0086] As illustrated, the housing 910 includes a potentiostat 991 that is communicably coupled to the electrodes 920, 922, 924 (or a combination thereof) using an optional multiplexer 990, or alternatively each of electrodes 920, 922, 924 can receive a direct dedicated connection to a potentiostat 991. In practical applications, the term “potentiostat” is to be interpreted broadly, and is not limited to any particular number of sensors. For instance, the potentiostat can be implemented as a bipotentiostat, polypotentiostat, etc., depending upon the sensor configuration provided by the wearable monitoring device 900.

[0087] Additionally, wearable monitoring device 900 includes a controller 993 that is communicably coupled to memory 992. The controller 993 is also communicably coupled to a communication interface 994.

[0088] The controller 993 includes necessary electronics that enable the controller 993 to carry out the intended functionality of the wearable monitoring device. For instance, the controller 993 can include a processor, bus interface, ports, registers, memory, etc., that enables the wearable monitoring device 900 to carry out the functionality described more fully herein. [0089] Also, as illustrated, the controller 992 is communicably coupled to one or more of the optional multiplexer 990, potentiostat 991, the memory 992, the transceiver s) 994, optional miscellaneous sensors 995, optional display/output 996, combinations thereof, etc.

[0090] The communication interface 994 may comprise, for example, at least one transceiver that communicates via Bluetooth, Wi-Fi, Ultrawideband, near field communication, combinations thereof, etc.

[0091] The optional display/output 996 can comprise a display screen, a dimensionally limited display screen, a touch screen, a haptic output, a light output, a speaker/alarm, or combinations thereof.

[0092] The controller 993 uses the potentiostat 991 to collect measurements from electrodes 920a, 920b, 920c, and stores the collected measurements in the memory 992. The controller 993 may further provide filtering, analysis, control, authorization, authentication, and other controller specific functions. The communication interface 994 facilitates coupling 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 can include one or more modalities, each with different data and/or authorizations. For instance, a patient may access data from the wearable monitoring device 900 on a smartphone 1006 as illustrated in FIG. 10, whereas a doctor may be able to access more detailed information from a cloud server and/or through electronic health records (see FIG. 9). In this regard, multiple modalities of communication may be utilized with wearable monitoring device 900.

[0093] In some embodiments, the adhesive 904 of the wearable heart 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, a gel electrode 950 could be the counter or reference electrode for the electrodes 920, 922, 924.

Working Electrode Configurations

[0094] With reference to FIG. 10, where like numerals refer to like features with respect to FIGS. 1-6, a more detailed description of one example physical layout of a plurality of working electrodes is illustrated. FIG. 10 shows that showing side-by-side positioning of electrodes can achieve the principles of the present invention. Support 1060 can be constructed for example of 10’s to 100’s pm of PET or Kapton or an electrically insulated metal foil. Exemplary supports 1060 may have similar dimensions to glucose monitor strips which for example can be 400 pm wide and 5 mm long. Support carries 10’s to 100’s to 1000’s of nm thick working electrode films such as 1020 and 1022 which for example may be a layer of titanium or chromium adhesion promoting layers followed by a coating of gold. These layers are then further coated by an electrical insulator 1055 than can be on the scale of nm’s to even >100 pm thick, such as screen printable dielectric pastes used for fabricating glucose sensors, photodefinable insulators such as Microchem SU-8, HD Microsystems PI2611, and Fujifilm Durimide 7020, other photo-defineable polymer coatings, adhesive tapes, patterned Parylene coatings, or other suitable film-based insulators. The remaining areas of exposed working electrodes 1020, 1022, can then be incubated with sensing chemistry and coated with other materials as needed, as taught herein. FIG. 10 shows an example of two depth-staggered working electrodes 1020, 1022, where electrode 1020 would have the deepest penetration into skin. This example taught for example 10 could use alternate patterning methods, alternate electrode geometries, alternate number of a plurality of working electrodes, and is not a limiting example of all the possible embodiments of the present invention. For example, films of PET or Kapton could also carry optical waveguides for optical sensing of molecular beacon aptamers bound to the waveguide surface, where instead of an electrical insulator was used as shown in FIG. 10 an optical cladding on a waveguide would serve a similar purpose and function to limit the area of exposure of an optical based sensor to a particular depth into skin. [0095] With reference to FIG. 11, where like numerals refer to like features with respect to FIGS. 1-6 and 10, a more detailed description of one example physical layout of a plurality of working electrodes is illustrated. FIG. 11 shows that layering of electrodes can also achieve the principles of the present invention. FIG. 11 simply shows a side-cross section of layers of solid support 1160, working electrode 1120, insulator 1555, working electrode 1122, and insulator 1155. Together, FIG. 10 and 11 illustrate that multiple methods for creating a plurality of working electrodes are possible, further including methods not specifically shown here such as electrode vias, coaxial wires and conductors, and other techniques as known in the fields of microelectronics, printed circuit boards, and other similar technologies.

Working Electrode Blood Access At a Depth That is Not Predetermined

With further reference to embodiments of the present invention, the present invention may also be applied to continuous blood measurement. Continuous blood measurement has not been strongly pursued because of difficulty in sensor placement in a vein or artery compared to simpler hypodermis placement. Continuous blood measurement may have advantages for example for vancomycin measurement during a blood infection, for narrow-therapeutic range drugs for which blood and/or tissue concentrations would provide a clear picture of pharmacokinetics, or for example large sized analytes that are so large (some antibodies, some proteins) that they have difficulty partitioning from blood to interstitial fluid. The motivation for blood measurement further includes cases where it will be more diagnostically accurate (for example, skin inflammation can skew inflammatory markers by generating them locally such that they do not represent blood concentrations). While methods exist for long-term blood intravenous line access, such as port-a-cath, peripherally inserted central catheters or a central line, they are all more invasive and typically require trained medical personnel for placement. A key aspect of the present invention when it is applied to continuous blood monitoring is that the device can be reliably inserted into blood by the user themselves without need for trained medical personel to do so. With reference to FIGS. 12, 13, 14, where like numerals refer to like procedures, the present invention can enable sensor placement in a blood arteries and veins, again like previous embodiments in a matter that is not predetermined in depth, because even arteriols and venioles close to the skin are only 30 pm in diameter which would make reliable sensor placement highly challenging if not impossible. In contrast, or more methods such as manual visual inspection, ultrasound, portable near-infrared vein finders can identify and enable insertion targeting of superficial arteries or veins such as the cephalic vein, cubital vein, basilic vein, or other veins or arteries. One of a plurality of working electrodes such as 1220, 1222, 1224 can then penetrate the vein 13 or and artery and remain in it allowing accurate blood access. While the present invention enables reliable insertion of a working electrode into a vein or artery, the greater remaining challenge is likely finding the vein or artery and aligning to the target vein or artery the working electrode inserter such as those used on continuous glucose meters. In a more detailed example, FIG. 13 shows a wearable sensor 1300 with an aperture for sensor insertion into skin 12 which is similar to that used with Abbott’s Libre 2 or Libre 3 glucose monitors. This same aperture can be used as a simple view port for a camera or for a red light source such as an LED or laser and as the device 1300 is moved horizontally over skin 12 a decrease in red light reflectance measured with a photodiode or spectrometer as shown in FIG. 14 indicates the presence of a vein due to deoxygenated hemoglobin. As a result, a more automated example of horizontal positioning over a vein is provided, after which the sensor can be inserted into the skin using one or more methods of insertion such as those used for continuous glucose monitor sensor insertion (for example Funderburk et al., US 7,381,184 B2). The method of alignment of a working electrode over an artery or vein is not a limiting factor for embodiments of the present invention for use with continuous blood monitoring, but the embodiments as taught herein for the ability to determine what tissue or fluid the sensor is in are critical. The ability to have multiple redundant working electrodes for the same analyte, and/or the ability to modulate the depth of insertion for a working electrode after application of the device to skin are also critical. In addition to previously described methods to identify that a working electrode is in a particular tissue location, blood measurement may also be differentiated between a working electrode in blood vs. a working electrode in tissue using as non-limiting examples a highest concentration in blood vs. tissue, a faster initial increase in concentration in blood vs. tissue, pulsatile pressure effects such as red-blood cells and turbulent flow on an exposed working electrode surface, or other suitable measures.

[0096] Embodiments of the present invention may therefore apply to direct sensing in blood, and the present invention may therefore include at least one of the plurality of sensors is a first sensor and at least of the plurality of sensors is a second sensor and at least one sensor is placed into blood at a depth that is not predetermined.

[0097] The present invention may therefore further include at least one component used to horizontally locate the position of a vein, artery, or capillary. [0098] Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

Claims

WHAT IS CLAIMED IS:

1. A wearable device for continuous monitoring of at least one analyte comprising: a. a plurality of aptamer sensors for a specific analyte, said aptamer sensors carried by a support; wherein at least a subset of said plurality of sensors are each capable of being placed in a distinct location with respect to depth into a user’s skin; b. a means to determine at least one location measurement; wherein said location measurement has a distinct measurement response between at least two skin tissues selected from the group consisting of epidermis, dermis, and hypodermis; and further, wherein at least one of the sensors is capable of being placed at a distinct location that is selected from one of the group consisting of epidermis, dermis, and hypodermis tissue.

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

3. The device of claim 1 wherein said at least one location measurement is an electrical impedance measurement.

4. The device of claim 3 wherein sensors are capable of being placed in a plurality of distinct locations and the same type of electrical impedance measurement is measured at a plurality of the distinct locations.

5. The device of claim 3 wherein said electrical impedance measurement has a threshold between a plurality of distinct locations and said threshold has at least 2X lower electrical impedance when the distinct location is the dermis.

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

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

8. The device of claim 6 wherein the first electrical waveform and the second electrical waveform are a square wave voltammetry waveform.

9. The device of claim 1 wherein said location measurement is an electrical impedance measurement that has a pre-associated measure of cellular density at the distinct location.

10. The device of claim 1 wherein said location measurement is an electrical impedance measurement that has a pre-associated correction factor for monitoring of the at least one analyte.

11. The device of claim 1 wherein said subset of the plurality of sensors is further comprised in part of a smaller subset of sensors, and the smaller subset of sensors are capable of all being placed within a skin tissue layer selected from the group consisting of epidermis, dermis, and hypodermis, and the monitoring of the analyte measurement is dominantly associated with the smaller subset of sensors.

12. The device of claim 1 wherein the at least one analyte is greater than 1 kDa in molecular weight.

13. The device of claim 1 wherein the distinct locations further include a separation distance between the distinct locations and the separation distance is a value from about 0.05 mm to about 5 mm.

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

15. The device of claim 1 wherein said support is plurality of distinct supports and at least a portion of said plurality of sensors are distributed across a plurality of distinct supports.

16. The device of claim 1, where said distinct location further comprises a blood vessel selected from the group consisting of a vein, artery, and capillary.

17. The device of claim 16, further comprising at least one component used to locate beneath the user’s skin a horizontal position of a vein, artery, or capillary.

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

19. The device of claim 18 wherein the location measurement is an electrical impedance measurement.

20. The device of claim 18 wherein the location measurement is a concentration measurement of said analyte.

21. The device of claim 18 wherein said electrical impedance measurement has a preassociated correction factor for the monitoring of the at least one analyte.

22. The device of claim 18 wherein the at least one analyte is greater than 1 kDa in molecular weight.

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

24. A method for using a wearable device to continuously monitor at least one analyte in a distinct location in a subject’s skin comprising; placing at least one aptamer sensor for the analyte in a distinct location in a subject’s skin, wherein the at least one aptamer sensor is in communication with the wearable device; performing at least one location measurement for the at least one aptamer sensor; performing an action selected from the group consisting of: a first action of adjusting sensor position into skin and therefore the distinct location such that it is the epidermis, dermis, or hypodermis, or blood in a vein or venous capillary; from a plurality of sensors for the same analyte, monitoring of the at least one analyte measurement being dominated by a subset of the plurality of sensors that may be as a small as one sensor; and a combination of both actions.

25. The method of claim 24 further comprising performing at least one location measurement to determine said distinct location of at least one aptamer sensor.

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

27. The method of claim 25 wherein said at least one location measurement is performed via the same type of electrical impedance measurement at a plurality of the distinct locations.

28. The method of claim 27 wherein said electrical impedance measurement has a threshold between a plurality of the distinct locations and said threshold has at least 2X lower electrical impedance when the location is in the dermis.

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

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

31. The method of claim 30 wherein the first electrical waveform and the second electrical waveform are a square wave voltammetry waveform.

32. The method of claim 25 wherein said location measurement is an electrical impedance measurement that has a pre-associated measure of cellular density at the location.

33. The method of claim 25 wherein said location measurement is an electrical impedance measurement that has a pre-associated correction factor for monitoring of the at least one analyte measurement.

34. The method of claim 25 wherein for said plurality of sensors for measuring the same analyte there is a subset of the plurality of sensors, and the smaller subset of sensors are all placed in a skin tissue layer selected from the group consisting of the epidermis, dermis, and hypodermis, and the at least one analyte measurement is only associated with the smaller subset of sensors.

35. The method of claim 1 wherein the at least one analyte is greater than 1 kDa in molecular weight.

36. The method of claim 24 wherein the distinct locations further comprise a separation distance between the distinct locations and the separation distance is a value from about 0.05 mm to about 5 mm.

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

38. The method of claim 24 wherein at least a portion of said plurality of sensors are distributed across a plurality of distinct supports.

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