Non-enzyme microneedle sensor and preparation method thereof

Abstract

The invention discloses an enzyme-free microneedle sensor and a preparation method thereof. The enzyme-free microneedle sensor comprises a flexible substrate, a working electrode, a counter electrode and a reference electrode, the flexible substrate is provided with a lead system and a signal acquisition system, and each of the working electrode, the counter electrode and the reference electrode comprises a microneedle base and a microneedle array formed on the microneedle base; microneedle bases in the working electrode, the counter electrode and the reference electrode are distributed on the flexible substrate and are connected with the signal acquisition system through the lead system, and a microneedle array of the working electrode comprises a microneedle body and an anion exchange type ionomer loaded on the microneedle body. The non-enzyme microneedle sensor is used for detecting the content of a physiological marker, has the performance which is almost good in a neutral environment and an alkaline environment, still has good performance after being placed for a long time, improves the detection stability and reproducibility, solves the problem that organic enzyme is easy to inactivate, and has stronger anti-interference capability.

Description

Technical Field

[0001] The invention relates to a physiological marker detection sensor, and in particular to an enzyme-free microneedle sensor and a preparation method thereof. Background Art

[0002] With economic progress and improved living standards, people are paying more and more attention to their health. However, the prevalence of chronic diseases such as diabetes, gout, and hyperlipidemia has increased year by year, and these diseases and their complications have had a significant impact on the daily lives of many people. Each of these diseases has a specific detection method: taking the current main clinical methods as an example, diabetes is diagnosed by measuring blood sugar levels, gout is diagnosed by detecting uric acid levels in the blood, and hyperlipidemia requires checking fat and triglyceride levels in the blood. However, due to individual living habits and physical differences, many patients may suffer from multiple diseases at the same time, so they need to frequently monitor different health indicators in the body. This method not only brings physical discomfort and inconvenience to patients, but also poses a risk of infection.

[0003] In addition to blood testing, there are also methods for measuring sweat and tears, but these methods have more or less problems such as difficulty in collection and poor measurement accuracy, and are not easy to promote. Microneedle transdermal testing is an emerging minimally invasive method for detecting physiological markers, which has the advantages of small wounds, imperceptible pain, and low risk of wound infection. However, how to increase the collection volume, reduce immune infection, and thus improve measurement accuracy remains the main challenge at present.

[0004] Blood glucose sensors based on glucose oxidase (GOx) include the following. In the first-generation sensor, glucose is oxidized to gluconolactone under the action of GOx and oxygen dissolved in the solution, and hydrogen peroxide is produced at the same time; in this process, the concentration of hydrogen peroxide produced is detected electrochemically, specifically by applying a specific potential to the working electrode and detecting the resulting current. The concentration of glucose in the solution has a linear relationship with the concentration of hydrogen peroxide produced. The second-generation sensor uses small molecule electron mediators (ferrocene and its derivatives, Prussian blue, quinones, nanomaterials, etc.) to replace the electron channel between the enzyme and the electrode, and detects the glucose concentration by the change in the oxidation current of the mediator. The third-generation sensor directly modifies the enzyme on a porous electropolymer electrode, and the active center of the enzyme is close to the electrode, which is conducive to the transfer of electrons.

[0005] Enzymes have some limitations in detection as biocatalysts. Their activity is greatly affected by environmental factors such as temperature and pH value, and experimental conditions need to be strictly controlled to maintain the activity of the enzyme. Enzyme-free detection methods overcome these shortcomings and provide better stability and reproducibility. Enzyme-free detection does not require the use of expensive and unstable enzymes, which significantly reduces costs. At the same time, enzyme-free detection methods do not rely on the catalytic action of enzymes, so they have stronger anti-interference capabilities and can adapt to more diverse sample types and environmental conditions.

[0006] 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

[0007] The invention provides an enzyme-free microneedle sensor and a preparation method thereof, which overcomes the easy inactivation of enzymes and their instability in electrochemical detection.

[0008] The present invention adopts the following technical solution:

[0009] In the first aspect, the present invention provides an enzyme-free microneedle sensor, comprising a flexible substrate, a working electrode, a counter electrode and a reference electrode; the flexible substrate has a lead system and a signal acquisition system, the working electrode, the counter electrode and the reference electrode all comprise a microneedle base and a microneedle array formed on the microneedle base, the microneedle bases in the working electrode, the counter electrode and the reference electrode are distributed on the flexible substrate and connected to the signal acquisition system through the lead system, and the microneedle array of the working electrode comprises a microneedle body and an anion exchange ionomer loaded on the microneedle body.

[0010] In a second aspect, the present invention provides a method for preparing the enzyme-free microneedle sensor described in the first aspect, comprising the following steps: (1) preparing a counter electrode, a working electrode and a reference electrode respectively; (2) fixing the microneedle bases of the counter electrode, the working electrode and the reference electrode on a flexible substrate and electrically connecting the microneedle bases of the counter electrode, the working electrode and the reference electrode to the signal acquisition system through the lead system in the flexible substrate.

[0011] The present invention has the following beneficial effects: the enzyme-free microneedle sensor of the present invention is used to detect the content of physiological markers. By loading an anion exchange ionomer on the working electrode, the enzyme-free microneedle sensor can have a performance in a neutral environment that is almost as good as that in an alkaline environment, and still has good performance after being placed for a long time, thereby improving the stability and reproducibility of detection, solving the problem of easy inactivation of organic enzymes, and having stronger anti-interference ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic top view of the enzyme-free microneedle sensor in a specific embodiment of the present invention;

[0013] Figure 2 is a side view schematic diagram of an enzyme-free microneedle sensor in a specific embodiment of the present invention;

[0014] Figure 3 is a schematic diagram of a microneedle of a working electrode of an enzyme-free microneedle sensor in a specific embodiment of the present invention;

[0015] Figure 4 The results of electrochemical It detection of the enzymatic microneedle sensor in Comparative Example 1;

[0016] Figure 5 The results of electrochemical CV detection of the enzymatic microneedle sensor in Comparative Example 2;

[0017] Figure 6 The results of electrochemical CV detection of the enzyme-free microneedle sensor in Example 1;

[0018] Figure 7 The results of electrochemical CV detection of the enzyme-free microneedle sensor in Example 2;

[0019] Figure 8 The results of electrochemical CV detection of the enzyme-free microneedle sensor in Example 3;

[0020] Fig. 9 The results of electrochemical CV detection of the enzyme-free microneedle sensor in Example 4;

[0021] Fig.10 The results of electrochemical CV detection of the enzyme-free microneedle sensor in Example 5;

[0022] Fig.11 The results of electrochemical CV detection of the enzyme-free microneedle sensor in Example 6 are shown in FIG. DETAILED DESCRIPTION

[0023] 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.

[0024] The specific embodiment of the present invention provides an enzyme-free microneedle sensor, such as Figure 1-3As shown, it includes a flexible substrate 1, a working electrode 2, a counter electrode 3 and a reference electrode 4; the flexible substrate 1 has a lead system 11 and a signal acquisition system 12, the working electrode 2 includes a microneedle base 21 and a microneedle array 22 formed on the microneedle base 21, the counter electrode 3 includes a microneedle base 31 and a microneedle array 32 formed on the microneedle base 31, and the reference electrode 4 includes a microneedle base 41 and a microneedle array 42 formed on the microneedle base 41. The microneedle bases 21, 31, 41 in each electrode are distributed on the flexible substrate 1 and connected to the signal acquisition system 12 through the lead system 11, and the microneedle array 21 of the working electrode 2 includes a microneedle body 211 and an anion exchange ionomer 212 loaded on the microneedle body 211.

[0025] In the present invention, the working electrode 2, the counter electrode 3 and the reference electrode 4 do not contain any enzyme. When used, the fine needle-shaped electrodes (such as Figure 2 As shown, the microneedle array area 5 of the working electrode 2, the counter electrode 3 and the reference electrode 4 penetrates the skin surface and epidermis and enters the deeper dermis area. The anion exchange type ionomer loaded on the working electrode can form an anion exchange membrane. This membrane not only provides a suitable microenvironment and promotes the electrochemical reaction, but also its ion selective transmission characteristics help to improve the selectivity and sensitivity of the sensor. The enzyme-free microneedle sensor is used to detect the content of physiological markers. The concentration of physiological markers is detected by measuring the potential difference between the working electrode and the reference electrode. For example, the enzyme-free microneedle sensor of the present invention can detect in vivo analytes such as glucose content in skin tissue fluid, and realizes efficient detection of physiological markers such as glucose. Through electrochemical testing, the enzyme-free microneedle sensor can effectively monitor the glucose concentration in subcutaneous tissue fluid under a pH environment (neutral environment) close to human skin tissue fluid. This enzyme-free detection method improves the stability and reproducibility of the detection, solves the problem of easy inactivation of organic enzymes, and has stronger anti-interference ability, providing a minimally invasive and real-time monitoring tool for diabetes management, and also provides ideas for other in vivo analyte detection.

[0026] Take the glucose content in the skin tissue fluid of the test body as an example. In the glucose oxidation reaction, the transmission of hydroxide ions helps to maintain a suitable pH environment and promote the oxidation of glucose. When these electrodes are inserted into the skin, the glucose molecules are oxidized on the working electrode, and the generated electrons are transmitted through the electrode to form a current signal. By matching the working electrode, reference electrode and counter electrode, current and voltage signal readings are given; the anion exchange ionomer loaded on the working electrode can form an anion exchange membrane. The ion exchange groups of the anion exchange membrane carry a positive charge and can interact strongly with anions (especially hydroxide ions OH-). These groups can capture anions (especially hydroxide ions OH-), allowing them to move through the membrane from high concentration areas to low concentration areas. That is, from cathode to anode, that is, the anion exchange ionomer The substance allows hydroxide ions to pass through, while It prevents other species that may interfere with the glucose oxidation reaction and promotes the electrochemical reaction. It increases the selectivity and sensitivity of the working electrode for the targeted electrochemical reaction of glucose by increasing the local concentration of anions (especially hydroxide ions OH-). Therefore, the enzyme-free microneedle sensor of the present invention can effectively detect the glucose level in the body under enzyme-free conditions, providing a potential minimally invasive monitoring tool for diabetes management.

[0027] In some embodiments, the microneedle body in the working electrode includes a metal-based microneedle or a polymer microneedle containing a metal coating.

[0028] In some embodiments, the metal-based microneedle is one of a silver-based, gold-based, platinum-based, nickel-based or silver-coated copper-based microneedle.

[0029] In some embodiments, the metal-based microneedle is made by the following preparation method: 50wt%-95wt% of metal powder and 5wt%-50wt% of curing agent are evenly mixed into a slurry, the slurry is transferred to a microneedle polymer mold, bubbles are removed under vacuum conditions, the mold is filled and then cured, and finally demolded; 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.

[0030] In some embodiments, the microneedle body in the working electrode is a silver-based microneedle, which is prepared by the following preparation method: 50wt%-95wt% of silver powder and 5wt%-50wt% of an epoxy resin precursor formula (bisphenol A resin and methyl nadic anhydride are mixed in a mass ratio of 1:1.1) are evenly mixed into a slurry, the slurry is transferred to a microneedle polymer mold, bubbles are removed under vacuum conditions, the mold is filled and then solidified, and finally demolded.

[0031] In some embodiments, the polymer microneedle containing a metal coating is made by the following preparation method: a layer of silver or copper is deposited as a base layer on a polymer substrate by magnetron sputtering or chemical plating, and the thickness of the base layer is 10nm-40μm, and then a silver layer is plated on the base layer by an electroplating process, and the thickness of the silver layer is 100nm-100μm, preferably 10μm-40μm.

[0032] like Figure 3 As shown, in some embodiments, the microneedle body 21 of the working electrode is a silver-based microneedle, and the silver-based microneedle also has a coating 213, and the anion exchange ionomer 22 is loaded on the coating 213, and the coating is a nickel layer, a gold layer or a copper layer. Preferably, the coating is a nickel layer or a gold layer. The silver-based microneedle is treated with nickel plating, gold plating or copper plating to improve the detection sensitivity of target molecules such as glucose, enhance the electrocatalytic activity of the microneedle, and thus improve the performance of the sensor. For example, the nickel layer or the copper layer can increase the hardness and wear resistance of the microneedle, make the microneedle more stable when penetrating the skin, reduce the risk of breakage, and extend the service life. At the same time, it provides corrosion protection to prevent the microneedle from being corroded in the biological environment and maintain its structural and functional stability. The gold layer can enhance conductivity and chemical stability. In other embodiments, when the microneedle body of the working electrode is a gold-based microneedle or a nickel-based microneedle, the above-mentioned coating can be omitted. Preferably, the microneedle body of the working electrode is a silver-based microneedle and a coating is provided on the silver-based microneedle.

[0033] In some embodiments, the microneedle bodies of the working electrode, the counter electrode, and the reference electrode are made of the same material, preferably all silver-based microneedles.

[0034] In some embodiments, the anion exchange ionomer includes at least one of Fumion FAA-3 and PiperION-A5-HCO3-EtOH; the thickness of the anion exchange ionomer loaded on the microneedle body is 100nm-100μm. Loading of anion exchange ionomer: the microneedles can be uniformly loaded by immersing them in a solution of ionomer dissolved in acetonitrile, or by using compressed air spray, ultrasonic spray or electrostatic spray technology at 80°C. The anion exchange ionomer can also be uniformly coated on the surface of the microneedle body by dripping, and the dripping volume is precisely controlled according to the size of the microneedles and the expected load.

[0035] 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: using a silver-based microneedle as a raw material, immersing it in a 0.05-0.3 mol / L (preferably 0.1-0.3 mol / L) FeCl3 solution, forming a layer of AgCl on the surface of the microneedle body through an electrochemical reaction, and filling a saturated potassium chloride KCl solution around the microneedle to form a complete Ag / AgCl electrode.

[0036] In some embodiments, the counter electrode is a silver-based microneedle or a polymer microneedle containing a metal coating, and preferably the counter electrode is a silver-based microneedle.

[0037] In some embodiments, the flexible substrate 1 includes a wire layer 13, a support layer 14 and an adhesive layer 15, wherein the wire layer 13 is embedded in the support layer 14, and the microneedle bases of the working electrode 2, the counter electrode 3 and the reference electrode 4 are distributed on the adhesive layer 15 and connected to the signal acquisition system 12 through the lead system 11 in the wire layer 13, and the adhesive layer 15 is used to fix the flexible substrate 1 to the skin surface during detection, and the support layer 14 is combined with the adhesive layer 15, and the support layer provides mechanical stability and support for the entire system. The flexible substrate can be firmly combined with the working electrode, the counter electrode and the reference electrode by physical bonding, intercalation or chemical linking. Preferably, silver paste curing is used as the connection method. The flexible substrate is flexible and can be bent within a certain range, and the lead system in the wire layer can be ensured not to be damaged. Preferably, the material of the flexible substrate is transparent.

[0038] In some embodiments, the length and width of the enzyme-free microneedle sensor do not exceed 75 mm.

[0039] In some embodiments, the thickness of the flexible substrate is 0.5 mm-3 mm.

[0040] In some embodiments, the material of the support layer is at least one of dimethylsiloxane, polysiloxane elastomer, PET, acrylic polymer, and silicone polymer.

[0041] In some embodiments, the microneedle array of the working electrode is a 7×10 to 10×15 array, the array length does not exceed 20 mm, the width does not exceed 20 mm, and the spacing between each microneedle ranges from 200 μm to 2000 μm.

[0042] In some embodiments, the microneedles in the microneedle array of the working electrode, the counter electrode, and the reference electrode have a shape of a pointed prism, a pyramid, a pointed cylinder, or a cone, and their height is between 150 μm and 1500 μm; the height of the microneedle base of the working electrode, the counter electrode, and the reference electrode is 0.2 mm to 3 mm.

[0043] The specific embodiment of the present invention also provides a method for preparing the enzyme-free microneedle sensor, which comprises the following steps:

[0044] (1) preparing a counter electrode, a working electrode and a reference electrode respectively;

[0045] (2) The microneedle bases of the counter electrode, the working electrode and the reference electrode are fixed on a flexible substrate and electrically connected to the signal acquisition system through a lead system in the flexible substrate.

[0046] The present invention uses agarose hydrogel as simulated skin, soaks the agarose hydrogel in glucose solutions of different concentrations to make it absorb glucose molecules in the solution to reach a desired specific concentration, and then inserts a microneedle sensor into the agarose hydrogel and closely attaches it to perform an electrochemical test to evaluate the measurement accuracy of the microneedle sensor. When performing the electrochemical test, a voltage of 100-800mV is applied, and the working time is 2-10min, preferably 5min.

[0047] The present invention is described in detail below through some embodiments and comparative examples.

[0048] Example 1

[0049] 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, and the mixed silver paste is transferred to a microneedle polymer mold. 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, which includes a microneedle base and a microneedle array.

[0050] 2. Preparation of working electrode (nickel-silver-based microneedle loaded with ionomer):

[0051] 2.1. Ni plating of silver-based microneedles: clean the plating tank thoroughly with pure water, inject part of the pure water into the tank, add 3.4 ml of commercial Ni-866A and 1.4 ml of Ni-866C under stirring; add pure water to the required volume, heat the plating solution to 38-41°C; check the pH value of the plating solution, and adjust it with 50% ammonia water or 10% sulfuric acid if necessary to keep it at 9.6-11.5; put the microneedle array of silver-based microneedles obtained in step 1 into the tank, immerse for 3-5 minutes, and obtain nickel-plated silver-based microneedles.

[0052] 2.2. Loading of ionomer (Fumion FAA-3): Plasma cleaning was performed on the nickel-silver-based microneedles obtained in step 2.1 to make them hydrophilic. The microneedle array of nickel-silver-based microneedles was immersed in a solution of Fumion FAA-3 (1 mg / mL dissolved in acetonitrile) for 120 s, taken out, washed with deionized water and dried to obtain nickel-silver-based microneedles loaded with ionomer as working electrodes. Some of the nickel-silver-based microneedles loaded with ionomer were placed for 24 hours, and some were placed for 240 hours.

[0053] 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.

[0054] 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 a flexible substrate by curing and bonding with silver paste to obtain an enzyme-free microneedle sensor.

[0055] 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.

[0056] 6. Insert the microneedle sensor into the agarose hydrogel with different glucose concentrations and close it. Insert the end of the flexible substrate (the end where the signal acquisition system is located) into the test device. The test device includes a lithium battery, a circuit, a data processing module, and a computing module. A double-sided substrate design can be adopted. The upper and lower layers are conductive copper substrates. The back wiring is connected to the printed circuit board (PCB), connected to the external terminal device via Bluetooth technology, receives the test instructions and sends the test results. The interface of the measuring device can be combined with the signal acquisition system on the microneedle sensor by conductive glue, silver paste bonding, contact pressing, welding, etc., preferably contact pressing. The intensity of the current signal is proportional to the concentration of ISF (Interstitial Fluid) in the sample being tested.

[0057] 7. Electrochemical test: The terminal device sends a command to apply voltage and measure the current response on the working electrode. At different glucose concentrations, the current response of the working electrode is recorded, and these data points are used to draw the concentration-response curve.

[0058] like Figure 6 As shown in Example 1, the performance of the nickel-plated silver-based microneedles loaded with ionomer after 24 hours and 240 hours is compared. The response current and linearity are not much different, indicating that there is no Enzyme detection is not affected by long-term storage. Maintain good performance, specifically, place 24 The curve of nickel-plated silver-based microneedles loaded with FAA-3 ionomer after 240 hours and the curve of nickel-plated silver-based microneedles loaded with FAA-3 ionomer after 240 hours are both linear, R 2 The value is close to 1, and the curve R after 24 hours 2 The curve R after 240 hours is 0.999. 2 It is 0.998, indicating that the enzyme-free microneedle sensor still maintains good performance after long-term placement.

[0059] Example 2

[0060] 1. Preparation of silver-based microneedles: same as in Example 1.

[0061] 2. Preparation of working electrode (gold-plated silver-based microneedle loaded with ionomer):

[0062] 2.1. Au plating of silver-based microneedles: The silver-based microneedles obtained in step 1 were cleaned and activated, 100 mL of gold (ammonium and gold sulfite complex solution, 100 g / L), 80 g / L of ammonium sulfite (free), and 30 g / L of ammonium sulfate were mixed evenly, and enough water was added to 1 L as the plating solution. The pretreated silver-based microneedle array was immersed in the plating solution at a pH of 6.8, a temperature of 56 °C, and a current density of 0.25 A / dm 2 Electroplating is carried out under the condition of 400 μm, and after completion, it is dried to obtain gold-plated silver-based microneedles.

[0063] 2.2. Loading of ionomer (Fumion FAA-3): Plasma cleaning was performed on the gold-plated silver-based microneedles obtained in step 2.1 to make them hydrophilic. The microneedle array of gold-plated silver-based microneedles was immersed in a solution of Fumion FAA-3 (1 mg / mL dissolved in acetonitrile) for 120 s, taken out, washed with deionized water and dried to obtain gold-plated silver-based microneedles loaded with ionomer as working electrodes. Some of the gold-plated silver-based microneedles loaded with ionomer were placed for 24 hours, and some were placed for 240 hours.

[0064] 3. Preparation of reference electrode: same as in Example 1.

[0065] 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 a flexible substrate by curing and bonding with silver paste to obtain an enzyme-free microneedle sensor.

[0066] 5-7. Same as 5-7 of Example 1.

[0067] like Figure 7 As shown, in Example 2, the curve of the gold-plated silver-based microneedle loaded with FAA-3 ionomer after 24 hours and the curve of the gold-plated silver-based microneedle loaded with FAA-3 ionomer after 240 hours are both linear, R 2 The value is close to 1, and the curve R after 24 hours 2 The curve R after 240 hours is 0.999. 2 It is 0.998, indicating that the enzyme-free microneedle sensor still maintains good performance after long-term placement.

[0068] Example 3

[0069] 1. Preparation of silver-based microneedles: same as in Example 1.

[0070] 2. Preparation of working electrode (nickel-silver-based microneedle loaded with ionomer):

[0071] 2.1. Ni plating of silver-based microneedles: same as step 2.1 of Example 1.

[0072] 2.2. Loading of ionomer (PiperION-A5-HCO3-EtOH): Plasma clean the nickel-silver-based microneedles obtained in step 2.1 to make them hydrophilic. Soak the microneedle array of nickel-silver-based microneedles in PiperION-A5-HCO3-EtOH (2 mg / mL dissolved in acetonitrile) solution for 200 seconds, take it out, wash it with deionized water and dry it to obtain nickel-silver-based microneedles loaded with ionomer as working electrodes. Some of the nickel-silver-based microneedles loaded with ionomer were placed for 24 hours, and some were placed for 240 hours.

[0073] 3. Preparation of reference electrode: same as in Example 1.

[0074] 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 a flexible substrate by curing and bonding with silver paste to obtain an enzyme-free microneedle sensor.

[0075] 5-7. Same as 5-7 of Example 1.

[0076] like Figure 8 As shown, in Example 3, after 24 hours of placement, the load

[0077] The curves of nickel-silver-based microneedles coated with PiperION-A5-HCO3-EtOH ionomer and the curves of nickel-silver-based microneedles coated with PiperION-A5-HCO3-EtOH ionomer after 240 hours of storage are both linear, R 2 The value is close to 1, and the curve R after 24 hours 2 The curve R after 240 hours is 0.999. 2 It is 0.998, indicating that the enzyme-free microneedle sensor still maintains good performance after long-term placement.

[0078] Example 4

[0079] 1. Preparation of silver-based microneedles: same as in Example 1.

[0080] 2. Preparation of working electrode (gold-plated silver-based microneedle loaded with ionomer):

[0081] 2.1. Au plating of silver-based microneedles: same as step 2.1 of Example 2.

[0082] 2.2. Loading of ionomer (PiperION-A5-HCO3-EtOH): Plasma clean the gold-plated silver-based microneedles obtained in step 2.1 to make them hydrophilic. Soak the microneedle array of gold-plated silver-based microneedles in PiperION-A5-HCO3-EtOH (2 mg / mL dissolved in acetonitrile) solution for 200 seconds, take it out, wash it with deionized water and dry it to obtain gold-plated silver-based microneedles loaded with ionomer as working electrodes. Some of the gold-plated silver-based microneedles loaded with ionomer were placed for 24 hours, and some were placed for 240 hours.

[0083] 3. Preparation of reference electrode: same as in Example 1.

[0084] 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 a flexible substrate by curing and bonding with silver paste to obtain an enzyme-free microneedle sensor.

[0085] 5-7. Same as 5-7 of Example 1.

[0086] like Fig. 9 As shown, in Example 4, the load after 24 hours

[0087] The curves of the gold-plated silver-based microneedles with PiperION-A5-HCO3-EtOH ionomer and the curves of the gold-plated silver-based microneedles loaded with PiperION-A5-HCO3-EtOH ionomer after 240 hours of storage are both linear, R 2 The value is close to 1, and the curve R after 24 hours 2 The curve R after 240 hours is 0.999. 2 It is 0.998, indicating that the enzyme-free microneedle sensor still maintains good performance after long-term placement.

[0088] Example 5

[0089] 1. Preparation of silver-based microneedles: same as in Example 1.

[0090] 2. Preparation of working electrode (nickel-plated microneedle loaded with ionomer):

[0091] 2.1. Ni plating of silver-based microneedles: same as step 2.1 of Example 1.

[0092] 2.2. Loading of ionomer: Plasma clean a portion of the nickel-plated silver-based microneedles prepared in step 2.1 to make them hydrophilic. Soak the microneedle array of nickel-plated silver-based microneedles in FAA-3 (1 mg / mL dissolved in acetonitrile) solution for 120 seconds, take it out, wash it with deionized water and dry it to obtain nickel-plated silver-based microneedles loaded with ionomer as working electrodes. A portion of the nickel-plated silver-based microneedles prepared in step 2.1 of this example are not treated in step 2.2 and used as working electrodes (which can be called "nickel-plated silver-based microneedles without ionomer loading").

[0093] 3. Preparation of reference electrode: same as in Example 1.

[0094] 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 a flexible substrate by curing and bonding with silver paste to obtain an enzyme-free microneedle sensor.

[0095] 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, 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 use. Use dilute HCl solution to adjust the pH to 6.5-7.4 in one part, and use NaOH solution to adjust the pH to 12.5-13.5 in the other part. Weigh a certain amount of agar powder and dissolve it in PBS solution (the mass fraction of agar powder is 0.7%-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 it into suitable sizes and demold it, soak it in PBS neutral solution and alkaline solution 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.

[0096] 6. The nickel-plated silver-based microneedle sensors without ionomer loading were inserted into neutral and alkaline agarose hydrogels with different glucose concentrations and stuck tightly together. The nickel-plated silver-based microneedle sensors loaded with ionomer were inserted into neutral agarose hydrogels with different glucose concentrations. The end of the flexible substrate was inserted into the test device, and the test device was connected to the external terminal device via Bluetooth.

[0097] 7. Electrochemical test: The terminal device sends a command to apply voltage and measure the current response on the working electrode. At different glucose concentrations, the current response of the working electrode is recorded, and these data points are used to draw the concentration-response curve.

[0098] like Fig.10 As shown, in Example 5, the fitting curve R of the nickel-plated silver-based microneedle sensor without ionomer loading in an alkaline environment is 2 The value is 0.998, which is linear, while the linearity of the fitting curve under neutral environment is low, R 2 The sensor performance is poor. The nickel-plated silver-based microneedle sensor loaded with ionomer Fitting curve R of the sensor in a neutral environment 2 is 0.996, indicating that Loaded ionomer improves thePerformance of the sensor under a neutral environment. It can be seen from Example 5 that the sensor using nickel-silver-based microneedles without ionomer loading as working electrodes has good performance under alkaline conditions and poor performance under neutral conditions. Under alkaline conditions, the electrocatalytic activity of the electrode material is enhanced, and it tends to form active sites or surface states on the electrode surface that are conducive to glucose oxidation, which helps the oxidation reaction of glucose, and some potential interfering substances may not undergo electrochemical reactions or their reaction potentials may differ greatly from the oxidation potential of glucose, thereby improving the performance of the sensor. However, under a neutral environment, these advantages may no longer exist and the sensor performance is significantly reduced. After the ionomer is loaded, the detection performance of the microneedle sensor under neutral conditions is as good as that under alkaline conditions, indicating that the limitations of detecting glucose under neutral conditions have been solved by loading the ionomer.

[0099] Example 6

[0100] 1. Preparation of silver-based microneedles: same as in Example 1

[0101] 2. Preparation of working electrode (gold-plated silver-based microneedle loaded with ionomer):

[0102] 2.1. Au plating of silver-based microneedles: same as step 2.1 of Example 2.

[0103] 2.2. Loading of ionomer (Fumion FAA-3): Plasma clean a portion of the gold-plated silver-based microneedles obtained in step 2.1 to make them hydrophilic. Soak the microneedle array of gold-plated silver-based microneedles in a solution of (Fumion FAA-3) (1 mg / mL dissolved in acetonitrile) for 120 seconds, take it out, wash it with deionized water, and then dry it to obtain gold-plated silver-based microneedles loaded with ionomer as working electrodes. A portion of the gold-plated silver-based microneedles prepared in step 2.1 of this example are not treated in step 2.2 and used as working electrodes (which can be called "gold-plated silver-based microneedles not loaded with ionomer").

[0104] 3. Preparation of reference electrode: same as in Example 1.

[0105] 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 a flexible substrate by curing and bonding with silver paste to obtain an enzyme-free microneedle sensor.

[0106] 5. Preparation of agarose gel: same as step 5 of Example 5.

[0107] 6. The gold-plated microneedle sensors without ionomer loading were inserted into neutral and alkaline agarose hydrogels with different glucose concentrations and attached tightly. The gold-plated microneedle sensors loaded with ionomer were inserted into neutral and alkaline agarose hydrogels with different glucose concentrations. Glucose concentration in agarose hydrogel The flexible substrate is inserted into the test device through theConnect to external terminal devices via Bluetooth.

[0108] 7. Electrochemical test: The terminal device sends a command to apply voltage and measure the current response on the working electrode. At different glucose concentrations, the current response of the working electrode is recorded, and these data points are used to draw the concentration-response curve.

[0109] like Fig.11 As shown in Example 6, the fitting curve R of the gold-plated microneedle sensor without ionomer loading in an alkaline environment 2 The value is 0.998, which is linear, while the linearity of the fitting curve under neutral environment is low, R 2 The fitting curve R of the gold-plated silver-based microneedle sensor loaded with ionomer in a neutral environment is 0.866, indicating poor sensor performance. 2 It is 0.997, which indicates that the performance of the sensor in a neutral environment is improved by loading the ionomer.

[0110] Comparative Example 1

[0111] 1. Preparation of silver-based microneedles: same as in Example 1.

[0112] 2. Preparation of working electrode: plasma clean the silver-based microneedles prepared in step 1, drop 20uL of glucose oxidase solution (concentration of 25mg / mL) on the microneedle array, place it in a cool place to dry, and set aside as a working electrode.

[0113] 3. Preparation of reference electrode: same as in Example 1.

[0114] 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 a flexible substrate by silver paste curing and bonding to obtain an enzymatic microneedle sensor.

[0115] 5-6. Same as 5-6 of Example 1.

[0116] 7. Electrochemical test: The terminal device issues a command to apply voltage and measures the current response on the working electrode. At different glucose concentrations, the current response of the working electrode is recorded, and these data points are used to draw a concentration-response curve. In this example, electrochemical It detection is performed, the detection voltage is 0.5V, and the detection time is 100 seconds.

[0117] like Figure 4As shown, in this comparative example 1, the curves of all concentrations show a similar downward trend, and the sensor has similar response dynamics to different concentrations of glucose. As time goes by, the slope of the curve gradually decreases, indicating that the enzyme is gradually consumed during the detection process, the sensor performance decreases, and the performance of the enzymatic microneedle sensor will be affected by time.

[0118] Comparative Example 2

[0119] 1. Preparation of silver-based microneedles: same as in Example 1.

[0120] 2. Preparation of working electrodes: Plasma clean the silver-based microneedles prepared in step 1, and drop 20uL of glucose oxidase solution (concentration of 25mg / mL) on the microneedle array, place it in a cool place to dry, and use it as a working electrode. Some microneedles are placed for 24 hours, and some are placed for 240 hours.

[0121] 3. Preparation of reference electrode: same as in Example 1.

[0122] 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 a flexible substrate by silver paste curing and bonding to obtain an enzymatic microneedle sensor.

[0123] 5-6. Same as 5-6 of Example 1.

[0124] 7. Electrochemical test: The terminal device sends a command to apply voltage and measure the current response on the working electrode. At different glucose concentrations, the current response of the working electrode is recorded, and these data points are used to draw the concentration-response curve. Electrochemical CV detection is performed with a detection voltage of 0.6V.

[0125] like Figure 5 As shown in the figure, in this comparative example 2, after being placed for 24 hours, GOx maintained a high activity, and the linear relationship between the response current and the glucose concentration was better, and its fitting R 2 The value is 0.999. After 240 hours, the enzyme activity decreased or inactivated, the sensitivity of the enzymatic microneedle sensor decreased, the linear relationship deteriorated, and the fitting R 2 The value is 0.937. Therefore, the response current of the enzymatic microneedle sensor loaded with GOx after 240 hours is weak and the linearity is poor, and the performance is significantly lower than that after 24 hours, indicating that the GOx enzyme will be inactivated after long-term storage. However, the detection of the enzyme-free microneedle sensor in the embodiment of the present invention is not affected by the storage time.

[0126] The enzyme-free microneedle sensor 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. The detected electrical signal is converted to directly reflect the change of blood glucose concentration. It adopts an enzyme-free detection method, has a long storage life and good electrochemical stability at room temperature, effectively solves the problem of enzyme inactivation and instability, and is suitable for use in home and medical environments. In addition, the pH value of human skin tissue fluid Usually close to neutral, by loading ionomers, the performance of enzyme-free microneedle sensors in neutral environments is improved. The enzyme-free microneedle sensor can be integrated into wearable devices to achieve real-time and continuous blood sugar monitoring, which helps to better manage diabetes.

[0127] 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 enzyme-free microneedle sensor, characterized in that: It includes a flexible substrate, a working electrode, a counter electrode and a reference electrode; the flexible substrate has a lead system and a signal acquisition system, the working electrode, the counter electrode and the reference electrode all include a microneedle base and a microneedle array formed on the microneedle base, the microneedle bases in the working electrode, the counter electrode and the reference electrode are distributed on the flexible substrate and connected to the signal acquisition system through the lead system, and the microneedle array of the working electrode includes a microneedle body and an anion exchange ionomer loaded on the microneedle body.

2. The enzyme-free microneedle sensor according to claim 1, characterized in that: The microneedle body in the working electrode includes a metal-based microneedle or a polymer microneedle containing a metal coating; Preferably, the metal-based microneedle is one of a silver-based, gold-based, platinum-based, nickel-based or silver-coated copper-based microneedle; preferably, the microneedle body in the working electrode is a silver-based microneedle; Preferably, the metal-based microneedle is prepared by the following preparation method: 50wt%-95wt% of metal powder and 5wt%-50wt% of curing agent are mixed evenly into slurry, the slurry is transferred to a microneedle polymer mold, bubbles are removed under vacuum conditions, the mold is filled and then cured, and finally demolded; 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; Preferably, the polymer microneedle containing a metal coating is made by the following preparation method: a layer of silver or copper is deposited as a base layer on a polymer substrate by magnetron sputtering or chemical plating, and the thickness of the base layer is 10nm-40μm, and then a silver layer is plated on the base layer by an electroplating process, and the thickness of the silver layer is 100nm-100μm, preferably 10μm-40μm.

3. The enzyme-free microneedle sensor according to claim 1, characterized in that: The microneedle body of the working electrode is a silver-based microneedle, and the silver-based microneedle also has a coating, and the anion exchange ionomer is loaded on the coating, and the coating is a nickel layer, a gold layer or a copper layer. Preferably, the coating is a nickel layer or a gold layer.

4. The enzyme-free microneedle sensor according to claim 1, characterized in that: The anion exchange ionomer includes at least one of Fumion FAA-3 and PiperION-A5-HCO3-EtOH; the thickness of the anion exchange ionomer loaded on the microneedle body is 100nm-100μm.

5. The enzyme-free microneedle sensor according to claim 1, characterized in that: The reference electrode is an Ag / AgCl electrode.

6. The enzyme-free microneedle sensor according to claim 1, characterized in that: The counter electrode is a silver-based microneedle or a polymer microneedle containing a metal coating.

7. The enzyme-free microneedle sensor according to claim 1, characterized in that: The flexible substrate comprises a wire layer, a support layer and an adhesive layer, wherein the wire layer is embedded in the support layer, the microneedle bases of the working electrode, the counter electrode and the reference electrode are distributed on the adhesive layer and connected to the signal acquisition system through the lead system in the wire layer, the adhesive layer is used to fix the flexible substrate to the skin surface during detection, and the support layer and the adhesive layer are combined together; preferably, the length and width of the enzyme-free microneedle sensor do not exceed 75 mm; the thickness of the flexible substrate is 0.5 mm-3 mm.

8. The enzyme-free microneedle sensor according to claim 7, characterized in that: The material of the support layer is at least one of dimethylsiloxane, polysiloxane elastomer, PET, acrylic polymer, and silicone polymer.

9. The enzyme-free microneedle sensor according to claim 1, characterized in that: The microneedle array of the working electrode is a 7×10 to 10×15 array, the array length does not exceed 20 mm, the width does not exceed 20 mm, and the spacing between each microneedle ranges from 200 μm to 2000 μm; The microneedles in the microneedle array of the working electrode, the counter electrode and the reference electrode have a shape of a pointed prism, a pyramid, a pointed cylinder or a cone, and their height is between 150μm-1500μm; the height of the microneedle base of the working electrode, the counter electrode and the reference electrode is 0.2mm-3mm.

10. A method for preparing an enzyme-free microneedle sensor according to any one of claims 1 to 9, characterized in that: The steps include: (1) preparing a counter electrode, a working electrode and a reference electrode respectively; (2) The microneedle bases of the counter electrode, the working electrode and the reference electrode are fixed on a flexible substrate and electrically connected to the signal acquisition system through a lead system in the flexible substrate.

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