Electrochemical microneedle patch, device and method for detecting physiological markers in skin tissue fluid

Abstract

The invention discloses an electrochemical microneedle patch, a device and a method for detecting a physiological marker in skin tissue fluid. The electrochemical microneedle patch comprises a patch as well as a counter electrode, a reference electrode and a working electrode which are fixed on the patch; the counter electrode, the reference electrode and the working electrode are converged on one side of the patch in a printed circuit mode to form a signal connecting port, and each of the counter electrode, the reference electrode and the working electrode comprises at least one conductive microneedle electrode; each conductive microneedle electrode is long enough to penetrate through the epidermal layer of the skin and reach the corium layer, the conductive microneedle electrode of the working electrode is provided with a detection component for detecting a physiological marker in skin tissue fluid, and the outer surface of the conductive microneedle electrode is covered with a hydrogel layer; the hydrogel layer has a net-shaped structure which can form a swelling layer without dissolution after absorbing skin tissue fluid in the corium layer, and the net-shaped structure allows the to-be-detected physiological marker to diffuse into or move away from the surface of the working electrode. According to the invention, the stability of the current signal can be improved, the problems of signal distortion and instability and the like are reduced, and the method is suitable for long-time online detection.

Description

Technical Field

[0001] The present invention relates to physiological marker detection and electrochemical technology, and in particular to an electrochemical microneedle patch, a device and a method for detecting physiological markers in skin tissue fluid. Background Art

[0002] Interstitial Skin Fluid (ISF) is a liquid that exists in the intercellular space. It flows between cells and tissues, provides nutrition to cells and removes waste. Its composition is similar to that of blood. It can provide information on physiological markers such as alcohol, uric acid, and glucose, and can reflect the physiological condition of the human body to a certain extent. Using ISF as a sampling object to analyze the health level of the human body can be implemented through microneedle technology. For example, electrochemical (such as enzyme-loaded electrodes) microneedles of micron size can detect the electrical signals of physiological markers (small molecules) in intercellular fluid. Microneedle technology has very small wounds and can replace blood sampling tests, which is beneficial to reduce human pain and infection risks. The sampling process does not require special operating skills and is a relatively safe, painless, and less damaging method. However, since intercellular fluid is located in the intercellular space, the total amount of intercellular fluid is small and it is not as easy to collect as blood. The currently known process of non-in situ electrochemical analysis of skin tissue fluid by microneedle testing is usually cumbersome, especially because the total volume of the collected samples is relatively small, and the process of transferring to the analysis equipment is prone to component loss and microbial contamination. In addition, some components are unstable in the air, resulting in changes in concentration, which in turn increases identification errors and even misdiagnosis.

[0003] In situ electrochemical microneedle sensing is a new type of ISF detection method developed to address the above problems. Electrochemical sensors are devices that convert chemical signals into electrical signals and perform sensing detection based on the electrochemical properties of samples. By combining electrochemical sensors with microneedle technology, not only can real-time signal detection be achieved inside the skin, but also the risk of false detection caused by the sample transfer process can be reduced. It has the advantages of high sensitivity, simple operation, rapid response, and low cost, and can be widely used in wearable electronic devices and other fields. Microneedle-based electrochemical sensors can analyze and detect biomarkers, metabolites in the body, drug concentrations, and other substances present in interstitial fluid. In addition, since electrochemical sensing is an extremely sensitive measurement method, it is particularly advantageous for the analysis of some volatile, unstable, and low-concentration components, and is a particularly promising detection method. However, electrochemical microneedle sensing technology still needs to be further developed and improved, especially in the online continuous monitoring of small molecules of physiological sign markers. It is not easy to achieve stable data collection, which limits its application in the diagnosis of a wide range of diseases.

[0004] It should be noted that the information disclosed in the above background technology section is only used for understanding the background of the present application, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the invention

[0005] The present invention provides an electrochemical microneedle patch, a device and a method for detecting physiological markers in skin tissue fluid.

[0006] The present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an electrochemical microneedle patch for detecting physiological markers in skin tissue fluid, comprising a patch and a counter electrode, a reference electrode and a working electrode fixed on the patch; the counter electrode, the reference electrode and the working electrode are converged into a signal connection port on one side of the patch in the form of a printed circuit; the counter electrode, the reference electrode and the working electrode each comprise at least one conductive microneedle electrode; the length of each conductive microneedle electrode is sufficient to penetrate the epidermis of the skin and reach the dermis, the conductive microneedle electrode of the working electrode has a detection component for detecting physiological markers in skin tissue fluid, and the outer surface of the conductive microneedle electrode of the working electrode is covered with a hydrogel layer, the hydrogel layer has a reticular structure that absorbs skin tissue fluid in the dermis to form a swollen layer without dissolving, and the reticular structure allows the physiological marker to be detected to diffuse into or move away from the surface of the working electrode.

[0008] In a second aspect, the present invention provides an electrochemical microneedle device for detecting physiological markers in skin tissue fluid, comprising the electrochemical microneedle patch, a circuit board and a control system as described in the first aspect, wherein the electrochemical microneedle patch is connected to the circuit board and to the control system via the signal connection port, the control system being configured to intermittently start the electrochemical microneedle patch at a predetermined intermittent time and a predetermined voltage to detect physiological markers in skin tissue fluid to obtain a current signal, and the control system being further configured to obtain the concentration of the physiological marker to be measured based on the current signal.

[0009] In a third aspect, the present invention provides a method for detecting physiological markers in skin tissue fluid using the electrochemical microneedle device described in the second aspect for non-diagnostic purposes, comprising the following steps:

[0010] (1) attaching the electrochemical microneedle patch to the skin surface of a subject, ensuring that the conductive microneedle electrodes penetrate the epidermis to reach the dermis;

[0011] (2) before measurement, applying a stabilization voltage to the electrochemical microneedle patch through the control system to allow current stabilization;

[0012] (3) intermittently starting the electrochemical microneedle patch with a predetermined intermittent time and a predetermined voltage through the control system to detect physiological markers in skin tissue fluid to obtain current signals;

[0013] (4) The concentration of the physiological marker to be measured is obtained according to the current signal through the control system.

[0014] The electrochemical microneedle patch for detecting physiological markers in skin tissue fluid and the device and method thereof of the present invention can improve the stability of the current signal, reduce problems such as signal distortion and instability, and are suitable for online detection for a long time, and specifically have the following beneficial effects:

[0015] 1. The intermittent measurement strategy improves the measurement accuracy, reduces the concentration polarization problem caused by continuous testing, reduces the energy consumption of the equipment, and prolongs the battery life.

[0016] 2. Reduce the complexity of data processing and improve the efficiency of data management.

[0017] 3. The acquisition frequency can be selected to ensure timely response during the critical period of changes in physiological markers (such as blood sugar) levels.

[0018] 4. It can provide more humane monitoring solutions and improve the patient experience.

[0019] 5. It can reduce the consumption rate of detection components (such as enzymes) and extend the working life of the microneedle patch. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A Schematic diagram of the top view of the structure of the electrochemical microneedle patch in a specific embodiment of the present invention;

[0021] Figure 1B It is a schematic structural diagram of a conductive microneedle electrode of a working electrode of an electrochemical microneedle patch in a specific embodiment of the present invention;

[0022] Figure 2 is the electrochemical it curve in Example 1;

[0023] Figure 3 is a graph showing the relationship between the current density (current density) and the glucose concentration (C) of the first generation microneedles loaded with MeHA in Example 1;

[0024] Figure 4 is the electrochemical it curve in Example 2;

[0025] Figure 5 is a graph showing the relationship between the current density and glucose concentration of the second-generation microneedles loaded with MeHA in Example 2;

[0026] Figure 6 is the electrochemical it curve in Example 3;

[0027] Figure 7 is a graph showing the relationship between the current density and glucose concentration of the third-generation microneedles loaded with MeHA in Example 3;

[0028] Figure 8 It is the electrochemical IT curve in Comparative Example 1. DETAILED DESCRIPTION

[0029] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope and application of the present invention. In the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other.

[0030] The specific embodiment of the present invention provides an electrochemical microneedle patch for detecting physiological markers in skin tissue fluid, such as Figure 1A and 1B As shown, it includes a patch 1 and a working electrode 2, a counter electrode 3 and a reference electrode 4 fixed on the patch 1; the counter electrode 3, the reference electrode 4 and the working electrode 2 are converged into a signal connection port 12 on one side of the patch 1 through a wire system 11 in the form of a printed circuit; the counter electrode, the reference electrode and the working electrode each include at least one conductive microneedle electrode; the length of each conductive microneedle electrode is sufficient to penetrate the epidermis of the skin and reach the dermis, the conductive microneedle electrode of the working electrode has a detection component for detecting physiological markers in skin tissue fluid, and the outer surface of the conductive microneedle electrode of the working electrode is covered with a hydrogel layer, the hydrogel layer 22 has a mesh structure that absorbs skin tissue fluid in the dermis to form a swelling layer without dissolving, and the mesh structure allows the physiological marker to be detected to diffuse into or move away from the surface of the working electrode 2.

[0031] The electrochemical microneedle patch can be directly inserted into the skin without the aid of a complex insertion device. It is easy to operate and painless during the insertion process, which reduces the patient's resistance. The duration of a single patch is not restricted by cost and can be optimized according to the user's living habits. The low cost of use makes it possible to continuously monitor or detect physiological markers in skin tissue fluid with smart microneedle patches. The electrochemical microneedle patch contains a specific working electrode that participates in the electrochemical reaction and interacts with the physiological marker to be measured. After the electrochemical microneedle patch is inserted into the epidermis of the skin and reaches the dermis, the counter electrode, reference electrode and working electrode are connected to form a measurement circuit to form an electrochemical reaction cell. The current signal is obtained through the electrochemical reaction occurring in the patch, and the concentration level of the physiological marker to be measured can be reflected by detecting the size of the current signal.

[0032] The detection component loaded on the working electrode interacts with the physiological marker in the skin tissue fluid to generate a current signal that can be detected, such as electrons or positive charges enriched on the working electrode; the physiological marker interacts with the detection component and the working electrode to enrich electrons or positive charges on the working electrode, and forms a loop with the reference electrode to convert the current signal that can be detected. The outer surface of the working electrode is covered with a hydrogel layer, and the hydrogel layer has a mesh structure that absorbs the skin tissue fluid in the dermis to form a swollen layer without dissolving. The mesh structure allows the physiological marker to diffuse into or move away from the surface of the working electrode, thereby promoting the stability of the detection component. That is, the present invention can achieve the stability of the detection component and the free diffusion of the physiological marker to be detected into and out of the working electrode through the hydrogel layer with a specific structure covered on the outer surface of the working electrode. When the physiological marker to be detected diffuses into the surface of the microneedle array, a current signal that can be detected is generated in the electrochemical reaction cell, and the concentration of the physiological marker to be detected is determined based on the quantitative relationship between the current signal and the physiological marker to be detected. When the electrochemical microneedle of the present invention is attached to the skin tissue fluid, the physiological marker to be measured can effectively contact with the detection component, and the detection component can be ensured to be stably present on the surface of the working electrode for a long time, thereby extending the use time of the electrochemical reaction cell and ensuring continuous monitoring.

[0033] In some embodiments, the areas of the counter electrode, the reference electrode and the working electrode on the patch are each 1-100 square millimeters; the spacing between the conductive microneedle electrodes of the counter electrode, the reference electrode and the working electrode is each 0.5-5 millimeters, and the length of the conductive microneedle electrode is 0.1-3 millimeters, preferably the length of the conductive microneedle electrode is 0.4-2.4 millimeters; the number of the conductive microneedle electrodes of the counter electrode, the reference electrode and the working electrode is each between 1-100.

[0034] In some embodiments, the shape of the conductive microneedle electrode includes, but is not limited to, a cone, a prism with a pointed top, a cylinder with a pointed top, a pyramid, or a combination of the two.

[0035] Preferably, the diameter of the conductive microneedle electrode at the maximum cross-sectional area is in the range of 50-700 μm, for example, in the range of 250-600 μm, 300-550 μm, 350-500 μm, and the values ​​are, for example, 400 μm, 650 μm, etc. The diameter of the conductive microneedle electrode at the maximum cross-sectional area includes but is not limited to the above numerical ranges and points.

[0036] By designing the length and / or diameter of the conductive microneedle electrode, the effect of the conductive microneedle electrode on the epidermis is a reversible deformation. After removal, the skin can return to its original state after a few hours, and the pain and resistance of the patient to be tested can be significantly reduced.

[0037] In some embodiments, the hydrogel in the hydrogel layer is at least one of vinyl functionalized hyaluronic acid, acryloyl hyaluronic acid polymer, sodium hyaluronate polymer, chitosan, alginate, sodium carboxymethyl cellulose, and polyethylene glycol. Preferably, the hydrogel is methacryloyl hyaluronic acid.

[0038] In some embodiments, the thickness of the hydrogel layer is 100 nanometers to 500 microns, and preferably the thickness of the hydrogel layer is 1 micron to 100 microns. Furthermore, when preparing the hydrogel layer, a crosslinking agent or initiator can be added to its formula to form a chemical bond under the action of ultraviolet light to achieve crosslinking. Since the reaction process of ultraviolet light-induced polymerization is prone to cause damage to enzyme molecules, the ultraviolet process must control the reaction time, preferably the light intensity is 100-5000Lx, the temperature is 25-50 degrees Celsius, and the polymerization time is 0.2-5 minutes. After polymerization, the microneedle patch can be shielded from light and refrigerated, and the refrigeration temperature is preferably -25 degrees Celsius to 5 degrees Celsius.

[0039] In some embodiments, the physiological marker in the skin tissue fluid is one of glucose, uric acid, acetone, alcohol, and drug metabolites; the detection component is at least one of an enzyme, a hormone, an antibody, DNA, RNA, and a modified enzyme. Preferably, the enzyme is at least one of glucose oxidase, catalase, amylase, creatine kinase, lactate oxidase, and cholesterol oxygenase; the hormone is growth hormone and / or thyroid stimulating hormone; preferably, the modified enzyme is glucose oxidase modified with an electron mediator; preferably, the electron mediator is at least one of metal oxides, gold nanoparticles, platinum nanoparticles, cobalt nanoparticles, carbon nanotubes, graphene, Prussian blue, ferrocene, titanium nitride, carbon nitride, and BiFeO3 particles. Wherein, the glucose oxidase modified with an electron mediator is a mixture of an electron mediator and glucose oxidase in a predetermined mass ratio (preferably, the mass ratio of the electron mediator to the glucose oxidase is 6:5).

[0040] In some embodiments, the physiological marker in the skin tissue fluid is glucose, the detection component is glucose oxidase modified with ferrocene, or the detection component is glucose oxidase modified with carbon nanotubes.

[0041] In some embodiments, the microneedle body of the conductive microneedle electrode of the counter electrode, the reference electrode and the working electrode is an inert conductor that does not dissolve in the electrochemical reaction; preferably, the inert conductor material of the microneedle body of the counter electrode, the reference electrode and the working electrode is the same; preferably, the material of the inert conductor is at least one of platinum, gold, copper, silver and carbon materials.

[0042] In some embodiments, the structure and material of the microneedle body of the counter electrode and the reference electrode are the same or similar to the structure and material of the microneedle body of the working electrode. Specifically, the conductive microneedle electrode of the counter electrode is composed of the microneedle body, such as Figure 1B As shown, the conductive microneedle electrode of the working electrode includes a microneedle body 21, a detection component 23 loaded on the microneedle body 21, and a hydrogel layer 22 covering the detection component 23 on the outer surface of the microneedle body 21, and the conductive microneedle electrode of the reference electrode includes a microneedle body and a layer of chloride modified on the surface of the microneedle body.

[0043] In some embodiments, the microneedle body is made by the following preparation method: mixing 50wt%-95wt% of metal powder and 5wt%-50wt% of curing agent to form a slurry, transferring the slurry to a microneedle polymer mold, removing bubbles under vacuum conditions, filling the mold and curing, and finally demolding; the curing agent is at least one of an epoxy resin curing agent, a polyurethane curing agent, and an acrylic resin curing agent, preferably an epoxy resin curing agent. More preferably, the microneedle body of the three electrodes is a silver-based microneedle, which is made by the following preparation method: mixing 50wt%-95wt% of silver powder and 5wt%-50wt% of epoxy resin precursor formula (bisphenol A resin and methyl nadic anhydride are mixed in a mass ratio of 1:1.1) to form a slurry, transferring the slurry to a microneedle polymer mold, removing bubbles under vacuum conditions, filling the mold and curing, and finally demolding.

[0044] In some embodiments, the reference electrode is a highly stable Ag / AgCl electrode that can provide a stable potential reference. The Ag / AgCl electrode can be prepared by the following method: a silver-based microneedle is used as a raw material, immersed in a 0.05-0.3 mol / L (preferably a concentration of 0.1-0.3 mol / L) FeCl3 solution, and a layer of AgCl is formed on the surface of the microneedle body through an electrochemical reaction, and a saturated potassium chloride KCl solution is filled around the microneedle to form a complete Ag / AgCl electrode.

[0045] In some embodiments, the number of conductive microneedle electrodes serving as working electrodes may be 2, 3, 4 or even more. The provision of multiple microneedles can not only increase the strength of the electrical signal, but also increase the stability of the microneedle patch.

[0046] A specific embodiment of the present invention also provides an electrochemical microneedle device for detecting physiological markers in skin tissue fluid, which includes the electrochemical microneedle patch, a circuit board and a control system. The electrochemical microneedle patch is connected to the circuit board and to the control system through a signal connection port 12. The control system is configured to intermittently start the electrochemical microneedle patch at a predetermined intermittent time and a predetermined voltage to detect physiological markers in skin tissue fluid to obtain a current signal. The control system is also configured to obtain the concentration of the physiological marker to be measured based on the current signal.

[0047] Since the total amount of skin tissue fluid is small, the signal obtained by the test decays rapidly as the components in the skin tissue fluid are decomposed by the current. The method of using hydrogel to cover the surface of the microneedle can improve the enrichment of the skin tissue fluid, which is beneficial to improving the stability of the electrochemical signal. However, the hydrogel layer covering the surface of the working electrode will make the diffusion of the skin tissue fluid more difficult to a certain extent, thereby increasing the equivalent series resistance, resulting in a decrease in the measured current signal, affecting the signal-to-noise ratio. In addition, since the volume of skin tissue fluid that the electrochemical microneedle can contact under the skin is relatively small, it is easily affected by concentration changes during continuous monitoring and affects the accuracy of the test results. In view of this, the control system of the electrochemical microneedle device of the specific embodiment of the present invention is configured to intermittently start the electrochemical microneedle patch with a predetermined intermittent time and a predetermined voltage to detect physiological markers in the skin tissue fluid to obtain a current signal, thereby reducing problems such as signal distortion and instability, improving the stability and accuracy of the detection signal, and being suitable for online detection for a long time, which is of great significance for achieving long-term and stable in vivo blood glucose monitoring.

[0048] In some embodiments, the predetermined intermittent time is 0-24 hours, preferably more than 40 minutes, and more preferably 3 hours.

[0049] In some embodiments, the predetermined voltage is 0-0.8V, preferably 0.1V.

[0050] In some embodiments, the continuous measurement time of the electrochemical microneedle patch after each start-up is more than 30 seconds, preferably 30-120 seconds.

[0051] In some embodiments, the signal connection port of the electrochemical microneedle patch is detachably connected to the circuit board through a plug connector or an elastic clip. The electrochemical microneedle patch and the circuit board are detachably connected. When the electrochemical microneedle patch needs to be replaced, the electrochemical microneedle patch can be directly removed from the circuit board and replaced with a new electrochemical microneedle patch to form a new detection device, and the control system can be reused multiple times. The control system can also be detachably connected to the circuit board. When a failure occurs and needs to be replaced, it can also be directly removed and replaced with a new device without affecting the use of other devices.

[0052] In some embodiments, the control system is disposed on the circuit board so that the electrochemical microneedle device is configured as a wearable device.

[0053] In some embodiments, the control system comprises:

[0054] A control unit, used to apply the predetermined voltage to the electrochemical microneedle patch at the predetermined intermittent time; further, the control unit includes a timer, used to determine the time interval for applying the predetermined voltage, that is, to determine the predetermined intermittent time;

[0055] A current sensor for measuring the current during the period when the predetermined voltage is applied to the electrochemical microneedle patch; further, the current sensor includes a signal amplifier for amplifying the current signal received from the electrochemical microneedle patch;

[0056] a data logger for storing data received by the current sensor;

[0057] The analysis unit is used to process the data stored in the data recorder to obtain the concentration of the physiological marker to be measured. Preferably, the analysis unit pre-stores a quantitative relationship between the input current signal and the level of the physiological marker to be measured.

[0058] In some embodiments, the control system further includes a wireless transmission module for transmitting information to a remote monitoring device (eg, a smart terminal including but not limited to a mobile phone, a tablet computer, etc.).

[0059] The specific embodiment of the present invention also provides a method for detecting physiological markers in skin tissue fluid using the electrochemical microneedle device for non-diagnostic purposes, which comprises the following steps:

[0060] (1) attaching the electrochemical microneedle patch to the skin surface of a subject, ensuring that the conductive microneedle electrodes penetrate the epidermis to reach the dermis;

[0061] (2) before measurement, applying a stabilization voltage to the electrochemical microneedle patch through the control system to allow current stabilization;

[0062] (3) intermittently starting the electrochemical microneedle patch with a predetermined intermittent time and a predetermined voltage through the control system to detect physiological markers in skin tissue fluid to obtain current signals;

[0063] (4) Obtaining the concentration of the physiological marker to be measured according to the current signal through the control system. Preferably, when using the electrochemical microneedle device, it can be worn on the user to achieve continuous detection or monitoring of the content or concentration of biological substances in the body, which may include the following process:

[0064] 1. Attachment: Attach the electrochemical microneedle patch to the skin surface (such as the arm) to ensure that the microneedles penetrate the epidermis to reach the dermis.

[0065] 2. Stabilization voltage application: Before measurement, a stabilization voltage is applied to the electrochemical microneedle patch to allow the current to stabilize.

[0066] 3. Receiving a measurement signal: The measurement signal can be received wirelessly to activate the electrochemical microneedle patch and change the electrochemical microneedle patch from a standby mode to an active mode.

[0067] 4. Current measurement: During the active mode, the current related to the physiological marker to be measured is measured. The time interval for signal acquisition is set through the user interface. For example, the initial setting is to collect signals once every specific time. The system automatically controls the signal acquisition and standby state of the microneedle electrode according to the time interval set by the user.

[0068] 5. Signal transmission: The measured current value is transmitted wirelessly.

[0069] 6. Intermittent measurement: Repeat steps 3 and 4 intermittently to reduce measurement errors and improve accuracy. The collected signals are transmitted to the control system for storage and preliminary analysis.

[0070] The control system performs data processing and analysis:

[0071] 7. Signal reception: Receive the measurement signal transmitted from the electrochemical microneedle patch.

[0072] 8. Signal processing: Convert the received electrical signal into a digital signal related to the level of the physiological marker to be measured.

[0073] 9. Data analysis: Calculate the concentration level of the physiological marker to be measured based on the quantitative relationship between the electrical signal and the concentration of the physiological marker to be measured.

[0074] 10. Result output: Users can view the monitoring results and historical data of blood sugar waiting to be measured physiological markers in real time through mobile devices or monitoring centers. When the system detects abnormal fluctuations in the levels of blood sugar waiting to be measured physiological markers, it automatically adjusts the collection frequency, increases the monitoring density, and reminds the user through the user interface or mobile device.

[0075] Taking the case where the physiological marker to be measured is glucose and the detection component is glucose oxidase as an example, the process of electrochemical reaction and electrical signal generation is as follows: the electrochemical microneedle patch pierces the epidermis of the skin and reaches the tissue fluid in the dermis. At this time, the hydrogel layer on the surface of the working electrode absorbs water from the skin tissue fluid and swells, and the glucose in the tissue fluid diffuses into the surface of the working electrode to undergo an electrochemical reaction. In the electrochemical reaction, glucose is adsorbed on the catalytic site of glucose oxidase and reacts to generate gluconolactone, which is then hydrolyzed into gluconic acid; the catalytic site of glucose oxidase is converted into a reduced state, and the reduced glucose oxidase is re-oxidized to an oxidized state with catalytic activity under the action of oxygen in the tissue fluid, while releasing a molecule of hydrogen peroxide; hydrogen peroxide loses two electrons on the surface of the working electrode to generate a molecule of oxygen and two hydrogen ions, and oxygen participates in the conversion of glucose oxidase, and the hydrogen ions become components of gluconic acid, and the products and reactants achieve local circulation.

[0076] Since an electrochemical reaction occurs on the working electrode, the reference electrode, as a reference for the working electrode potential, does not undergo an electrochemical reaction. The counter electrode assists the working electrode in forming an electron / ion conduction circuit. When voltage is applied, the working electrode and the counter electrode are electrically connected to form a circuit, and current is generated between them. The glucose concentration level is estimated through the quantitative relationship between the current and the physiological marker (glucose).

[0077] The effect of glucose oxidase on glucose is the presence of hydrogen peroxide, and there is an interference current. In a preferred embodiment, by adding an electron mediator (such as ferrocene) for modification, the active center of the modified glucose oxidase is directly connected to ferrocene and then connected to the working electrode, and the reaction mechanism changes. When ferrocene-modified glucose oxidase catalyzes glucose, it oxidizes glucose to gluconolactone, and itself becomes a reduced state. The reduced ferrocene-modified glucose oxidase is oxidized on the working electrode and becomes an oxidized state again, and forms two molecules of hydrogen ions and two electrons. The generated electrons are detected by the working electrode to form a corresponding current signal. Therefore, by adding an electron mediator and glucose oxidase to compound as a detection component, glucose oxidase can be modified, and the glucose concentration can be detected by the change in the oxidation current of the electron mediator, so that glucose detection can be performed at a lower point, avoiding the oxidation current of the interfering substance. In a preferred embodiment, glucose oxidase can also be modified by adding carbon nanotubes, and glucose oxidase is modified on carbon nanotubes, which is conducive to the transfer of electrons.

[0078] The present invention is described in detail below through some embodiments.

[0079] Example 1

[0080] 1. Preparation of silver-based microneedles: Commercial silver powder and epoxy resin precursor formula (bisphenol A resin and methyl nadic anhydride are mixed in a mass ratio of 1:1.1) are evenly mixed in a mass ratio of 9:1 to form a silver paste. The mixed silver paste is transferred to a microneedle polymer mold, and bubbles are removed under vacuum conditions. After filling the mold, it is placed in a bake at 150°C for curing, and finally demolded to obtain a silver-based microneedle.

[0081] 2. Preparation of working electrode:

[0082] 2.1. Silver-based microneedles loaded with glucose oxidase (GOx): The silver-based microneedles obtained in step 1 were plasma cleaned, 20 uL of GOx solution (concentration of 25 mg / mL) was drop-coated on the microneedle array, and the microneedle array was placed in a cool place to dry, thereby obtaining silver-based microneedles loaded with GOx.

[0083] 2.2. Loading of hydrogel: The silver-based microneedles loaded with GOx obtained in step 2.1 are immersed in a monomer or prepolymer of MeHA hydrogel and an aqueous solution of an initiator, taken out and fully cured with ultraviolet light to obtain silver-based microneedles loaded with hydrogel and GOx as working electrodes.

[0084] 3. Preparation of reference electrode: Plasma cleaning is performed on the silver-based microneedles prepared in step 1 to make them hydrophilic. The cleaned silver-based microneedle array is immersed in a solution containing ferric chloride, and a layer of silver chloride (AgCl) is formed on the surface of the silver-based microneedle array through a chemical reaction. Saturated potassium chloride (KCl) solution is filled around the microneedles to form a complete Ag / AgCl electrode.

[0085] 4. Sensor assembly: The working electrode, counter electrode (silver-based microneedle obtained in step 1) and reference electrode (Ag / AgCl electrode obtained in step 3) prepared in step 2 are fixed on the patch by curing and bonding with silver paste to obtain an electrochemical microneedle patch.

[0086] 5. Preparation of agarose gel: Weigh 4.023g of NaCl, 0.101g of KCl, 0.717g of Na2HPO4 and 0.123g of KH2PO4 and dissolve them in 500ml of deionized water. Prepare some PBS solutions according to this formula, and then dissolve glucose in the PBS solution to prepare solutions with glucose concentrations of 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM and 20mM for later use. Weigh a certain amount of agar powder and dissolve it in PBS solution (the mass fraction of agar powder is 0.7% to 2%), and use a microwave to heat it until the agarose is completely dissolved. Pay attention to the process to avoid overflow. Place the dissolved agarose solution on a cooling plate or ice water bath, let it cool slightly, but still keep it above 60°C, pour the slightly cooled agarose solution into the gel mold until it covers the bottom of the mold, and place it at room temperature or 4°C to cool until the agarose is completely solidified. Cut into suitable sizes and demold, soak in PBS solutions with different glucose concentrations, and change the buffer solution of corresponding concentrations several times to allow the gel to fully absorb glucose molecules and reach the accurate glucose concentration inside.

[0087] 6. Insert the electrochemical microneedle patch into the agarose hydrogel with different glucose concentrations and stick it tightly. Insert the end of the patch (the end where the signal connection port is located) into the circuit board and connect it to the control system. The circuit board adopts a double-sided substrate design, with the upper and lower layers being conductive copper substrates. The back wiring is connected to the printed circuit board (PCB). The control system can be connected to an external terminal device via Bluetooth technology, receive test instructions and send test results. The signal connection port of the electrochemical microneedle patch can be detachably connected to the circuit board via a plug connector or an elastic clip. The intensity of the current signal is proportional to the concentration of ISF (Interstitial Fluid) in the sample being tested.

[0088] 7. The microneedle with glucose oxidase loaded on the working electrode in Example 1 was named as the first-generation microneedle. The first-generation microneedles loaded with and without hydrogel MeHA were tested in agarose hydrogels with different glucose concentrations. The area of ​​the working electrode was 1 cm 2 The test object is agarose gel with glucose concentration of 0mM, 4mM, and 8mM. The test is intermittent, the test voltage is 0.1V, the test time is 120s, and the test is performed every 3h. The test is performed using Chenhua CHI660E electrochemical workstation. The results are as follows: Figure 2 , Figure 3 As shown in Table 1, Figure 3 The data comes from Figure 2 At 60s of the first test of 0mM+MeHA, 4mM+MeHA, and 8mM+MeHA, the data of “current density of first-generation microneedles + no MeHA” and “current density of first-generation microneedles + MeHA” in Table 1 are from Figure 2 Current density at 60s of three intermittent tests of 4mM without MeHA and 4mM + MeHA.

[0089] Table 1:

[0090]

[0091] from Figure 2 , Figure 3 From Table 1, we can see that:

[0092] 1) Compared with the case without MeHA hydrogel (first generation microneedles + no MeHA), the current signal was weakened but more stable after adding MeHA hydrogel; 2) The fitting equation of the current density and glucose concentration of the first generation microneedles loaded with MeHA gel (first generation microneedles + MeHA) is:

[0093] I (μA / cm 2 )=3.45C(glucose)+5.64,R 2 =0.972. The formation of hydrogen peroxide, the product of the first generation of microneedles + MeHA, leads to the appearance of interference current. The current signal increases with the increase of glucose concentration, but the linear relationship is not good. R 2 =0.972. The first generation of microneedle test data showed that the average signal value decreased after adding MeHA, the current variance of three tests decreased, and the data stability was better.

[0094] Example 2

[0095] 1. Preparation of silver-based microneedles: same as in Example 1.

[0096] 2. Preparation of working electrode:

[0097] 2.1. Silver-based microneedles loaded with modified glucose oxidase: The silver-based microneedles obtained in step 1 were plasma cleaned, and 20 uL of a mixed solution of glucose oxidase and ferrocene (glucose concentration was 25 mg / mL, ferrocene concentration was 30 mg / mL) was drop-coated on the microneedle array, and placed in a cool condition to dry to obtain silver-based microneedles loaded with modified GOx.

[0098] 2.2. Loading of hydrogel: The silver-based microneedles loaded with modified GOx obtained in step 2.1 are immersed in a monomer or prepolymer aqueous solution of MeHA hydrogel, taken out and fully cured with ultraviolet light to obtain silver-based microneedles loaded with hydrogel and modified GOx as working electrodes.

[0099] 3. Preparation of reference electrode: same as in Example 1.

[0100] 4. Sensor assembly: The working electrode, counter electrode (silver-based microneedle obtained in step 1) and reference electrode (Ag / AgCl electrode obtained in step 3) prepared in step 2 are fixed on the patch by curing and bonding with silver paste to obtain an electrochemical microneedle patch.

[0101] 5-6. Same as 5-6 of Example 1.

[0102] 7. The microneedle with modified glucose oxidase loaded on the working electrode in Example 2 was named as the second-generation microneedle. The second-generation microneedles loaded with and without hydrogel MeHA were tested in agarose hydrogels with different glucose concentrations. The area of ​​the working electrode was 1 cm 2 The test object is agarose gel with glucose concentration of 0mM, 4mM, and 8mM. The test voltage is 0.1V, the test time is 120s, and the test is performed every 3h. The test is performed using Chenhua CHI660E electrochemical workstation. The results are as follows Figure 4 , 5 As shown in Table 2, Figure 5 The data comes from Figure 4 At 60s of the first test of 0mM+MeHA, 4mM+MeHA, and 8mM+MeHA, the data of “current density of second-generation microneedles + no MeHA” and “current density of second-generation microneedles + MeHA” in Table 2 are from Figure 4 Current density at 60s of three intermittent tests of 4mM without MeHA and 4mM + MeHA.

[0103] Table 2:

[0104]

[0105] from Figure 4 , Figure 5 From Table 2, we can see that:

[0106] 1) Compared with the case without MeHA hydrogel (second-generation microneedles + no MeHA), the current signal was weakened but more stable after adding MeHA hydrogel; 2) The current density of the second-generation microneedles loaded with MeHA gel (second-generation microneedles + MeHA) maintained a good linear relationship with the glucose concentration, and the fitting equation was I (μA / cm 2 )=2.95C(glucose)+6.69,R 2 =0.999. Compared with the first-generation microneedles in Example 1, the second-generation microneedles added ferrocene to avoid the interference current caused by the formation of hydrogen peroxide. The current signal increased linearly with the glucose concentration, and the current signal showed a good positive correlation with the concentration. R 2 =0.999. The second-generation microneedle test data showed that the average signal value decreased after adding MeHA, the current variance of three tests decreased, and the data stability was better.

[0107] Example 3

[0108] 1. Preparation of silver-based microneedles: same as in Example 1.

[0109] 2. Preparation of working electrode:

[0110] 2.1. Silver-based microneedles loaded with modified glucose oxidase: The silver-based microneedles prepared in step 1 were plasma cleaned, and 20uL of a mixed solution of glucose oxidase and single-walled carbon nanotubes (glucose concentration was 25mg / mL, single-walled carbon nanotube concentration was 30mg / mL) was drop-coated on the microneedle array and placed in a cool place to dry to obtain silver-based microneedles loaded with modified GOx.

[0111] 2.2. Loading of hydrogel: The silver-based microneedles loaded with modified GOx obtained in step 2.1 are immersed in a monomer or prepolymer aqueous solution of MeHA hydrogel, taken out and fully cured with ultraviolet light to obtain silver-based microneedles loaded with hydrogel and modified GOx as working electrodes.

[0112] 3. Preparation of reference electrode: same as in Example 1.

[0113] 4. Sensor assembly: The working electrode, counter electrode (silver-based microneedle obtained in step 1) and reference electrode (Ag / AgCl electrode obtained in step 3) prepared in step 2 are fixed on the patch by curing and bonding with silver paste to obtain an electrochemical microneedle patch.

[0114] 5-6. Same as 5-6 of Example 1.

[0115] 7. The microneedle with modified glucose oxidase loaded on the working electrode in Example 3 was named as the third-generation microneedle. The third-generation microneedles loaded with and without hydrogel MeHA were tested in agarose hydrogels with different glucose concentrations. The area of ​​the working electrode was 1 cm 2 The test object is agarose gel with glucose concentration of 0mM, 4mM, and 8mM. The test voltage is 0.1V, the test time is 120s, and the test is performed every 3h. The test is performed using Chenhua CHI660E electrochemical workstation. The results are as follows Figure 6 , 7 As shown in Table 3, Figure 7 The data comes from Figure 6 The current density at 60s of the first test of 0mM+MeHA, 4mM+MeHA, and 8mM+MeHA. The data of “current density of the third-generation microneedles + no MeHA” and “current density of the third-generation microneedles + MeHA” in Table 3 are from Figure 6 Current density at 60s of three intermittent tests of 4mM without MeHA and 4mM + MeHA.

[0116] Table 3:

[0117]

[0118]

[0119] from Figure 6 , Figure 7 As shown in Table 3, compared with the second-generation microneedles, carbon nanotubes are further added to the third-generation microneedles, which is beneficial to electron transfer and increases the response current (current density*area of ​​the working electrode). The third-generation microneedle test data reading shows that the average signal value decreases after adding MeHA, the current variance of three tests decreases, and the data stability is better.

[0120] from Figure 7 It can be seen that the current density of the third-generation microneedles loaded with MeHA gel (third-generation microneedles + MeHA) maintains a good linear relationship with the glucose concentration, and the fitting equation is I (μA / cm 2 )=7.68C(glucose)+10.81,R 2 =0.999, sensitivity is 7.68μA·cm -2 mM -1 .

[0121] Comparative Example 1

[0122] 1-6. Same as Example 3.

[0123] 7. The difference from Example 3 is that the three-generation microneedles loaded with and without hydrogel MeHA were tested in agarose hydrogels with different glucose concentrations. The test object was agarose gel with a glucose concentration of 4mM, the test voltage was 0.1V, the test time was 600s, and the test was performed using Chenhua CHI660E electrochemical workstation. The results are shown in Figure 8As shown, in a 4mM glucose PBS solution, three generations of microneedles were used to continuously test for ten minutes to observe the stability of the signal. The initial signal of the microneedle without MeHA was large, but the decay was fast. The microneedle response current of the MeHA-using microneedle was stable, but it would also decay slowly for a long time, that is, the current was relatively stable within about 120, but the current signal gradually decayed after a certain period of time. Therefore, compared with Comparative Example 1, the intermittent test in the embodiment is better, and the test signal maintains good consistency and stability. Its signal variance is shown in Tables 1, 2, and 3, and the data stability is excellent. This is because when the measurement circuit is disconnected, the detected molecules in the electrochemical reaction are re-enriched on the surface of the working electrode and maintain a certain amount of stability in the area where the polymer hydrogel is located. As the electrochemical reaction continues, the detected substances such as glucose pre-stored in the hydrogel continue to undergo electrochemical reactions at the working electrode. In each unit reaction, the oxidized and reduced states of glucose oxidase, as well as hydrogen peroxide and oxygen, self-circulate during the reaction. Therefore, the current in the disconnected circuit can be recorded, read and processed intermittently to achieve continuous monitoring of blood sugar values. Furthermore, the glucose concentration can be obtained under the condition of known current through the empirical formula between current and glucose concentration. Based on the kinetic relationship between glucose concentration and current value, the current value in the circuit is measured and the glucose concentration is accurately calculated, which can better ensure the accuracy of the glucose concentration measurement value and ensure the practicality of the microneedle patch.

[0124] The electrochemical microneedle patch in the specific embodiment of the present invention can complete the detection of blood glucose concentration without puncturing blood vessels and nerves, effectively reducing the risk of infection and pain. By introducing the intermittent testing method, the electrochemical microneedle patch can monitor blood glucose in a more intelligent and energy-saving way, while ensuring the continuity and accuracy of the monitoring data, reducing the adverse factors of electrical signal distortion caused by the rapid decrease in glucose concentration, and the detected electrical signal is converted to directly reflect the change in blood glucose concentration, effectively solving the problem of uncertainty, suitable for use in home and medical environments, and providing a more convenient and comfortable blood glucose monitoring method for diabetic patients. In addition, this electrochemical microneedle patch can also be integrated into wearable devices to achieve real-time and continuous blood glucose monitoring, which helps to better manage diabetes.

[0125] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, it can also make several substitutions or modifications to these described embodiments, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention. In the description of this specification, the description of the reference terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily target the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In the absence of mutual contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. An electrochemical microneedle patch for detecting physiological markers in skin tissue fluid, characterized in that: It includes a patch and a counter electrode, a reference electrode and a working electrode fixed on the patch; the counter electrode, the reference electrode and the working electrode are gathered into a signal connection port on one side of the patch in the form of a printed circuit; The counter electrode, the reference electrode and the working electrode each comprise at least one conductive microneedle electrode; The length of each conductive microneedle electrode is sufficient to penetrate the epidermis of the skin and reach the dermis. The conductive microneedle electrode of the working electrode has a detection component for detecting physiological markers in skin tissue fluid, and the outer surface of the conductive microneedle electrode of the working electrode is covered with a hydrogel layer. The hydrogel layer has a mesh structure that forms a swollen layer without dissolving after absorbing skin tissue fluid in the dermis. The mesh structure allows the physiological marker to be detected to diffuse into or move away from the surface of the working electrode.

2. The electrochemical microneedle patch according to claim 1, characterized in that: The areas of the counter electrode, the reference electrode and the working electrode on the patch are each 1-100 square millimeters; the spacing between the conductive microneedle electrodes of the counter electrode, the reference electrode and the working electrode is each 0.5-5 millimeters, and the length of the conductive microneedle electrode is 0.1-3 millimeters, preferably the length of the conductive microneedle electrode is 0.4-2.4 millimeters; the number of the conductive microneedle electrodes of the counter electrode, the reference electrode and the working electrode is each between 1-100.

3. The electrochemical microneedle patch according to claim 1, characterized in that: The hydrogel in the hydrogel layer is at least one of vinyl functionalized hyaluronic acid, acrylated hyaluronic acid polymer, sodium hyaluronate polymer, chitosan, alginate, sodium carboxymethyl cellulose, and polyethylene glycol. Preferably, the hydrogel is methacrylated hyaluronic acid.

4. The electrochemical microneedle patch according to claim 1, characterized in that: The thickness of the hydrogel layer is 100 nanometers to 500 micrometers, and preferably the thickness of the hydrogel layer is 1 micrometer to 100 micrometers.

5. The electrochemical microneedle patch according to claim 1, characterized in that: The physiological marker in the skin tissue fluid is one of glucose, uric acid, acetone, alcohol, and drug metabolites; the detection component is at least one of enzymes, hormones, antibodies, DNA, RNA, and modified enzymes; Preferably, the enzyme is at least one of glucose oxidase, catalase, amylase, creatine kinase, lactate oxidase, and cholesterol oxygenase; the hormone is growth hormone and / or thyroid stimulating hormone; Preferably, the modified enzyme is glucose oxidase modified with an electron mediator; preferably, the electron mediator is at least one of metal oxides, gold nanoparticles, platinum nanoparticles, cobalt nanoparticles, carbon nanotubes, graphene, Prussian blue, ferrocene, titanium nitride, carbon nitride, and BiFeO3 particles; Preferably, the physiological marker in the skin tissue fluid is glucose, and the detection component is glucose oxidase modified with ferrocene, or the detection component is glucose oxidase modified with carbon nanotubes.

6. The electrochemical microneedle patch according to claim 1, characterized in that: The microneedle bodies of the conductive microneedle electrodes of the counter electrode, the reference electrode and the working electrode are all inert conductors that do not dissolve in electrochemical reactions; preferably, the inert conductor materials of the microneedle bodies of the counter electrode, the reference electrode and the working electrode are the same; preferably, the material of the inert conductor is at least one of platinum, gold, copper, silver and carbon materials.

7. An electrochemical microneedle device for detecting physiological markers in skin tissue fluid, characterized in that: The invention comprises the electrochemical microneedle patch, the circuit board and the control system as described in any one of claims 1 to 6, wherein the electrochemical microneedle patch is connected to the circuit board and the control system through the signal connection port, the control system is configured to intermittently start the electrochemical microneedle patch at a predetermined intermittent time and a predetermined voltage to detect physiological markers in skin tissue fluid to obtain current signals, and the control system is also configured to obtain the concentration of the physiological marker to be measured according to the current signal.

8. The electrochemical microneedle device according to claim 7, characterized in that The predetermined intermittent time is 0-24h, preferably more than 40 minutes, more preferably 3h; the predetermined voltage is 0-0.8V, preferably 0.1V; preferably, the continuous measurement time of the electrochemical microneedle patch after each startup is more than 30s, preferably the continuous measurement time is 30-120 seconds; preferably, the signal connection port of the electrochemical microneedle patch is detachably connected to the circuit board through a plug connector or an elastic clip; preferably, the control system is arranged on the circuit board so that the electrochemical microneedle device is constituted as a wearable device.

9. The electrochemical microneedle device according to claim 7, characterized in that: The control system comprises: A control unit, for applying the predetermined voltage to the electrochemical microneedle patch at the predetermined intermittent time; preferably, the control unit includes a timer, for determining the time interval for applying the predetermined voltage, that is, determining the predetermined intermittent time A current sensor for measuring the current during the period when the predetermined voltage is applied to the electrochemical microneedle patch; preferably, the current sensor comprises a signal amplifier for amplifying the current signal received from the electrochemical microneedle patch; a data logger for storing data received by the current sensor; The analysis unit is used to process the data stored in the data recorder to obtain the concentration of the physiological marker to be measured; preferably, the analysis unit pre-stores a quantitative relationship between the input current signal and the level of the physiological marker to be measured.

10. A method for detecting physiological markers in skin tissue fluid using the electrochemical microneedle device according to any one of claims 7 to 9 for non-diagnostic purposes, characterized in that: The steps include: (1) attaching the electrochemical microneedle patch to the skin surface of a subject, ensuring that the conductive microneedle electrodes penetrate the epidermis to reach the dermis; (2) before measurement, applying a stabilization voltage to the electrochemical microneedle patch through the control system to allow current stabilization; (3) intermittently starting the electrochemical microneedle patch with a predetermined intermittent time and a predetermined voltage through the control system to detect physiological markers in skin tissue fluid to obtain current signals; (4) The concentration of the physiological marker to be measured is obtained according to the current signal through the control system.

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