Device and method for monitoring based on aptamer-assisted microneedles for biomarkers

Abstract

[0003] Methods, apparatus, systems, and methods are described for microneedle-assisted aptamer-based electrochemical sensing for label-free, continuous, real-time monitoring of biomarkers in biological fluids. One example of a device for electrochemical monitoring of one or more analytes in a biological fluid includes a substrate and at least two microneedles coupled to the substrate. Each microneedle in the at least two microneedles includes a protruding needle structure and an electrode probe structure. The electrode probe structure of a first microneedle in the at least two microneedles includes an aptamer sequence specific to a first analyte, and the electrode probe structure of the first microneedle is operable as a working electrode for detection of the first analyte using a first electrochemical detection technique.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on Provisional Application No. 62 / 947,399, entitled "DEVICES AND METHODS FOR APTAMER - ASSISTED MICRONEEDLE - BASED MONITORING OF PROTEIN BIOMARKERS", filed on December 12, 2019. The entire content of the above - mentioned provisional application is incorporated by reference as part of the disclosure of this document.

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

Background Art

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

Summary of the Invention

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

[0005] One aspect of the disclosed technology relates to a device that includes a substrate. The device also includes at least two micro-needles coupled to the substrate. Each micro-needle of the at least two micro-needles includes a protruding needle structure and an electrode probe structure. The protruding needle structure includes an outer wall that extends outward from the surface of the substrate, the outer wall enclosing an internal volume of the protruding needle structure and forming a tip at the end of the outer wall. The electrode probe structure is configured to generate a signal in response to one or more chemical or biological substances in the biological fluid that contacts the electrode probe structure. The electrode probe structure of the first micro-needle of the at least two micro-needles includes an aptamer sequence specific to a first analyte, and the electrode probe structure of the first micro-needle is operable as a working electrode for the detection of the first analyte using a first electrochemical detection technique. The electrode probe structure of the second micro-needle of the at least two micro-needles is operable as an electrochemical counter electrode or an electrochemical reference electrode.

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

Brief Description of the Drawings

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

[0009] The analysis is performed at an aptamer-modified electrode, which is in the form of a micro-needle (e.g., a polymeric hollow micro-needle) and acts as an electrochemical sensor. The aptamer functionalized with a redox reporter molecule (e.g., methylene blue or anthraquinone) binds specifically and reversibly to the analyte of interest, causing folding of the three-dimensional structure of the aptamer bound to the electrode. This folding induced by the binding corresponds to the concentration of the target biomarker and results in a change in the electron transfer properties of the redox molecule, which is detected using electrochemical techniques, such as square wave voltammetry (SWV).

[0010] This document describes the application of the disclosed technology to the real-time aptamer-assisted measurement of biomarkers for cortisol and insulin as model analytes, but the technology disclosed in this application can be easily extended to the continuous monitoring of any biomarker, such as hormones and proteins of interest, by binding to a biorecognition element based on their related aptamers.

[0011] The in vivo stable electrochemical detection of the target analyte present in the ISF can be achieved by the disclosed technology without surface attachment by using an effective anti-biofouling self-assembled monolayer on the surface of the micro-needle electrode or hydrogel coating. Furthermore, the aptamer can be rationally manipulated in vitro to perform the detection of the analyte in the desired concentration range.

[0012] Different from micro-needle-based enzyme sensing, the disclosed technology extends the capabilities of micro-needle sensors to broaden the range of biomarkers that can be detected. The sensing of such biomarkers usually relies on immunoassays that are not always feasible to perform with micro-needle chips.

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

[0014] Currently, there are no available devices for minimally invasive or non-invasive continuous monitoring of various biomarkers, and the only way available to measure biomarkers is based on the traditional method of quantification using external measuring instruments / techniques in a centralized laboratory following blood sampling with a hypodermic needle (mostly using the very time-consuming and expensive ELISA method). Such methods have significant disadvantages including patient discomfort, the need for a relatively large sample volume, and the generation of sharp waste. More importantly, they cannot track the trends and fluctuations in biomarker levels and thus cannot provide real-time information regarding biomarker concentrations.

[0015] Currently, there is no feasible technology for providing real-time on-body information regarding the concentrations of different biomarkers, and the development of wearable multi-sensors that can simultaneously and continuously monitor the dynamically changing levels of different disease markers would be highly important for disease diagnosis, prognosis, and treatment.

[0016] There are many examples of sensors for detecting physiologically relevant biomarkers in vitro, but there are still no examples of biosensors for in vivo detection of ISF biomarkers. Wearable on-body biosensing has the potential to bring about a major revolution in health management by enabling personalized medicine. By providing clinically relevant health information in a continuous basis using wearable non-invasive sensors, it is possible to fully understand an individual's health status and provide a platform for disease diagnosis, prognosis, and management. This document shows examples of wearable on-body sensor devices for continuous real-time monitoring of various biomarkers that eliminate the cost and complexity of traditional immunoassay-based approaches. Examples of microneedle devices based on the technology disclosed in this application can be used for multiplex monitoring of different biomarkers, such as cortisol, insulin, ketone bodies, and glucose for diabetic patients. Such devices can be in the form of fully integrated wearable microneedle patches that can be used for self-monitoring and / or by providers of health management. The aptamer-based microneedle sensing protocol described herein can be easily extended to the detection of other biomarker targets. This represents a leap forward in the fields of disease diagnosis and personalized health management.

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

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

[0019] The application of microneedles for diagnostic and monitoring purposes has received much attention over the past decade due to the rich molecular information contained in interstitial fluid. However, the direct sensing of ISF markers with microneedle chips is typically performed using enzyme-modified microneedle electrode transducers. These rely on the reversible biocatalytic reaction of the corresponding substrate.

[0020] In contrast, the implementation of conventional bioaffinity assays, such as immunoassays, with microneedle chips faces obstacles due to the strong interaction between the target antigen and the capture antibody, which, along with the need for several washing and incubation steps, makes the regeneration of antibody receptors extremely difficult. Such assays often also require additional incubation steps with related tagging reagents to perform competitive or sandwich assays, which may not always be feasible to perform on microneedle chips.

[0021] The only reports on antibody-based biomarker analysis with microneedle assistance are based on sampling of ISF containing biomarkers, or selective capture of specific biomarkers by their binding to functionalized antibodies on the surface of microneedles, followed by off-body analysis procedures. These reports do not have the ability to adapt to direct continuous monitoring of biomarkers with microneedle chips. As a result, the use of microneedle ISF sensors has not been reported for in vivo biocompatibility monitoring of ISF biomarkers, and the realization of in situ real-time biocompatibility assays in microneedle chips for ISF monitoring of various markers remains an important issue.

[0022] This document describes the realization of real-time biocompatibility assays in microneedle chip electrodes by using reversibly binding conformation-dependent aptamers.

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

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

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

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

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

[0028] In examples of various embodiments, the electrode probe structure is disposed (fully or partially), for example, inside or within the cavity of each micro-needle structure. In examples of other embodiments, the electrode probe structure is incorporated into or attached to each micro-needle structure and is located (fully or partially) inside or outside the cavity of each micro-needle structure. In examples of still other embodiments, the electrode probe structure is incorporated into or attached to each micro-needle structure, and each micro-needle structure does not have the mentioned internal or cavity. FIG. 1 shows the electrode probe structures 117, 127, and 137 of the micro-needle electrodes 110, 120, and 130, respectively. For example, the electrode probe structure of the electrodes of the micro-needle sensor device 100 can include a wire or film of silver (Ag), gold (Au), or platinum (Pt), and / or carbon paste (CP).

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

[0030] In some embodiments, the height of the microneedle structure (e.g., from the base to the top) can be in the range of 1 mm to 2 mm, for example, preferably about 1.5 mm. In some embodiments, the diameter or width of the microneedle structure can be 200 μm to 500 μm. The microneedle structures can be spaced at various intervals and arrangements, which can be selected based on the application of the microneedle sensor device 100. In some embodiments, the microneedle structures are arranged with the bases tens or hundreds of microns apart from each other, for example, in some embodiments, the microneedle structures are arranged with the tops about 1 mm apart from each other. In some embodiments, the microneedle structures are arranged on the substrate 105 in a linear array, but in some embodiments, the microneedle structures are arranged on the substrate 105 in a circular array, a rectangular array, a triangular array, or any other pattern or non-pattern arrangement.

[0031] The microneedle structure and the electrode can be configured in other arrangements, such as those described in U.S. Patent No. 9,737,247, which is hereby incorporated by reference in its entirety as part of the disclosure of this application.

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

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

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

[0035] An aptamer functionalized with a redox reporter molecule (e.g., methylene blue or anthraquinone) can specifically and reversibly bind to a target compound, and folding of the three-dimensional structure of the aptamer bound to the electrode occurs. This binding-induced folding corresponds to the concentration of the target compound of interest and can result in a change in the electron transfer properties of the redox molecule, which is detected using an electrochemical detection technique, such as square wave voltammetry (SWV), performed on the aptamer-functionalized electrode of the sensor device 100. Multiplex detection of different biomarkers can be achieved by labeling the aptamer with different redox reporters having different redox properties or by spatially localized measurement of the current signal of a single redox tag.

[0036] Electrochemical detection modes or measurement techniques other than square wave voltammetry can be used for the detection of a target analyte at either the micro-needle working electrode 120 or 130 of the sensor device 100. Among the electrochemical measurement techniques that can potentially be used by the sensor device 100, the following can be mentioned.

[0037] The potential difference measurement technique is one in which the open circuit potential of an electrochemical cell is directly measured. This potential can be measured between a reference electrode (e.g., reference electrode 110 of sensor device 100) and a working electrode (e.g., working electrode 120 or working electrode 130 of sensor device 100).

[0038] The potential difference measurement technique is contrasted with measurements of a family of current measurements that measure current while controlling the potential of the cell. For example, chronoamperometry is a powerful tool for measuring diffusion-limited reactions. In chronoamperometry, the potential is stepped at the start of the measurement and then held constant throughout the measurement. The current resulting from this stimulus is plotted as a function of time.

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

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

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

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

[0043] Figure 2 shows a block diagram representing an example of an embodiment of the electrochemical micro-needle sensor device 100 of FIG. 1 communicating with an electronic device 200 according to the present technology.

[0044] For simplicity of explanation, the same reference numbers are used to identify some elements in different figures (e.g., electrodes 110, 120, and 130 in FIGS. 1 and 2), but it should be noted that these designations do not necessarily mean that these elements are identical. In particular, one or more characteristics of the elements (e.g., physical, chemical, electrochemical, material, dimensions, etc.) are contemplated to be modified based on the particular configuration of the micro-needle sensor device according to the technology disclosed in this application and / or based on its desired performance characteristics.

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

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

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

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

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

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

[0051] The above examples include electrodes of the device 100 configured to detect two different target analytes, but the techniques disclosed in this application can be applied to the detection of any number of target analytes. Thus, the device 100 can have any number of electrodes configured to detect any number of target analytes.

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

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

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

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

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

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

[0058] In some embodiments, I / O unit 230 includes a wireless communication interface 231, such as a wireless transmitter configured to transmit stored and / or processed data, or a wireless transceiver (Tx / Rx) configured to transmit and receive data. For example, in some embodiments, I / O unit 230 can also include a wireless communication interface 232 that can be used to exchange electrical signals, for example, between elements of electronic device 200 and between electronic device 200 and device 100. I / O unit 230 can utilize various types of wired or wireless interfaces that are compatible with typical data communication standards. For example, this can include, but is not limited to, Bluetooth, Bluetooth Low Energy, Zigbee, IEEE 802.11, Wireless Local Area Network (WLAN), Wireless Personal Area Network (WPAN), Wireless Wide Area Network (WWAN), WiMAX, IEEE 802.16 (Worldwide Interoperability for Microwave Access (WiMAX)), 3G / 4G / LTE / 5G cellular communication methods, NFC (Near Field Communication), and parallel interfaces, and can be used in the communication of electronic device 200. In some embodiments, electronic device 200 uses I / O unit 230 for the purpose of transferring data to another device and for receiving data (e.g., a voltage profile applied across one or more electrodes of sensor device 100 during its operation) from another device.

[0059] In some embodiments, electronic device 200 includes or is otherwise interfaced with a display unit 240, which can include visual displays, such as display screens, auditory displays, such as speakers, or any other type of display, or combinations thereof.

[0060] The I / O unit 230 of the electronic device 200 can be interfaced with other external interfaces, data sources, data storage devices, and / or image or auditory display devices, etc., to receive and transfer data and information that can be processed by the processor 220, stored in the memory 210, or presented in the output unit of the electronic device 200 or an external device (e.g., the display unit 240). For example, the display unit 240 can be configured to communicate data with the electronic device 200, for example, via the I / O unit 230, to provide image display, auditory display, and / or other sensory displays that generate a user interface of a software application. In some examples, the display unit 240 can include, but is not limited to, various types of screen displays, speakers, or printing interfaces, such as light-emitting diodes (LEDs), or liquid crystal display (LCD) monitors or screens, cathode ray tubes (CRTs) as image displays, audio signal transducer devices as auditory displays, and / or printing devices using toner, liquid inkjet, solid ink, sublimation dyes, inkless (e.g., thermal or UV, etc.).

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

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

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

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

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

[0066] Note that the target analytes mentioned above can share a biomarker associated with a particular disease, such as diabetes, or a common property indicating the state of one or more physiological systems and / or organs of the body.

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

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

[0069] The functional materials 125, 135, 145, and 155 of the microneedle sensor device 100 shown in FIG. 6 can include either the functional materials described above or any other functional materials suitable for the detection of a target analyte, which can provide information regarding the assessment of a particular condition of the body (e.g., diabetes) with any of the functionalized working electrodes 120, 130, 140, and / or 150 using any of the electrochemical measurement techniques mentioned above. For example, the functional material 145 of the electrode 140 can enable detection based on an antibody to a target analyte (e.g., insulin), and the functional material 155 of the electrode 150 can provide detection based on an aptamer to the same target analyte (insulin).

[0070] In an implementation example, the functionalized material 145 is configured to facilitate an electrochemical immunoassay (e.g., including a label-free immunoassay) for detecting the concentration of an analyte in a biological fluid. In some embodiments, an electrochemical micro-needle sensor device 100 having working electrodes based on at least three or four micro-needles for multiplex sensing of one or more target analytes includes a micro-needle electrode 140 disposed within a hollow interior formed as a channel in which a functionalized electrode probe structure 147 extends between two openings of the micro-needle structure 143 of the electrode 140. Within the channel, the electrode probe structure 147 is functionalized with a functionalized material 145 that includes capture antibodies (e.g., anti-analyte capture antibodies and / or detection antibodies) covalently connected to a self-assembled monolayer (SAM) linked to the electrode probe structure 147. The channel enables a fluid (e.g., ISF) to flow through the channel within the interior of the micro-needle structure where the functionalized immunoassay electrodes are present to facilitate a detectable reaction. In some implementation examples, the anti-analyte antibody-functionalized material 145 can interact with a target analyte for detection at the working electrode (e.g., after covalent attachment to a capture antibody for the analyte (e.g., insulin) and / or subsequent binding of a detection antibody). The immunoassay reaction can cause an electrical signal to be detected at the electrode 140.

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

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

[0073] The functional materials 125, 135, 145, and 155 of the micro-needle sensor device 100 represented in FIG. 7 can include either the functional materials described above or any other functional materials suitable for the detection of target analytes, which can provide information about a particular condition of the body (e.g., diabetes) with any of the functionalized working electrodes 120, 130, 140, and / or 150 using any of the electrochemical measurement techniques mentioned above.

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

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

[0076] The micro-needle electrode 140 filled with carbon paste for monitoring the concentration of ketone bodies shown in FIG. 7 can be constructed by incorporating an appropriate mediator into the carbon paste, followed by encapsulating stable HBD / NAD+ enzymes / cofactors, and droplet casting an outer polymer film that enables continuous monitoring of ketone bodies.

[0077] Similarly, the micro-needle electrode 150 filled with carbon paste shown in FIG. 7 and used for monitoring glucose concentration relies on the reduction of hydrogen peroxide (H2O2) generated on a graphite / mineral oil carbon paste modified with immobilized glucose oxidase (GOx) enzyme and Prussian blue (PB). The GOx enzyme and PB can be easily incorporated into the carbon paste without the problem of leaching during long-term use in a fluid matrix.

[0078] FIG. 8 illustrates an example of an embodiment of a method 200 for manufacturing an electrochemical sensing device. Method 200 includes a step 210 of preparing a substrate. Method 200 further includes a step 220 of connecting at least two micro-needles to the substrate.

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

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

[0081] In some example embodiments, the device includes at least two electrically conductive channels, and each channel in the at least two electrically conductive channels is electrically coupled to an electrode probe structure of a micro-needle in at least two micro-needles to transmit a signal from or apply a control signal to the electrode probe structure. In other example embodiments, the electrode probe structure of a third micro-needle in at least two micro-needles includes an aptamer sequence specific for a second analyte, and the electrode probe structure of the third micro-needle is operable as a working electrode for the detection of the second analyte using a second electrochemical detection technique. In still other example embodiments, the second analyte is different from the first analyte. According to an example embodiment, the second electrochemical detection technique is different from the first electrochemical detection technique.

[0082] In some example embodiments, for at least one micro-needle in at least two micro-needles, the internal volume of the protruding needle structure of the micro-needle includes a hollow interior defined by an inner wall, the outer wall of the protruding needle structure of the micro-needle includes an opening to the hollow interior, and the electrode probe structure of the micro-needle is at least partially disposed within the hollow interior. In still other example embodiments, the electrode probe structure of the micro-needle in at least two micro-needles includes a metal film. In some example embodiments, the electrode probe structure of the micro-needle in at least two micro-needles includes a metal wire. According to some example embodiments, the metal is gold. In other example embodiments, the electrode probe structure of a second micro-needle in at least two micro-needles includes silver / silver chloride (Ag / AgCl).

[0083] In another example of embodiments, the electrode probe structure of the fourth microneedle in at least two microneedles includes a coating on the surface of the electrode probe structure, the coating is imparted with a first enzyme, the coating is configured to interact with a third analyte, and the electrode probe structure of the fourth microneedle is operable as a working electrode for detecting a third analyte using a third electrochemical detection technique. According to examples of some embodiments, the electrode probe structure of the fifth microneedle in at least two microneedles includes a coating on the surface of the electrode probe structure, the coating is imparted with a second enzyme, the coating is configured to interact with a fourth analyte, and the electrode probe structure of the fifth microneedle is operable as a working electrode for detecting a fourth analyte using a fourth electrochemical detection technique. In an example of certain embodiments, the second enzyme is different from the first enzyme.

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

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

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

[0087] Yet another aspect of the disclosed technology is a method of electrochemical-based sensing, the method comprising the step of providing a device according to the technology disclosed in the present application, the device including at least two electrical conduction channels, each channel of the at least two electrical conduction channels being electrically coupled to an electrode probe structure of a microneedle of at least two microneedles of the device to transmit a signal (a sensing signal, e.g., a signal generated in response to a compound in a biological fluid or tissue) from the electrode probe structure or to apply a control signal to the electrode probe structure. The method further comprises the step of transmitting a signal from an electrode probe structure of a first microneedle of the at least two microneedles using a first electrical conduction channel of the at least two electrical conduction channels.

[0088] Aspects of the disclosed technology are methods of electrochemical sensing, including the step of providing a device according to the technology disclosed in this application, the device including at least two electrical conduction channels, each channel in the at least two electrical conduction channels being electrically connected to the electrode probe structure of a micro-needle in at least two micro-needles of the device to transmit a signal (e.g., a sensing signal, e.g., a signal generated in response to a compound in a biological fluid or tissue) from the electrode probe structure or to apply a control signal to the electrode probe structure. The method further includes the step of using a first electrical conduction channel in the at least two electrical conduction channels to apply a first control signal to the electrode probe structure of a first micro-needle in the at least two micro-needles. The method also includes the step of using a second electrical conduction channel in the at least two electrical conduction channels to transmit a signal from the electrode probe structure of a second micro-needle in the at least two micro-needles. In some embodiments, the method of electrochemical sensing further includes the step of determining the concentration of an analyte using the signal transmitted from the electrode probe structure of the second micro-needle.

[0089] In some example embodiments of the method of electrochemical-based sensing, the first micro-needle and the second micro-needle refer to the same micro-needle among at least two micro-needles. In other example embodiments of the method, the first micro-needle and the second micro-needle refer to different micro-needles among at least two micro-needles. In still other example embodiments of the method, the first electrical conduction channel and the second electrical conduction channel refer to the same electrical conduction channel among at least two electrical conduction channels. In certain example embodiments of the method, the first electrical conduction channel and the second electrical conduction channel refer to different electrical conduction channels among at least two electrical conduction channels. According to some example embodiments of the method, the electrical conduction channels among at least two electrical conduction channels are wires made of a conductive material or trace amounts of conductive material on a circuit board.

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

[0091] This application includes many details, which should not be construed as limitations to the scope of what may be described in the scope of any invention or claims, but rather should be construed as descriptions of features that may be specific to particular embodiments of a particular invention. Certain features described in this application in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment can also be implemented separately in multiple embodiments or in any suitable sub-combination. Also, features are described above as acting in a particular combination and may even be initially claimed in the claims as such, but one or more features from the combination recited in the claims may, in some cases, be deleted from the combination, and the combination recited in the claims may be directed to a sub-combination or a modification of a sub-combination.

[0092] Similarly, although operations are presented in a particular order in the drawings, this should not be understood as requiring that such operations be performed in the particular shown order or in a sequential order to achieve the desired result, or that all of the illustrated operations be performed. Also, the separation of the various system components in the embodiments described in this application should not be understood as requiring such separation in all embodiments.

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

[0094] The various information and data processing operations described herein may, in one embodiment, be executed by a computer program product embedded in a computer-readable medium containing computer-executable instructions, such as program code, to be executed by a computer in a networked environment. The computer-readable medium may include removable and non-removable storage devices including, but not limited to, read only memory (ROM), random access memory (RAM), compact disc (CD), digital versatile disc (DVD), etc. Thus, the computer-readable medium described in this application includes non-transitory storage media. Generally, program modules may include routines, programs, objects, components, data structures, etc. that perform particular tasks or execute particular abstract data types. Computer-executable instructions, associated data structures, and program modules represent examples of program code for performing the steps of the methods disclosed herein. A particular arrangement of such executable instructions or associated data structures represents an example of corresponding acts for performing the functions described in such steps or processes.

[0095] Only a few implementations and examples are described, and other implementations, enhancements, and variations can be made based on what is described and illustrated herein. This application provides the following. 1. A substrate, and at least two microneedles coupled to the substrate A device comprising: each microneedle of the at least two microneedles has a protruding needle structure, and an electrode probe structure comprising the protruding needle structure includes an outer wall extending outward from the surface of the substrate, the outer wall surrounds the internal volume of the protruding needle structure, and forms a top at the end of the outer wall. The electrode probe structure is configured to generate a signal in response to one or more chemical or biological substances in a biological fluid that contacts the electrode probe structure. The electrode probe structure of the first micro-needle in at least two micro-needles includes an aptamer sequence specific for a first analyte, and the electrode probe structure of the first micro-needle is operable as a working electrode for the detection of the first analyte using a first electrochemical detection technique. The electrode probe structure of the second micro-needle in at least two micro-needles is operable as an electrochemical counter electrode or an electrochemical reference electrode. Device. 2. The device according to 1 above, wherein the electrode probe structure is incorporated into or attached to a protruding needle structure. 3. The device according to 1 above, wherein the aptamer sequence is linked to the electrode probe structure of the first micro-needle via a 5'-thiol. 4. The device according to 1 above, wherein the aptamer sequence is functionalized with a redox reporter molecule. 5. The device according to 4 above, wherein the redox reporter molecule is methylene blue. 6. The device according to 4 above, wherein the redox reporter molecule is anthraquinone. 7. The device according to 4 above, wherein the functionalization is at the 3'-end of the aptamer sequence. 8. The device according to 4 above, wherein the functionalization is at the 5'-end of the aptamer sequence. 9. The device according to 1 above, comprising at least two electrical conduction channels, wherein each channel in the at least two electrical conduction channels is electrically connected to the electrode probe structure of the micro-needle in at least two micro-needles to transmit a signal from the electrode probe structure or apply a control signal to the electrode probe structure. 10. The electrode probe structure of the third microneedle in at least two microneedles includes an aptamer sequence specific to a second analyte, and the electrode probe structure of the third microneedle is operable as a working electrode for the detection of the second analyte using a second electrochemical detection technique, the device according to 1 above. 11. The device according to 10 above, wherein the second analyte is different from the first analyte. 12. The device according to 10 above, wherein the second electrochemical detection technique is different from the first electrochemical detection technique. 13. For at least one microneedle in at least two microneedles, the internal volume of the protruding needle structure of the microneedle includes a hollow interior defined by an inner wall, the outer wall of the protruding needle structure of the microneedle includes an opening to the hollow interior, and the electrode probe structure of the microneedle is at least partially disposed within the hollow interior, the device according to 1 above. 14. The device according to 1 above, wherein the electrode probe structure of the microneedle in at least two microneedles includes a metal film. 15. The device according to 13 above, wherein the electrode probe structure of the microneedle includes a metal wire. 16. The device according to 14 or 15 above, wherein the metal is gold. 17. The device according to 1 above, wherein the electrode probe structure of the second microneedle in at least two microneedles includes silver / silver chloride (Ag / AgCl). 18. The electrode probe structure of the fourth microneedle in at least two microneedles includes a coating on the surface of the electrode probe structure, the coating is functionalized with a first enzyme, the coating is configured to interact with a third analyte, and the electrode probe structure of the fourth microneedle is operable as a working electrode for the detection of the third analyte using a third electrochemical detection technique, the device according to 10 above. 19. The electrode probe structure of the fifth micro-needle in at least two micro-needles includes a coating on the surface of the electrode probe structure, the coating is imparted with a function by a second enzyme, the coating is configured to interact with a fourth analyte, and the electrode probe structure of the fifth micro-needle is operable as a working electrode for detecting a fourth analyte using a fourth electrochemical detection technique, the device according to 18 above. 20. The device according to 19 above, wherein the second enzyme is different from the first enzyme. 21. The electrode probe structure of the sixth micro-needle in at least two micro-needles includes a coating on the surface of the electrode probe structure, the coating is imparted with a function by an ionophore receptor, the coating is configured to interact with a fifth analyte, and the electrode probe structure of the sixth micro-needle is operable as a working electrode for detecting a fifth analyte using a fifth electrochemical detection technique, and the fifth analyte is an electrolyte, the device according to 18 or 19 above. 22. Any electrochemical detection technique from the first, second, third, and fourth electrochemical detection techniques is one of a cyclic voltammetry technique, a fast scan cyclic voltammetry technique, a square wave voltammetry technique, a potentiometry technique, or a chronoamperometry technique, the device according to 19 above. 23. The fifth electrochemical detection technique is one of a cyclic voltammetry technique, a fast scan cyclic voltammetry technique, a square wave voltammetry technique, a potentiometry technique, or a chronoamperometry technique, the device according to 21 above. 24. The electrode probe structure of at least one micro-needle in at least two micro-needles includes a conformal coating, the device according to 1 above. 25. The device according to 24 above, wherein the conformal coating includes an electrically insulating polymer or a dielectric material. 26. The device according to 24 above, wherein the conformal coating comprises at least one of a poly(p-xylene) polymer, a polyethyleneimine polymer, or SiO2. 27. The device according to 1 above, wherein the first analyte is a protein. 28. The device according to 27 above, wherein the protein is a cytokine. 29. The device according to 27 above, wherein the protein is one of insulin, interleukin-6 protein, tumor necrosis factor protein, or C-reactive protein. 30. The device according to 18 above, wherein the third analyte is a small molecule compound. 31. The device according to 19 above, wherein the fourth analyte is a small molecule compound. 32. The device according to 30 or 31 above, wherein the small molecule compound is one of lactose, lactic acid, alcohol, glucose, glutamic acid, or a ketone body. 33. The device according to 1 above, wherein the first analyte is a drug. 34. The device according to 10 above, wherein the first analyte is cortisol and the second analyte is insulin. 35. The device according to 18 above, wherein the first enzyme is glucose oxidase and the third analyte is glucose. 36. The device according to 19 above, wherein the second enzyme is β-hydroxybutyrate dehydrogenase and the fourth analyte is a ketone body. 37. The device according to 21 above, wherein the electrolyte is one of sodium, potassium, or lithium. 38. The device according to 18 above, wherein the third analyte is different from the first analyte and the third analyte is different from the second analyte. 39. The device according to 19 above, wherein the fourth analyte is different from the first analyte, the fourth analyte is different from the second analyte, and the fourth analyte is different from the third analyte. 40. The device according to 1 above, wherein the device is configured to be placed on human skin. 41. A method of manufacturing an electrochemical sensing device, comprising A step of preparing a substrate, and A step of connecting at least two microneedles to the substrate comprising each microneedle among at least two microneedles has a protruding needle structure, and an electrode probe structure comprising the protruding needle structure includes an outer wall extending outward from the surface of the substrate, the outer wall surrounds the internal volume of the protruding needle structure, and forms a top at the end of the outer wall, the electrode probe structure is configured to generate a signal in response to one or more chemical or biological substances in the biological fluid in contact with the electrode probe structure, the electrode probe structure of the first microneedle among at least two microneedles includes an aptamer sequence specific to the first analyte, and the electrode probe structure of the first microneedle is operable as a working electrode for the detection of the first analyte using a first electrochemical detection technique, the electrode probe structure of the second microneedle among at least two microneedles is operable as an electrochemical counter electrode or an electrochemical reference electrode, Method. 42. A method for electrochemical sensing, comprising A step of preparing the device according to any one of the above items 1 to 8 or 10 to 40, wherein the device includes at least two electrical conduction channels, and each channel among the at least two electrical conduction channels is electrically connected to the electrode probe structure of the microneedle among at least two microneedles to transmit a signal from the electrode probe structure or apply a control signal to the electrode probe structure, and A step of transmitting a signal from the electrode probe structure of the first microneedle among at least two microneedles using the first electrical conduction channel among at least two electrical conduction channels comprising 43. A method of sensing based on electrochemistry, comprising: preparing the device according to any one of items 1 to 8 or 10 to 40 above, wherein the device includes at least two electrical conduction channels, and each channel in the at least two electrical conduction channels is electrically connected to the electrode probe structure of the micro-needles in at least two micro-needles to transmit a signal from the electrode probe structure or apply a control signal to the electrode probe structure; using a first electrical conduction channel in the at least two electrical conduction channels to apply a first control signal to the electrode probe structure of the first micro-needle in the at least two micro-needles; and using a second electrical conduction channel in the at least two electrical conduction channels to transmit a signal from the electrode probe structure of the second micro-needle in the at least two micro-needles. A method comprising the above steps. 44. A method according to item 43 above, further comprising determining the concentration of the analyte using the signal transmitted from the electrode probe structure of the second micro-needle. A method according to item 43 above, further comprising the above step. 45. A method according to item 43 or item 44 above, wherein the first micro-needle and the second micro-needle refer to the same micro-needle in the at least two micro-needles. 46. A method according to item 43 or item 44 above, wherein the first micro-needle and the second micro-needle refer to different micro-needles in the at least two micro-needles. 47. A method according to item 43 or item 44 above, wherein the first electrical conduction channel and the second electrical conduction channel refer to the same electrical conduction channel in the at least two electrical conduction channels. 48. A method according to item 43 or item 44 above, wherein the first electrical conduction channel and the second electrical conduction channel refer to different electrical conduction channels in the at least two electrical conduction channels. 49. The method according to any one of items 42 to 48 above, wherein the electrical conduction channels in at least two electrical conduction channels are wires made of a conductive material or trace amounts of conductive material on a circuit board.

Claims

1. A substrate, and at least three microneedles coupled to the substrate comprising a device, each of the at least three microneedles having, a protruding needle structure, and an electrode probe structure comprising, the protruding needle structure including an outer wall extending outward from the surface of the substrate, the outer wall surrounding the internal volume of the protruding needle structure and forming a top at the end of the outer wall, the electrode probe structure being configured to generate a signal in response to one or more chemical or biological substances in a biological fluid contacting the electrode probe structure, the electrode probe structure of the first microneedle among the at least three microneedles including a first aptamer sequence specific to a first analyte, the electrode probe structure of the first microneedle being operable as a first working electrode for detection of the first analyte using a first electrochemical detection technique, the electrode probe structure of the second microneedle among the at least three microneedles being operable as an electrochemical counter electrode or an electrochemical reference electrode, the electrode probe structure of the third microneedle among the at least three microneedles including a second aptamer sequence specific to a second analyte, the electrode probe structure of the third microneedle being operable as a second working electrode for detection of the second analyte using a second electrochemical detection technique, the device.

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

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

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

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

6. The device according to claim 4, wherein the redox reporter molecule is anthraquinone.

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

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

9. The device according to claim 1, comprising at least two electrically conductive channels, wherein each channel in the at least two electrically conductive channels is electrically connected to an electrode probe structure of a micro-needle in at least three micro-needles to transmit a signal from the electrode probe structure or to apply a control signal to the electrode probe structure.

10. The device according to claim 1, wherein the second analyte is different from the first analyte.

11. The device according to claim 1, wherein the second electrochemical detection technique is different from the first electrochemical detection technique.

12. For at least one micro-needle in at least three micro-needles, the internal volume of the protruding needle structure of the micro-needle includes a hollow interior defined by an inner wall, the outer wall of the protruding needle structure of the micro-needle includes an opening to the hollow interior, and the electrode probe structure of the micro-needle is at least partially disposed within the hollow interior. The device according to claim 1.

13. The device according to claim 1, wherein the electrode probe structure of the micro-needle in at least three micro-needles includes a metal film.

14. The device according to claim 13, wherein the electrode probe structure of the micro-needle includes a metal wire.

15. The device according to claim 13 or claim 14, wherein the metal is gold.

16. The device according to claim 1, wherein the electrode probe structure of the second micro-needle in at least three micro-needles includes silver / silver chloride (Ag / AgCl).

17. The device according to claim 1, wherein the electrode probe structure of the fourth micro-needle in at least three micro-needles includes a coating on the surface of the electrode probe structure, the coating is functionalized with a first enzyme, the coating is configured to interact with a third analyte, and the electrode probe structure of the fourth micro-needle is operable as a third working electrode for the detection of a third analyte using a third electrochemical detection technique.

18. The electrode probe structure of the fifth micro-needle in at least three micro-needles includes a coating on the surface of the electrode probe structure, the coating is functionalized with a second enzyme, the coating is configured to interact with a fourth analyte, and the electrode probe structure of the fifth micro-needle is operable as a fourth working electrode for the detection of a fourth analyte using a fourth electrochemical detection technique, the device according to claim 17.

19. The device according to claim 18, wherein the second enzyme is different from the first enzyme.

20. The electrode probe structure of the sixth micro-needle in at least three micro-needles includes a coating on the surface of the electrode probe structure, the coating is functionalized with an ionophore receptor, the coating is configured to interact with a fifth analyte, and the electrode probe structure of the sixth micro-needle is operable as a fifth working electrode for the detection of a fifth analyte using a fifth electrochemical detection technique, and the fifth analyte is an electrolyte, the device according to claim 17 or claim 18.

21. The device according to claim 18, wherein any electrochemical detection technique from the first, second, third, and fourth electrochemical detection techniques is one of a cyclic voltammetry technique, a fast scan cyclic voltammetry technique, a square wave voltammetry technique, a potentiometry technique, or a chronoamperometry technique.

22. The device according to claim 20, wherein the fifth electrochemical detection technique is one of a cyclic voltammetry technique, a fast scan cyclic voltammetry technique, a square wave voltammetry technique, a potentiometry technique, or a chronoamperometry technique.

23. The device according to claim 1, wherein the electrode probe structure of at least one micro-needle in at least three micro-needles includes a conformal coating.

24. The device according to claim 23, wherein the conformal coating includes an electrically insulating polymer or a dielectric material.

25. The conformal coating comprises at least one of a poly(p-xylene) polymer, a polyethyleneimine polymer, or SiO 2 The device of claim 23, wherein the conformal coating comprises at least one of a poly(p-xylene) polymer, a polyethyleneimine polymer, or SiO

26. The device according to claim 1, wherein the first analyte is a protein.

27. The device according to claim 26, wherein the protein is a cytokine.

28. The device according to claim 26, wherein the protein is one of insulin, interleukin-6 protein, tumor necrosis factor protein or C-reactive protein.

29. The device according to claim 1, wherein the first analyte is cortisol.

30. The device according to claim 17, wherein the third analyte is a small molecule compound.

31. The device according to claim 18, wherein the fourth analyte is a small molecule compound.

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

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

34. The device according to claim 1, wherein the first analyte is cortisol and the second analyte is insulin.

35. The device according to claim 17, wherein the first enzyme is glucose oxidase and the third analyte is glucose.

36. The device according to claim 35, wherein the first analyte is cortisol.

37. The device according to claim 35, wherein the first analyte is cortisol and the second analyte is cortisol.

38. The device according to claim 18, wherein the second enzyme is β-hydroxybutyrate dehydrogenase and the fourth analyte is a ketone body.

39. The device according to claim 20, wherein the electrolyte is one of sodium, potassium or lithium.

40. The device according to claim 17, wherein the third analyte is different from the first analyte and the third analyte is different from the second analyte.

41. The device according to claim 18, wherein the fourth analyte is different from the first analyte, the fourth analyte is different from the second analyte, and the fourth analyte is different from the third analyte.

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

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