A uric acid sensor and its preparation method and application

Abstract

The present invention discloses a uric acid sensor and its preparation method and application, the uric acid sensor comprises a flexible substrate and a waterproof layer which are stacked; three electrodes and a conductive circuit are arranged between the flexible substrate and the waterproof layer; a detection port is arranged on the waterproof layer, and the three electrodes are located at the detection port; the three electrodes include a working electrode; a graphene-polyacrylonitrile electrostatic spinning film layer is arranged on the working electrode; uricase and a film-forming aid are adsorbed in the graphene-polyacrylonitrile electrostatic spinning film layer; a waterproof insulating layer is arranged at the contact part between the edge of the detection port and the waterproof layer. The sensor in the present invention has high sensitivity and wide detection range, can meet the detection of uric acid in different concentration ranges, has high detection result accuracy, will not be interfered by other substances, and can detect the concentration of uric acid efficiently and specifically.

Description

Technical Field

[0001] The present invention relates to the field of detection, and in particular to a uric acid sensor and a preparation method and application thereof. Background Art

[0002] Uric acid is the final metabolite of purine and purine derivatives in the body, with a chemical formula of C5H4N4O3. The main methods for detecting uric acid include phosphotungstic acid reduction method, spectrophotometry, high performance liquid chromatography, uric acid sensor method, etc. Electrochemical uric acid biosensors currently mainly use uricase as a biorecognition element. Uricase can consume oxygen and catalyze uric acid to produce allantoin, carbon dioxide and hydrogen peroxide. Hydrogen peroxide generates electron transfer through oxidation or reduction to generate an electrical signal on the electrode or use an electron mediator for electron transfer, thereby achieving the purpose of detecting uric acid. Using materials such as silk membrane and eggshell membrane to adsorb and fix urate oxidase, an enzyme membrane electrode for detecting uric acid can be prepared. The introduction of nanomaterials such as gold nanoparticles and modified nano-silica gel can improve the performance of enzyme membrane electrodes. CN107462617A discloses a uricase biosensor based on calcium hydrogen phosphate-reduced graphene / nano-gold particles. The calcium hydrogen phosphate-reduced graphene / nano-gold particle composite material prepared by a one-step method is loaded on the surface of a glassy carbon electrode by a drop coating method, and then the uricase solution is dripped after drying, thereby improving the problem of easy inactivation of the enzyme sensor. However, the sensor is not a flexible sensor. The flexible wearable uric acid sensor in the prior art still generally has the following problems: the flexible insulating materials such as polyethylene terephthalate and polyimide used are tightly structured and lack air permeability. Wearing airtight sensors for a long time will hinder the evaporation of skin sweat and the release of organic components, and easily cause inflammation of the skin. The breathable flexible fabric is not waterproof. When used as a flexible sensor substrate, sweat easily penetrates and disperses therein, making it difficult to control and collect, affecting the test results. During the detection of uric acid, the dripped uric acid solution will flow along the circuit of the painting, contaminating the circuit connector and affecting the test results. In order to solve the problems existing in the above-mentioned prior art, it is urgent to develop a wearable uric acid sensor with good air permeability and flexibility. Summary of the invention

[0003] In order to overcome the problems existing in the above-mentioned prior art, one of the objectives of the present invention is to provide a uric acid sensor.

[0004] A second object of the present invention is to provide a method for preparing a uric acid sensor.

[0005] A third object of the present invention is to provide a uric acid sensor for use in uric acid detection.

[0006] A fourth object of the present invention is to provide a method for detecting uric acid.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a uric acid sensor, comprising a stacked flexible substrate and a waterproof layer; three electrodes and a conductive circuit are arranged between the flexible substrate and the waterproof layer; a detection port is provided on the waterproof layer, and the three electrodes are located at the detection port; the three electrodes include a working electrode; a graphene-polyacrylonitrile electrospun film layer is provided on the working electrode; uricase and a film-forming aid are adsorbed in the graphene-polyacrylonitrile electrospun film layer; a waterproof insulating layer is provided at the edge of the detection port and the contact portion with the waterproof layer.

[0009] Preferably, the flexible substrate comprises a polytetrafluoroethylene film layer and a polyurethane medical tape layer stacked. A polyurethane medical tape layer is arranged on the polytetrafluoroethylene film layer, and then three electrodes and a conductive circuit are drawn on the polyurethane medical tape layer using a conductive silver paste. If the three electrodes and the conductive circuit are drawn directly on the polytetrafluoroethylene film layer, the conductive circuit is discontinuous, easily broken and unable to conduct electricity. The present invention draws three electrodes and a conductive circuit on the polyurethane medical tape layer on the polytetrafluoroethylene film layer, and a continuous conductive circuit can be formed. The present invention adopts a waterproof, breathable, and high-temperature resistant polytetrafluoroethylene film layer, and cooperates with a waterproof, breathable, anti-allergic polyurethane medical tape layer. There is a synergistic effect between the polytetrafluoroethylene film layer and the polyurethane medical tape layer. The presence of the polytetrafluoroethylene film layer can avoid the problem of melting and shrinking of the polyurethane medical tape during heating and curing; and the polyurethane medical tape layer can avoid the problem of the conductive circuit being broken and discontinuous when the three electrodes and the conductive circuit are directly drawn on the polytetrafluoroethylene film layer.

[0010] The graphene-polyacrylonitrile electrostatic spinning film layer in the present invention has a large specific surface area and a rich network structure, which can make uricase attached and fixed, greatly improving the stability of the fixation of uricase. Because graphene has high conductivity, the graphene-polyacrylonitrile electrostatic spinning film layer can quickly conduct the electrons produced after uricase catalyzes uric acid to the electrode, improving the sensitivity and detection speed of detection. In addition, the specific surface area and volume of the graphene-polyacrylonitrile electrostatic spinning film layer prepared can be improved by mixing graphene and polyacrylonitrile with electrostatic spinning, so that it has higher porosity and air permeability.

[0011] Preferably, in the graphene-polyacrylonitrile electrostatic spinning film layer, the mass ratio of graphene and polyacrylonitrile is (0.5-5):100; further preferably, in the graphene-polyacrylonitrile electrostatic spinning film layer, the mass ratio of graphene and polyacrylonitrile is (1-5):100; further preferably, in the graphene-polyacrylonitrile electrostatic spinning film layer, the mass ratio of graphene and polyacrylonitrile is (1-3):100.

[0012] Preferably, the waterproof layer is made of polyurethane medical tape.

[0013] Preferably, the three electrodes further include a counter electrode and a reference electrode.

[0014] Preferably, among the three electrodes, the counter electrode is located at one side of the detection port, and the working electrode and the reference electrode are located at the other side of the detection port. The above arrangement of the three electrodes can reduce the possibility of short circuit between the counter electrode and the working electrode.

[0015] Preferably, the graphene-polyacrylonitrile electrospun film layer is adhered to the working electrode via a conductive glue or a conductive tape.

[0016] Preferably, the area of ​​the working electrode is smaller than the area of ​​the graphene-polyacrylonitrile electrospinning film layer; further preferably, the area of ​​the working electrode is half the area of ​​the graphene-polyacrylonitrile electrospinning film layer. When the area of ​​the graphene-polyacrylonitrile electrospinning film layer is larger than the area of ​​the working electrode, the graphene-polyacrylonitrile electrospinning film layer can be firmly attached to the working electrode, avoiding the problem of the graphene-polyacrylonitrile electrospinning film layer falling off during the uric acid test.

[0017] The present invention pastes the graphene-polyacrylonitrile electrospun film layer on the working electrode before the curing step, so that the graphene-polyacrylonitrile electrospun film layer and the uncured conductive silver paste can be better combined. Otherwise, if the conductive silver paste is heated and cured and then the graphene-polyacrylonitrile electrospun film layer is pasted, the graphene-polyacrylonitrile electrospun film layer is easily fallen off due to long-term immersion in uric acid during the uric acid detection process.

[0018] Preferably, the counter electrode is drawn using conductive silver paste.

[0019] Preferably, the conductive circuit is drawn using conductive silver paste.

[0020] Preferably, the counter electrode and the conductive circuit are both provided with a gold coating, which can make the counter electrode and the conductive circuit more stable and more conductive.

[0021] Preferably, the gold coating is obtained by electroplating.

[0022] Preferably, the reference electrode is a silver / silver chloride reference electrode.

[0023] Preferably, the reference electrode is prepared by first drawing the reference electrode region with conductive silver paste, then adding ferric chloride solution to the reference electrode region, and the ferric chloride solution reacts with the silver in the reference electrode region to form a silver / silver chloride reference electrode.

[0024] The present invention uses conductive silver paste to draw three electrodes and a conductive circuit. Compared with the traditional techniques of preparing three electrodes and a conductive circuit by photolithography, vapor deposition, sputtering and the like, the present invention uses conductive silver paste to draw three electrodes and a conductive circuit, does not require expensive equipment, has a simple operation process, is low in cost, and is flexible and convenient.

[0025] Preferably, the method for preparing the conductive silver paste used in the present invention comprises the following steps:

[0026] (1) mixing a silver nitrate solution with polyacrylic acid and diethanolamine to react, cooling, then mixing with ethanol, and centrifuging to separate nanosilver particles;

[0027] (2) preparing a nano silver suspension, filtering it, and then mixing it with a hydroxyethyl cellulose solution to obtain the conductive silver paste.

[0028] Preferably, the mass ratio of silver nitrate, polyacrylic acid and diethanolamine is (8-12):1:(16-24); further preferably, the mass ratio of silver nitrate, polyacrylic acid and diethanolamine is (9-11):1:(18-22).

[0029] Preferably, in the method for preparing the conductive silver paste, the temperature of the mixing reaction is 50-70°C.

[0030] Preferably, in the preparation method of the conductive silver paste, the mixing method of the mixing reaction is at least one of ultrasonic mixing and mechanical mixing; further preferably, in the preparation method of the conductive silver paste, the mixing method of the mixing reaction is a combination of ultrasonic mixing and mechanical mixing; even further preferably, in the preparation method of the conductive silver paste, the mixing method of the mixing reaction is first mechanical mixing for 18 to 30 hours; and then ultrasonic mixing for 1 to 3 hours.

[0031] Preferably, the mechanical mixing is magnetic stirring.

[0032] Preferably, in the method for preparing the conductive silver paste, the mass percentage of hydroxyethyl cellulose in the hydroxyethyl cellulose solution is 1-3%.

[0033] Preferably, the solvent used in the hydroxyethyl cellulose solution is a mixture of water and methanol.

[0034] Preferably, in the hydroxyethyl cellulose solution, the volume ratio of water to methanol is 1:(0.5-2).

[0035] Preferably, in the method for preparing the conductive silver paste, the water is ultrapure water.

[0036] Preferably, the film-forming aid is a perfluorosulfonic acid polymer solution. The perfluorosulfonic acid polymer solution is a nafion solution. The pH of human sweat is 4.5-5.5, uric acid is a cation, and the formed nafion membrane can prevent anions in sweat from passing through, thereby avoiding interference with uric acid detection.

[0037] Preferably, the waterproof insulating layer is prepared by coating a polyvinyl butyral resin solution on the edge of the detection port and drying to form a waterproof insulating layer. The waterproof insulating layer is provided at the detection port to prevent the uric acid solution dripped onto the working electrode of the detection port from penetrating into the junction of the waterproof layer and the flexible electrode and flowing along the conductive circuit, thereby contaminating the circuit joint and the conductive circuit and affecting the accuracy of the detection result.

[0038] Preferably, the solvent in the polyvinyl butyral resin solution is ethanol.

[0039] Preferably, the mass ratio of the polyvinyl butyral resin to ethanol is 1:(8-15); further preferably, the mass ratio of the polyvinyl butyral resin to ethanol is 1:(8-12).

[0040] Preferably, in the method for preparing the waterproof insulating layer, the drying temperature is 35-45°C.

[0041] The second aspect of the present invention is to provide a method for preparing the uric acid sensor provided in the first aspect of the present invention, comprising the following steps:

[0042] S1: forming three electrodes and a conductive circuit on a flexible substrate, and then pasting a graphene-polyacrylonitrile electrospun film layer on the working electrode and heating and curing it;

[0043] S2: first adding uricase to the graphene-polyacrylonitrile electrospun film layer and drying it, and then adding the film-forming aid and drying it;

[0044] S3: Pasting a waterproof layer on the flexible substrate, and then forming a waterproof insulating layer at the edge of the detection port to obtain the uric acid sensor.

[0045] Preferably, in step S1, the step of forming three electrodes and a conductive circuit on the flexible substrate is specifically: using conductive silver paste to draw a working electrode, a counter electrode, a reference electrode region and a conductive circuit on the flexible substrate; then adding a silver chloride solution to the reference electrode region to react the silver chloride solution with the silver in the reference electrode region, thereby forming a silver / silver chloride reference electrode;

[0046] Preferably, in step S1, the temperature of heating and curing is 130-140°C. The present invention performs heating and curing within the above temperature range, which greatly reduces the resistance of the three electrodes and the conductive circuit drawn. If the curing temperature is lower than 130°C, the resistance of the three electrodes and the conductive circuit drawn by the conductive silver paste is large, and the resistance value difference is large, and the uniformity is poor. If the heating and curing temperature exceeds 140°C, the preparation cost increases and has a certain destructive effect on the flexible substrate.

[0047] Preferably, the heating and curing time is 25 to 60 minutes; more preferably, the heating and curing time is 30 to 35 minutes.

[0048] Preferably, the drying step in step S2 is: drying at 1-5°C; more preferably, the drying step in step S2 is: drying at 3-5°C.

[0049] Preferably, the graphene-polyacrylonitrile electrospinning film layer is prepared by mixing a polyacrylonitrile solution and a graphene solution and electrospinning the mixture, followed by pre-oxidation and calcination.

[0050] Preferably, the solvent in the polyacrylonitrile solution is N,N-dimethylformamide.

[0051] Preferably, the solvent in the graphene solution is N,N-dimethylformamide.

[0052] Preferably, in the mixed solution obtained by the step of mixing the polyacrylonitrile solution and the graphene solution, the mass ratio of polyacrylonitrile, graphene and N,N-dimethylformamide is 100:(0.5-5):(800-1500).

[0053] Preferably, the step of mixing the polyacrylonitrile solution and the graphene solution is specifically: stirring and mixing the polyacrylonitrile solution and the graphene solution at a stirring speed of 300 to 800 rpm and a temperature of 30 to 40° C. for 10 to 15 hours.

[0054] Preferably, the electrospinning step is specifically as follows: the voltage is 18-20 kV, and the perfusion speed is 0.6-0.8 mL / h.

[0055] Preferably, the pre-oxidation step is specifically: in an oxygen-containing atmosphere, the temperature is raised to 250-280°C at a heating rate of 1-10°C / min for oxidation; further preferably, the pre-oxidation step is specifically: in an oxygen-containing atmosphere, the temperature is raised to 250-280°C at a heating rate of 3-8°C / min for oxidation; further preferably, the pre-oxidation step is specifically: in an oxygen-containing atmosphere, the temperature is raised to 250-280°C at a heating rate of 4-6°C / min for oxidation.

[0056] Preferably, the oxidation time is 1 to 5 hours; more preferably, the oxidation time is 1 to 3 hours.

[0057] Preferably, the calcination step is specifically: in a mixture of inert gas and hydrogen, the temperature is raised to 800-1000°C at a heating rate of 1-10°C / min for calcination; further preferably, the calcination step is specifically: in a mixture of inert gas and hydrogen, the temperature is raised to 800-1000°C at a heating rate of 3-8°C / min for calcination; further preferably, the calcination step is specifically: in a mixture of inert gas and hydrogen, the temperature is raised to 800-1000°C at a heating rate of 4-6°C / min for calcination.

[0058] Preferably, in the mixed gas, the volume ratio of the inert gas to the hydrogen is 8 to 10:1; further preferably, in the mixed gas, the volume ratio of the inert gas to the hydrogen is 9:1.

[0059] Preferably, the inert gas is at least one of argon, nitrogen and helium; more preferably, the inert gas is argon.

[0060] Preferably, the calcination time is 1 to 5 hours; more preferably, the calcination time is 2 to 3 hours.

[0061] Preferably, the preparation method further comprises the step of preparing a gold coating on the counter electrode among the three electrodes and the conductive circuit by electroplating.

[0062] Preferably, the electroplating voltage is 6-8V.

[0063] The third aspect of the present invention provides an application of the uric acid sensor provided by the first aspect of the present invention in uric acid detection.

[0064] The fourth aspect of the present invention provides a method for detecting uric acid, comprising the following steps: connecting the uric acid sensor provided by the first aspect of the present invention to an electrochemical workstation, dripping a uric acid sample solution onto a working electrode, detecting the peak current value of uric acid using differential pulse voltammetry, and obtaining the concentration of uric acid based on a uric acid detection standard curve.

[0065] Preferably, the differential pulse voltammetry method is specifically as follows: the scanning voltage is -200 to 800 mV, the step height is 5 mV, the pulse height is 50 mV, the pulse interval is 400 ms, the pulse width is 100 ms, and the acquisition time is 20 ms.

[0066] Preferably, the method for obtaining the uric acid detection standard curve is: connecting the uric acid sensor provided in the first aspect of the present invention to an electrochemical workstation, dripping a uric acid standard solution on the working electrode, using differential pulse voltammetry to detect the peak current value of uric acid, and constructing the relationship between uric acid concentration and peak current value, thereby calculating the uric acid standard curve.

[0067] Preferably, the preparation method of the uric acid standard solution is: using 0.01 mol / L acetate buffer solution with a pH of 5.0 to prepare uric acid solutions with uric acid contents of 0 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L, and 800 μmol / L, respectively.

[0068] The beneficial effect of the present invention is that the sensor of the present invention has high sensitivity, and its sensitivity can reach 0.13μA / (μmol / L*cm 2 ), the detection linear range is 0-800 μmol / L, the detection range is wide, it can meet the detection of uric acid in different concentration ranges, the detection result is highly accurate, it will not be interfered by other substances, and the concentration of uric acid can be detected efficiently and specifically. In addition, the sensor in the present invention has excellent stability.

[0069] In addition, the sensor of the present invention has good air permeability. When it is worn on the body to monitor the concentration of uric acid in sweat in real time, it is not easy to cause skin allergies, and the user experience is better. The sensor is waterproof to prevent sweat in the detection area from penetrating into the non-detection area of ​​the sensor, thereby causing inaccurate uric acid detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is a process flow chart for preparing a breathable, flexible, wearable uric acid sensor in Examples 1 to 2 of the present invention.

[0071] Figure 2 This is a physical picture of the sensor in Example 1 of the present invention after being connected to the electrochemical workstation.

[0072] Figure 3 This is a diagram showing the differential pulse voltammetry results of the sensor in Example 1 of the present invention detecting uric acid of different concentrations.

[0073] Figure 4 This is a standard curve diagram of uric acid detection by the sensor in Example 1 of the present invention.

[0074] Figure 5 This is a physical picture of the electrodes and circuit diagram drawn on the polytetrafluoroethylene film.

[0075] Figure 6 This is a physical picture of the electrodes and circuit diagram drawn on the polytetrafluoroethylene film containing polyurethane medical tape.

[0076] Figure 7 This is a test diagram of the anti-interference performance of the sensor in Example 1 of the present invention.

[0077] Figure 8 This is a stability test diagram of the sensor in Example 1 of the present invention. DETAILED DESCRIPTION

[0078] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0079] The sources of the raw materials used in Examples 1 and 2 of the present invention are:

[0080] The trade name of the polytetrafluoroethylene film is Tianjin Linghang Microporous Filter Membrane, model number is NMF28TS, purchased from Tianjin Linghang Experimental Equipment Co., Ltd.;

[0081] The trade name of the polyurethane film medical tape is 3M microporous breathable tape, model number is 1530C-1, and it was purchased from Jiangxi Shilaikang Industrial Co., Ltd.

[0082] Example 1

[0083] The structure of the breathable, flexible, wearable uric acid sensor in this example includes a stacked polytetrafluoroethylene film layer, a first polyurethane medical tape layer and a second polyurethane medical tape layer; a working electrode, a counter electrode, a reference electrode and a conductive circuit drawn with a conductive silver paste pen are provided between the first polyurethane medical tape layer and the second polyurethane medical tape layer; a gold-plated layer is provided on the conductive circuit and the counter electrode; a graphene-polyacrylonitrile electrospun film is provided on the working electrode; uricase and perfluorosulfonic acid type polymer are adsorbed in the graphene-polyacrylonitrile electrospun film; a detection port is provided on the second polyurethane medical tape layer, and the working electrode, counter electrode and reference electrode are located in the detection port, and a waterproof insulating ring is provided at a position adjacent to the detection port and the second polyurethane medical tape layer.

[0084] The breathable, flexible, wearable uric acid sensor in this example is prepared by the following preparation method, which specifically includes the following steps:

[0085] 1. Prepare a conductive silver paste pen. The conductive silver paste pen is prepared by the following steps:

[0086] 20 grams of silver nitrate are dissolved in 15 milliliters of ultrapure water, and the silver nitrate solution is added to a solution containing 2 grams of polyacrylic acid, 40 grams of diethanolamine, and 50 milliliters of ultrapure water under magnetic stirring at 500 rpm, and magnetic stirring is performed for 24 hours; then the solution is placed in a 60°C water bath for ultrasonic treatment for 1.5 hours, and then cooled; the supernatant is poured out, and ethanol is added to the remaining solution at a volume ratio of 1:3 to condense the nanosilver particles, and then the solution is centrifuged at 800g for 15 minutes in a centrifuge, the supernatant is removed, ultrapure water is added to resuspend the nanosilver particles, and the resuspended solution is filtered using a 0.22 micron filter membrane, and a 2% by mass hydroxyethyl cellulose solution (the solvent is a mixed solution of ultrapure water and methanol, wherein the volume ratio of ultrapure water to methanol is 1:1) is added and mixed, and the fluidity of the nanosilver particle solution is adjusted to obtain a nano-conductive silver paste, and the obtained nano-conductive silver paste is loaded into an empty pen to obtain a conductive silver paste pen.

[0087] 2. Preparation of graphene-polyacrylonitrile electrospinning film

[0088] The polyacrylonitrile was dried at 80°C in a vacuum oven for 4 hours, 1 gram of polyacrylonitrile was dissolved in 4.5 grams of N,N-dimethylformamide, stirred at room temperature for 12 hours, and then ultrasonicated for 10 minutes to obtain a polyacrylonitrile solution. A graphene dispersion with a solid-liquid ratio of 1% was taken and freeze-dried at -60°C for 48 hours using a vacuum freeze dryer to obtain dried graphene. The dried graphene was weighed at 2% of the weight of polyacrylonitrile, added to 5.5 grams of N,N-dimethylformamide, and then ultrasonicated for 3 hours to obtain a graphene solution. The polyacrylonitrile solution and the graphene solution were mixed, and magnetically stirred at 35°C for 12 hours at a stirring speed of 500rpm to obtain a graphene-polyacrylonitrile electrospinning stock solution. Tin foil was placed on the rotating shaft of the electrospinning machine, and the graphene-polyacrylonitrile electrospinning stock solution was loaded into a syringe with a needle diameter of 0.5 mm and fixed in the electrospinning machine. The voltage was set to 20 kV and the infusion rate was set to 0.6 ml / h for electrospinning, so that a graphene-polyacrylonitrile electrospinning membrane was formed on the surface of the tin foil. The graphene-polyacrylonitrile electrospinning membrane was placed in a tubular furnace, and in an air atmosphere, the temperature was raised to 250°C at a rate of 5°C / min and maintained for 2 hours for pre-oxidation; then, under the protection of a mixed gas with a volume ratio of argon:hydrogen of 9:1, the tubular furnace was heated to 900°C at a rate of 5°C / min and maintained for 2 hours to obtain a carbonized graphene-polyacrylonitrile electrospinning membrane.

[0089] 3. Prepare a breathable, flexible, wearable uric acid sensor as follows:

[0090] (1) A waterproof, breathable, and high-temperature resistant polytetrafluoroethylene film is used as a substrate, and a layer of waterproof, breathable, anti-allergic polyurethane film medical tape is pasted on it. Electrodes and conductive circuits are drawn on the polyurethane medical tape with a conductive silver paste pen. The prepared graphene-polyacrylonitrile electrospun film is pasted on the conductive tape and pasted on the painted working electrode area to obtain a graphene electrospun flexible silver three-electrode.

[0091] (2) The graphene electrospun flexible silver three-electrode was placed in a drying oven and heated and cured at 130°C for 30 minutes.

[0092] (3) Take out the graphene electrospun flexible silver three-electrode after heating and curing, and add 0.1 mol / L ferric chloride solution to the reference electrode area, so that the ferric chloride reacts with the silver in the reference electrode area for 1 minute to form a silver / silver chloride reference electrode. Remove the ferric chloride solution, wash the electrode, and absorb the water.

[0093] (4) Using a gold plating pen, 24k gold is electroplated on the silver on the counter electrode and the conductive circuit at 8 volts. This makes the electrode and circuit more stable and more conductive. The gold plating solution is washed off and dried to obtain a graphene electrospinning flexible gold three-electrode.

[0094] (5) 1 μL of 2 mg / mL uricase was added to the graphene-polyacrylonitrile electrospun film located at the working electrode and placed in a refrigerator at 4°C to dry. The uricase attached to the graphene-polyacrylonitrile electrospun film can efficiently and specifically catalyze the oxidation of uric acid.

[0095] (6) 1 μL of 0.5% by mass nafion solution (perfluorosulfonic acid polymer solution) was continued to be dripped onto the graphene-polyacrylonitrile electrospun film for encapsulation, and the film was placed in a refrigerator at 4° C. to dry, thereby obtaining a working electrode sensitive to uric acid.

[0096] (7) A layer of waterproof, breathable, anti-allergic polyurethane film medical tape is pasted outside the detection area for waterproofing.

[0097] (8) Polyvinyl butyral resin was dissolved in anhydrous ethanol at a weight ratio of 1:10, and a waterproof insulating circle was drawn on the outer edge of the detection area to prepare a sensor. The sensor was placed in a drying oven and dried at 40°C to obtain the breathable, flexible, wearable uric acid sensor in this example.

[0098] Example 2

[0099] The breathable, flexible, wearable uric acid sensor in this example has the same structure as the sensor in Example 1.

[0100] The breathable, flexible, wearable uric acid sensor in this example is prepared by the following preparation method, which specifically includes the following steps:

[0101] 1. Preparation of conductive silver paste pen, the steps are as follows:

[0102] Dissolve 20 grams of silver nitrate in 15 milliliters of ultrapure water, stir magnetically at a stirring speed of 500rpm, then add the silver nitrate solution to a solution containing 2 grams of polyacrylic acid, 40 grams of diethanolamine, and 50 milliliters of ultrapure water, stir magnetically for 24 hours, then place in a 65°C water bath for ultrasonication for 1.5 hours, and place to cool. Pour out the supernatant, add ethanol to the remaining solution at a volume ratio of 1:3 to condense the nanosilver particles. Centrifuge again at 800g for 15 minutes in a centrifuge, remove the supernatant, add ultrapure water to resuspend the nanosilver particles. Filter the resuspended solution using a 0.22 micron filter membrane, add a solution of 2% hydroxyethyl cellulose by mass percentage (the solvent is a mixture of ultrapure water and methanol, wherein the volume ratio of ultrapure water to methanol is 1:1) and mix well, adjust the fluidity of the nanosilver particle solution, and prepare a nano-conductive silver paste, and put the prepared nano-conductive silver paste into an empty pen to prepare a conductive silver paste pen.

[0103] 2. Preparation of graphene-polyacrylonitrile electrospinning film

[0104] The polyacrylonitrile was dried at 80°C in a vacuum oven for 4 hours, 1 gram of polyacrylonitrile was dissolved in 4.5 grams of N, N-dimethylformamide, stirred at room temperature for 12 hours, and then ultrasonicated for 10 minutes to obtain a polyacrylonitrile solution. A graphene dispersion with a solid-liquid ratio of 1% was taken and freeze-dried at -60°C for 48 hours using a vacuum freeze dryer to obtain dried graphene. The dried graphene was weighed at 2% of the weight of polyacrylonitrile, added to 5.5 grams of N, N-dimethylformamide, and then ultrasonicated for 3 hours to obtain a graphene solution. The polyacrylonitrile solution and the graphene solution were mixed, and magnetically stirred at 35°C for 12 hours at a stirring speed of 500 rpm to obtain a graphene-polyacrylonitrile electrospinning stock solution. Tin foil was placed on the rotating shaft of the electrospinning machine, and the graphene-polyacrylonitrile electrospinning stock solution was loaded into a syringe with a needle diameter of 0.5 mm and fixed in the electrospinning machine. The voltage was set to 18 kV and the infusion rate was set to 0.8 ml / h for electrospinning, so that a graphene-polyacrylonitrile electrospinning membrane was formed on the surface of the tin foil. The graphene-polyacrylonitrile electrospinning membrane was placed in a tubular furnace, heated to 280°C at a heating rate of 5°C / min in an air atmosphere, and maintained for 2 hours for pre-oxidation. Under the protection of a mixed gas with an argon:hydrogen volume ratio of 9:1, the tubular furnace was heated to 1000°C at a rate of 5°C / min and maintained for 2 hours to obtain a carbonized graphene-polyacrylonitrile electrospinning membrane.

[0105] 3. Prepare a breathable, flexible, wearable uric acid sensor as follows:

[0106] (1) A waterproof, breathable, and high-temperature resistant polytetrafluoroethylene film is used as a substrate, and a waterproof, breathable, and anti-allergic polyurethane film medical tape is pasted on it. Electrodes and conductive circuits are drawn on the polyurethane medical tape using a conductive silver paste pen. The prepared graphene-polyacrylonitrile electrospun film is pasted on the conductive tape and pasted on the painted working electrode area to obtain a graphene electrospun flexible silver three-electrode.

[0107] (2) The graphene electrospun flexible silver three-electrode was placed in a drying oven and heated and cured at 140°C for 30 minutes.

[0108] (3) Take out the graphene electrospun flexible silver three-electrode after heating and curing, and add 0.1 mol / L ferric chloride solution to the reference electrode area, so that the ferric chloride reacts with the silver in the reference electrode area for 2 minutes to form a silver / silver chloride reference electrode. Remove the ferric chloride solution, wash the electrode, and absorb the water.

[0109] (4) Using a gold plating pen, 24k gold is electroplated on the silver on the counter electrode and the conductive circuit at a voltage of 7 volts. This makes the electrode and circuit more stable and more conductive. The gold plating solution is washed off and dried to obtain a graphene electrospinning flexible gold three-electrode.

[0110] (5) 0.5 μL of 2 mg / mL uricase was added to the graphene-polyacrylonitrile electrospun film located at the working electrode and placed in a refrigerator at 4°C to dry. The uricase attached to the graphene-polyacrylonitrile electrospun film can efficiently and specifically catalyze the oxidation of uric acid.

[0111] (6) 1 μL of 0.5% by mass nafion solution was further added to the graphene-polyacrylonitrile electrospun film for encapsulation, and the film was dried again in a refrigerator at 4° C. to obtain a working electrode sensitive to uric acid.

[0112] (7) A layer of waterproof, breathable, anti-allergic polyurethane film medical tape is pasted outside the detection area for waterproofing.

[0113] (8) Dissolve the polyvinyl butyral resin in anhydrous ethanol at a weight ratio of 1:10, and draw a waterproof insulating circle on the outer edge of the detection area. Place the sensor in a drying oven and dry it at 40°C to obtain the breathable, flexible, wearable uric acid sensor in this example.

[0114] The process flow chart of preparing the breathable flexible wearable uric acid sensor in Example 1 and Example 2 of the present invention is as follows: Figure 1 shown.

[0115] In order to draw a standard curve of the sensor detecting uric acid, the sensor in Example 1 of the present invention was used to test the peak current value of the uric acid standard solution with different concentrations. The test method was as follows: the breathable, flexible, wearable uric acid sensor prepared in Example 1 was connected to the electrochemical workstation. The actual connection diagram is as shown in FIG. Figure 2 As shown, 60 μL of uric acid standard solution was dripped onto the working electrode in the sensor detection area. The uric acid solution was detected using differential pulse voltammetry, and the settings of differential pulse voltammetry were: scanning voltage of -0.1 to 0.75 V, step height of 5 mV, pulse height of 50 mV, pulse interval of 400 msec, pulse width of 100 msec, and acquisition time of 20 msec. The test results of uric acid standard solutions of different concentrations are shown in Figure 3 As shown, a standard curve for detecting uric acid is drawn based on the peak current value and uric acid concentration obtained in the test, as shown in FIG. Figure 4 As shown, the standard curve of uric acid is y=0.009x-0.3372, where R 2 The detection sensitivity is 0.13μA / (μmol / L*cm 2 ), the detection linear range is 0~800μmol / L, Figure 4 It can be seen that the peak current value obtained by the sensor of the present invention for detecting different concentrations of uric acid is linearly correlated with the uric acid concentration. When the sensor of the present invention is used to detect a uric acid sample, the actual uric acid concentration in the sample can be obtained according to the uric acid concentration corresponding to the detected peak current value. The detection method is simple, fast, and highly sensitive, and can achieve real-time monitoring of uric acid. The concentration of uric acid in human sweat is about 35.7 μmol / L. Within the linear detection range of the present invention, the sensor of the present invention can meet the detection requirements of uric acid in sweat. Therefore, the sensor of the present invention can be worn on the body for real-time monitoring of the uric acid concentration in human sweat.

[0116] Example 3

[0117] In this example, the breathable flexible wearable uric acid sensor in Example 1 is used to detect uric acid. The specific detection method is as follows:

[0118] The breathable, flexible, wearable uric acid sensor prepared in Example 1 was connected to an electrochemical workstation, and 60 μL of the uric acid solution to be tested was dripped onto the working electrode in the sensor detection area. The uric acid solution was detected using differential pulse voltammetry, and the settings of the differential pulse voltammetry were as follows: the scanning voltage was -0.1 volt to 0.75 volt, the step height was 5 millivolts, the pulse height was 50 millivolts, the pulse interval was 400 milliseconds, the pulse width was 100 milliseconds, and the acquisition time was 20 milliseconds.

[0119] The sensor in this example detected a solution containing 200 μmol / L of uric acid as 181 μmol / L.

[0120] Example 4

[0121] In this example, the breathable flexible wearable uric acid sensor in Example 2 is used to detect uric acid. The specific detection method is as follows:

[0122] The breathable, flexible, wearable uric acid sensor prepared in Example 2 was connected to an electrochemical workstation, and 60 μL of the uric acid solution to be tested was dripped onto the working electrode in the sensor detection area. The uric acid solution was detected using differential pulse voltammetry, and the settings of the differential pulse voltammetry were as follows: the scanning voltage was -0.1 volt to 0.75 volt, the step height was 5 millivolts, the pulse height was 50 millivolts, the pulse interval was 400 milliseconds, the pulse width was 100 milliseconds, and the acquisition time was 20 milliseconds.

[0123] The sensor detected 184 μmol / L of uric acid in a solution containing 200 μmol / L.

[0124] Performance Testing:

[0125] The conductive silver paste pen in Example 1 is used to directly draw electrodes and circuit diagrams on the polytetrafluoroethylene film. The physical pictures of the drawn electrodes and circuit diagrams are as shown in FIG. Figure 5 As shown by Figure 5 It can be seen that if the electrode and circuit diagram are directly drawn on the polytetrafluoroethylene film, the conductive circuit is easily disconnected due to the large pores of the polytetrafluoroethylene film. In Example 1 of the present invention, a layer of polyurethane medical adhesive tape is pasted on the polytetrafluoroethylene film, and then the conductive silver paste pen in Example 1 is used to draw the electrode and circuit diagram on the polyurethane medical adhesive tape. The actual picture of the electrode and circuit diagram drawn on the polytetrafluoroethylene film containing the polyurethane medical adhesive tape in Example 1 is as shown in FIG. Figure 6 As shown by Figure 6 It can be seen that the conductive silver paste presents a continuous circuit on the polyurethane medical tape, and there is no circuit disconnection.

[0126] In order to test the effect of curing temperature on the resistance value of the circuit drawn by the conductive silver paste pen in the process of preparing the breathable, flexible and wearable uric acid sensor in step 3, five parallel experimental groups were set up, and each parallel experimental group was divided into five experimental groups. During the preparation of the sensor, the five experimental groups were cured at room temperature (25°C), 110°C, 120°C, 130°C and 140°C for 30 minutes, and then the resistance value of the circuit in all experimental groups was tested. The specific test results are shown in Table 1.

[0127] Table 1 Resistance test results of the circuits of five parallel experimental groups at different curing temperatures

[0128]

[0129]

[0130] It can be seen from Table 1 above that when the curing temperature is lower than 130°C, the resistance of the circuit drawn by the conductive silver paste is relatively large, and the resistance value is inconsistent, and the resistance value difference is relatively large. However, the present invention adopts a curing temperature of 130°C, and the resistance value of the circuit drawn by the conductive silver paste is relatively uniform, and the resistance value difference is relatively small.

[0131] In order to test the anti-interference performance of the sensor, the sensor in Example 1 of the present invention was used to test the concentration of uric acid in a 100 μmol / L uric acid (UA) solution, a mixed solution containing 100 μmol / L uric acid and 100 μmol / L lactic acid (LA), a mixed solution containing 100 μmol / L uric acid and 100 μmol / L urea (UR), a mixed solution containing 100 μmol / L uric acid and 100 μmol / L glucose (GLU), and a 200 μmol / L uric acid solution. The specific test results are as follows: Figure 7 As shown. Figure 7 It can be seen that the sensor of the present invention has specificity for detecting uric acid because it contains uricase. At the same time, combined with the ability of differential pulse voltammetry to distinguish different substances, it can achieve the ability to resist interference from lactic acid, urea, and glucose and selectively detect uric acid, and the accuracy of the test result is not interfered by lactic acid, urea, and glucose. Therefore, the sensor of the present invention has a high degree of specificity for detecting uric acid, can selectively detect uric acid from many secretions in sweat, and the accuracy of the detection is not interfered by other secretions.

[0132] In order to test the stability of the sensor, the sensor prepared in Example 1 of the present invention was divided into five batches. The five batches of sensors were placed in a refrigerator at 4°C for 1d, 2d, 3d, 4d, and 5d, respectively, and then taken out to test 100 μmol / L uric acid solution. The specific test results are shown in FIG. Figure 8 As shown by Figure 8 It can be seen that as the sensor is placed in the refrigerator for a longer time, the detection efficiency decreases slightly, but the overall test results have little deviation. After being placed in the refrigerator for 5 days, the sensor still maintains a detection efficiency of about 75%. Therefore, the sensor in Example 1 of the present invention has good stability.

[0133] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A uric acid sensor, characterized in that: It comprises a flexible substrate and a waterproof layer which are stacked; three electrodes and a conductive circuit are arranged between the flexible substrate and the waterproof layer; a detection port is arranged on the waterproof layer, and the three electrodes are located at the detection port; the three electrodes comprise a working electrode; a graphene-polyacrylonitrile electrostatic spinning film layer is arranged on the working electrode; uricase and a film-forming aid are adsorbed in the graphene-polyacrylonitrile electrostatic spinning film layer; a waterproof insulating layer is arranged at the contact position between the edge of the detection port and the waterproof layer; the flexible substrate comprises a polytetrafluoroethylene film layer and a polyurethane medical adhesive tape layer which are stacked.

2. The uric acid sensor according to claim 1, characterized in that: The material of the waterproof layer is polyurethane medical adhesive tape.

3. The uric acid sensor according to claim 1, characterized in that: The three electrodes also include a counter electrode and a reference electrode; The counter electrode and the conductive circuit are both provided with a gold coating.

4. The method for preparing the uric acid sensor according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1: forming three electrodes and a conductive circuit on a flexible substrate, and then pasting a graphene-polyacrylonitrile electrospun film layer on the working electrode and heating and curing it; S2: first adding uricase to the graphene-polyacrylonitrile electrospun film layer and drying it, and then adding the film-forming aid and drying it; S3: Pasting a waterproof layer on the flexible substrate, and then forming a waterproof insulating layer at the edge of the detection port to obtain the uric acid sensor.

5. The method for preparing a uric acid sensor according to claim 4, characterized in that: In the step S1, the temperature of heating and curing is 130-140°C.

6. The method for preparing a uric acid sensor according to claim 4, characterized in that: The preparation method of the graphene-polyacrylonitrile electrostatic spinning film layer is: mixing a polyacrylonitrile solution and a graphene solution and electrostatic spinning, and then pre-oxidizing and calcining to obtain the film layer.

7. The method for preparing a uric acid sensor according to claim 4, characterized in that: The preparation method further comprises the step of preparing a gold coating on the counter electrode of the three electrodes and the conductive circuit by electroplating.

8. Use of the uric acid sensor according to any one of claims 1 to 3 in uric acid detection.

9. A method for detecting uric acid, characterized in that: The following steps are involved: The uric acid sensor according to any one of claims 1 to 3 is connected to an electrochemical workstation, a uric acid sample solution is dripped onto the working electrode, the peak current value of uric acid is detected by differential pulse voltammetry, and the concentration of uric acid is obtained according to a uric acid detection standard curve.

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