Enzyme-free glucose biosensor, preparation method thereof and application of enzyme-free glucose biosensor in intelligent wearable electronic equipment

Abstract

The invention provides an enzyme-free glucose biosensor, a preparation method thereof and application of the enzyme-free glucose biosensor in intelligent wearable electronic equipment. The non-enzyme glucose biosensor comprises a flexible material layer, a sensor layer and a perfluorosulfonic polymer layer which are sequentially stacked. The sensor layer comprises a metal sensing material substrate layer and a three-electrode modification layer which is arranged on the outer surface, far away from the perfluorosulfonic polymer layer, of the substrate layer; the three-electrode modification layer comprises a working electrode modification layer, a counter electrode modification layer and a reference electrode modification layer, and materials of the working electrode modification layer comprise copper oxide nanoparticles, calcium titanate nanoparticles and a perfluorosulfonic acid polymer. The glucose biosensor does not need the participation of glucose oxidase, is not easily influenced by external environmental factors, and can improve the stability of the device and the test sensitivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of glucose sensor, in particular to an enzyme-free glucose biosensor, a preparation method thereof and application thereof in intelligent wearable electronic devices. BACKGROUND

[0002] Glucose is an indispensable nutrient in human physiological activities, which is not only the energy source of living cells, but also the intermediate product of metabolism. Since the measurement of blood glucose concentration requires the extraction of patient blood, it will cause damage to the patient's body, while the glucose in sweat can indirectly feedback the corresponding blood glucose concentration and is easy to obtain without harming the patient's body. Therefore, the detection of glucose in sweat has become an effective means for detecting human health.

[0003] The traditional glucose sensor involves glucose oxidase, and the enzyme in the enzyme electrode must be in an environment with suitable temperature, pH, etc. Therefore, the conditions for maintaining enzyme activity are high. When the environment changes, the enzyme is easy to be inactivated, which will cause a series of problems such as poor electrode stability and limited detection accuracy of the device. SUMMARY

[0004] In order to solve the above technical problems, the purpose of the present application is to provide an enzyme-free glucose biosensor, a preparation method thereof and application thereof in intelligent wearable electronic devices. The glucose biosensor does not need the participation of glucose oxidase and is not easily affected by external environmental factors, which can improve the stability of the device and the test sensitivity.

[0005] In order to achieve the above purpose, the present application provides an enzyme-free glucose biosensor, which comprises a flexible material layer, a sensor layer and a perfluorosulfonic acid-based polymer layer which are sequentially stacked. The sensor layer is arranged on the outer surface of the flexible material layer; the perfluorosulfonic acid-based polymer layer is arranged on the outer surface of the sensor layer away from the flexible material layer. The sensor layer comprises a metal sensing material base layer and at least one group of three-electrode modification layers arranged on the outer surface of the base layer away from the perfluorosulfonic acid-based polymer layer; each group of three-electrode modification layers comprises a working electrode modification layer, a counter electrode modification layer and a reference electrode modification layer, the material of the working electrode modification layer comprises copper oxide nanoparticles, calcium titanate nanoparticles and perfluorosulfonic acid-based polymer, and the weight ratio of copper oxide nanoparticles, calcium titanate nanoparticles and perfluorosulfonic acid-based polymer is 0.5-1.5:0.5-1.5:1.5-2.5.

[0006] Firstly, the sensor layer of the present application can realize glucose monitoring without the participation of glucose oxidase, and has strong anti-interference ability. The present application adopts a modified working electrode based on copper oxide nanoparticles, calcium titanate nanoparticles and perfluorosulfonic acid-based polymer. The calcium titanate nanoparticles dispersed on the surface of the copper oxide nanoparticles can improve the electron transfer rate in the detection process and broaden the linear range of detection. The perfluorosulfonic acid-based polymer can increase the conductivity and reduce the interference of other substances in the sweat, thereby enabling the present application to realize glucose monitoring without the participation of glucose oxidase, so as to be less affected by external environmental factors, improve the stability of the device, and improve the test sensitivity.

[0007] The principle involved in the process is that the monovalent copper Cu(I) of CuO is first electrochemically oxidized to divalent copper Cu(II), and then the obtained Cu(OH)2 will continue to be electrochemically oxidized to trivalent copper Cu(III), that is, CuOOH, on the working electrode. Subsequently, in the presence of trivalent copper Cu(III), glucose is catalytically oxidized to obtain gluconolactone, and glucose acid is generated by the hydrolysis of gluconolactone. In this process, Cu(III) trivalent copper compound is further reduced to Cu(II) divalent copper compound.

[0008] The reaction formula involved is as follows:

[0009] CuO+OH - →CuOOH+e - ;

[0010] CuOOH+glucose→Cu(OH)2+gluconic acid;

[0011] Cu(III)+glucose→Cu(II)+gluconolactone;

[0012] Gluconolactone→gluconic acid;

[0013] Secondly, the present application sets a perfluorosulfonic acid-based polymer layer on the sensor layer to form a cation exchange polymer membrane layer, so as to selectively remove anions from the electrode surface. Furthermore, the present application can not only more stably fix the sensor and protect the electrode modification material, but also can increase the overall conductivity of the sensor while increasing its anti-interference ability to other interfering substances (such as lactic acid, uric acid, fatty acid, sodium chloride, etc.) in the sweat, thereby reducing the signal interference caused by other substances to glucose detection.

[0014] Furthermore, the glucose biosensor of the present application also has good biocompatibility, and can be safely attached to the human skin for subsequent sweat glucose testing. At the same time, the present application uses a flexible material layer in cooperation with the above-mentioned sensor layer, so that the device is easy to bend, has high ductility, is convenient to store, and can better fit the skin in subsequent applications.

[0015] Further, the material of the metal sensing material base layer is copper.

[0016] Further, the material of the flexible material layer is polyimide.

[0017] Further, the thickness of the flexible material layer is 0.102-0.106 mm; the thickness of the sensor layer is 0.03-0.07 mm; and the thickness of the perfluorosulfonic acid-based polymer layer is 0.01-0.03 mm.

[0018] In a preferred embodiment, the material of the counter electrode modification layer is selected from one or more than two combinations of gold, silver and platinum.

[0019] In a preferred embodiment, the material of the reference electrode modification layer is silver and silver chloride, and the weight ratio of silver to silver chloride is 1.8-2.2:1. Further, a solid electrolyte layer is further provided on the outer surface of the reference electrode modification layer, and the material of the solid electrolyte layer comprises polyvinyl butyral and sodium chloride, and the weight ratio of polyvinyl butyral to sodium chloride is 1.5-2.2:1.

[0020] To achieve the above object, the present application further provides a preparation method of the above-mentioned enzyme-free glucose biosensor, which comprises the following steps:

[0021] providing a sample preparation substrate, which comprises a flexible material layer and a metal sensing material layer provided on the outer surface of the flexible material layer;

[0022] providing a paper pre-product printed with a carbon-based sensor image;

[0023] transferring the carbon-based sensor image from the paper pre-product to the outer surface of the metal sensing material layer of the sample preparation substrate by heat transfer printing;

[0024] removing the metal sensing material on the sample preparation substrate which is not covered by the carbon-based sensor image;

[0025] removing the carbon-based sensor image on the sample preparation substrate to obtain a flexible material layer provided with a metal sensing material base layer on the outer surface thereof;

[0026] providing at least one set of three-electrode modification layers on the outer surface of the metal sensing material base layer to form a sensor layer;

[0027] providing a perfluorosulfonic acid-based polymer layer on the outer surface of the sensor layer away from the flexible material layer to obtain an enzyme-free glucose biosensor.

[0028] Based on the foregoing reasons, the non-enzyme glucose biosensor obtained by the present application is easy to bend, has high ductility, is convenient to store, can better adhere to the skin, does not need the participation of glucose oxidase, is not easily affected by external environmental factors, can improve the stability of the device and the test sensitivity, and has excellent conductivity and strong anti-interference ability.

[0029] In particular, the present application uses a heat transfer printing technology to prepare the above-mentioned glucose biosensor, and such a preparation method has the advantages of cost saving, flexible design, environmental friendliness and the like. First, heat transfer printing can reduce material waste, labor input and printing equipment cost through highly customized computer patterns. Second, the heat transfer printing technology has the advantages of easy replacement, fast pattern replacement, flexible design and the like, and is suitable for small-batch, multi-change creative categories. Third, the heat transfer printing technology is convenient, easy to copy, has high definition and good accuracy. In addition, the above-mentioned preparation method of the present application is more environmentally friendly and requires less chemical solvent.

[0030] Further, the processing temperature of heat transfer printing is 180-220℃. In some optional embodiments, the above-mentioned heat transfer printing technology of the present application can be realized only by using some simple instruments, such as an iron, a portable heat transfer printer, or any device that can adjust the temperature, heat the surface flatly and uniformly, that is, the entire sensor shape can be printed, or the electrode shape can be printed according to the pre-designed pattern for subsequent processing.

[0031] Further, the three-electrode modification layer is arranged on the outer surface of the metal sensing material substrate layer by the following steps:

[0032] The working electrode modification step: a dispersion liquid containing copper oxide nanoparticles, calcium titanate nanoparticles and perfluorosulfonic acid-based polymer is coated on the outer surface of the metal sensing material substrate layer corresponding to the working electrode, and a working electrode modification layer is obtained after drying;

[0033] The counter electrode modification step: a counter electrode modification layer is arranged on the outer surface of the metal sensing material substrate layer corresponding to the working electrode by using a magnetron sputtering method;

[0034] The reference electrode modification step: a reference electrode material is coated on the outer surface of the metal sensing material substrate layer corresponding to the reference electrode, and a reference electrode modification layer is obtained after drying; a solid electrolyte material is coated on the outer surface of the reference electrode modification layer, and a solid electrolyte layer is obtained after drying;

[0035] The working electrode modification layer, the counter electrode modification layer and the reference electrode modification layer are assembled to form a three-electrode modification layer.

[0036] Further, the copper oxide nanoparticles are prepared by the following steps: mixing copper sulfate aqueous solution and urea, and then performing hydrothermal reaction at 130-150 ℃ for 18-22 h to obtain the copper oxide nanoparticles; the molar ratio of copper sulfate to urea in the copper sulfate aqueous solution is 1:8.0-9.0.

[0037] Further, the calcium titanate nanoparticles are prepared by the following steps: heat treating calcium nitrate tetrahydrate and titanium dioxide P25 at 550-650 ℃ for 9-11 h to obtain the calcium titanate nanoparticles; the weight ratio of calcium nitrate tetrahydrate to titanium dioxide P25 is 1:1-1.3.

[0038] Further, the present application removes the metal sensing material on the sample preparation substrate which is not covered by the carbon-based sensor image by corrosion. Alternatively, the person skilled in the art can place the sample to be treated in a corrosion liquid for corrosion. Specifically, the PCB circuit board etchant (a conventional commercial reagent in the art, without special limitation) can be dissolved in water to form a corrosion liquid for application. In the corrosion liquid, the weight amount of the PCB circuit board etchant is 150-200 g per 1 L of water. Further, the treatment temperature for corrosion is 35-45 ℃, and the treatment time is 15-25 min.

[0039] Specifically, in a preferred embodiment, the person skilled in the art can prepare the above-mentioned enzyme-free glucose biosensor by the following steps:

[0040] S1, using a printer (for example, a digital printer using carbon powder as printing material, which can print the designed shape with high precision, and the heat transfer process will not cause a decrease in precision), the pre-designed sensor image is printed on the heat transfer paper (for example, A4 size heat transfer paper).

[0041] S2, prepare a flexible material-metal sensing material composite film (with a flexible material as the substrate, the surface of which is covered with a thin film of metal sensing material) of the same size. Place the flexible material-metal sensing material composite film on a flat surface, and then place the pre-printed paper with the sensor image on the flexible material-metal sensing material composite film, with the side with the sensor image facing the side of the metal sensing material film, so as to facilitate subsequent heat transfer.

[0042] S3, place the heat transfer equipment on the sample to be transferred, adjust the heating temperature of the heat transfer equipment, and press for 8-12 min after the temperature reaches the above-mentioned treatment temperature and stabilizes. Then, when the sample is reduced to room temperature, carefully tear off the A4 size heat transfer paper, and at this time the sensor pattern is transferred to the flexible material-metal sensing material composite film.

[0043] S4, compare the pattern after heat transfer with the design pattern, the missing part can be filled with carbon pen (carbon pen containing carbon material), complete the pattern. Then put the processed flexible material-metal sensing material composite film into a container with pre-added etching solution, heat the container to about 35-45℃ and keep shaking for 15-25 min, when the flexible material-metal sensing material composite film is etched except the metal sensing material on the sensor pattern, the etching is completed.

[0044] S5, use sandpaper to wipe off the excess carbon powder on the etched flexible material-metal sensing material composite film, after wiping off, the previously designed sensor pattern can be clearly seen. According to the needs of subsequent processes, cut the sensor pattern into multiple complete three-electrode sensor substrate layers or cut into multiple separate working electrode substrate layers, reference electrode substrate layers and counter electrode substrate layers, so as to modify individual electrodes later.

[0045] S51, modification of working electrode: under ultrasonic conditions, disperse calcium titanate and copper oxide in a perfluorosulfonic acid-based polymer solution to obtain a mixed solution of copper oxide, calcium titanate and perfluorosulfonic acid-based polymer. Coat the mixed solution on the surface of the aforementioned pre-heat transferred working electrode, and after drying, the above working electrode is obtained.

[0046] S52, modification of counter electrode: cut the pre-heat transferred counter electrode and put it into a magnetron sputtering machine, and use magnetron sputtering technology (sputtering conditions: vacuum degree less than 1.8×10 -3 Pa, sputtering power 60-100W, sputtering time 3-8min) to plate gold on its surface to obtain the above counter electrode.

[0047] S53, modification of reference electrode: cut the pre-heat transferred reference electrode, coat silver and silver chloride paste on the outer surface of the reference electrode, and after drying, continue to add polyvinyl butyral mixed solution (obtained by dissolving polyvinyl butyral and sodium chloride in methanol) to the surface, and after drying, the above reference electrode is obtained.

[0048] For the synthesis of copper oxide, weigh copper sulfate pentahydrate and dissolve it in deionized water and stir, then add urea and continue to stir to obtain a solution system. Then use hydrothermal method to put the solution system into a hydrothermal reactor and continue to heat to 135-145℃ for 15-25h to obtain the reaction product, which is dried to obtain powder-like copper oxide for subsequent modification application.

[0049] For the synthesis of calcium titanate, weigh calcium nitrate tetrahydrate and titanium dioxide P25 and heat to 550-650℃, and keep the temperature for 9-11h, then cool to room temperature to obtain nanoparticle-like calcium titanate for subsequent modification application.

[0050] To achieve the above objectives, the present invention also provides an application of the aforementioned enzyme-free glucose biosensor in a smart wearable electronic device.

[0051] Based on the reasons stated above, the enzyme-free glucose biosensor of the present invention can be effectively used for continuous glucose monitoring (e.g., sweat), and can thus be applied in glucose monitoring sensors and smart wearable electronic devices. Its application in glucose monitoring offers the following advantages: it is flexible, highly ductile, easy to store, and conforms better to the skin; it does not require the participation of glucose oxidase, making it less susceptible to external environmental factors, thus improving both device stability and testing sensitivity; simultaneously, it exhibits excellent conductivity and strong anti-interference capabilities. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the glucose sensor in one embodiment of the present invention;

[0053] Figure 2 This is a scanning electron microscope image of the calcium titanate-copper oxide-perfluorosulfonic acid polymer mixed solution in Example 1 of the present invention;

[0054] Figure 3 This is a schematic diagram of the modified working electrode, counter electrode, and reference electrode, and the sensor layer formed by assembling the three in Embodiment 1 of the present invention.

[0055] Figure 4 The above is a response curve of the glucose sensor in Embodiment 1 of the present invention when detecting glucose concentrations of 0.01 to 0.05 mM.

[0056] Figure 5 This is a calibration curve of glucose detection by the glucose sensor in Embodiment 1 of the present invention;

[0057] Figure 6 The above is a response curve of the glucose sensor in Embodiment 1 of the present invention when detecting glucose concentrations of 0.01 to 2 mM.

[0058] Figure 7 The H&E image of the tissue surrounding the glucose sensor at day 14 of Embodiment 1 of the present invention is shown.

[0059] Figure 8 This is a cytotoxicity test diagram of the glucose sensor in Embodiment 1 of the present invention;

[0060] Figure 9 This is a graph showing the migration test of L929 cells by the leaching solution of the glucose sensor in Embodiment 1 of the present invention.

[0061] Figure 10 This is a graph showing the glucose concentration in the sweat of volunteers before and after riding a stationary bike in Test Example 4 of this invention.

[0062] Figure 11 This is a graph showing the glucose concentration in the sweat of volunteers before and after climbing a hill, as shown in Test Example 4 of this invention.

[0063] Figure 12 These are photographs of each group of sensor layers in Test Example 5 of this invention;

[0064] Figure 13 This is a stability test diagram of the glucose sensor in Embodiment 1 of the present invention;

[0065] Figure 14 This is a test diagram of the anti-interference performance of the glucose sensor in Embodiment 1 of the present invention.

[0066] Reference numerals: 10 - Flexible material layer; 20 - Sensor layer; 30 - Perfluorosulfonic acid polymer layer. Detailed Implementation

[0067] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0068] Example 1

[0069] like Figure 1 As shown, the glucose sensor in this embodiment includes a flexible material layer 10, a sensor layer 20 (with a set of three electrodes), and a perfluorosulfonic acid-based polymer layer 30, which are stacked sequentially. The flexible material layer 10 has a thickness of 0.104 mm; the sensor layer 20 has a thickness of 0.05 mm; and the perfluorosulfonic acid-based polymer layer 30 has a thickness of 0.02 mm.

[0070] This embodiment provides a method for preparing a glucose sensor:

[0071] (1) Use a digital printer with toner as the printing material to print the pre-designed sensor image onto A4-sized thermal transfer paper. Also prepare a polyimide-copper film of the same A4 size (a thin film of copper covering a polyimide substrate).

[0072] (2) Place the polyimide-copper film on a flat surface, and then place the pre-printed A4-sized heat transfer paper with the sensor image on the polyimide-copper film, ensuring that the side with the sensor image faces the copper side to facilitate subsequent heat transfer.

[0073] (3) Place the iron on the sample to be transferred, adjust the iron heating temperature, and press for 10 minutes after the temperature reaches 200℃ and stabilizes. Then, when the sample cools down to room temperature, carefully peel off the A4-sized heat transfer paper. At this time, the sensor pattern has been successfully heat transferred to the polyimide-copper film.

[0074] (4) The pattern after thermal transfer is observed to see if it is consistent with the design pattern. Missing parts can be filled in with carbon pen to complete the pattern. Then put the treated polyimide-copper film into a container with pre-added etching solution (prepared by adding 180 g of commercial PCB etching agent to 1 L of deionized water) and heat the container to about 40°C while keeping it shaking for 20 min. When the copper on the polyimide-copper film except the sensor pattern is etched, the etching is completed.

[0075] (5) Use sandpaper to remove the excess carbon powder on the etched polyimide-copper film. After wiping, the previously designed sensor pattern can be clearly seen.

[0076] (6) Cut the above sensor pattern into multiple individual working electrode substrate layers, reference electrode substrate layers, and counter electrode substrate layers for subsequent modification.

[0077] Copper oxide nanoparticle synthesis:

[0078] Weigh 1.6 g of copper sulfate pentahydrate and dissolve it in 100 mL of deionized water, stirring on a magnetic stirrer for 10 min, then add 3.2 g of urea and continue stirring for 60 min. Then use the hydrothermal method to place the solution in a hydrothermal reactor and continue heating to 140°C for 20 h, and finally dry the obtained copper oxide nanoparticles at 80°C for later use.

[0079] Calcium titanate nanoparticle synthesis:

[0080] Weigh 2 g of calcium nitrate tetrahydrate and 2.5 g of titanium dioxide P25 and place them in a crucible. Heat the crucible to 600°C in a muffle furnace, keep it at this temperature for 10 h, then cool it to room temperature. Finally, collect the calcium titanate nanoparticles for subsequent use.

[0081] Modification of working electrode: Under ultrasonic conditions, disperse 1.0 mg of calcium titanate nanoparticles synthesized above and 1.0 mg of copper oxide nanoparticles synthesized above in 1.0 mL of 0.5% volume concentration of perfluorosulfonic acid-based polymer aqueous solution to form a calcium titanate-copper oxide-perfluorosulfonic acid-based polymer mixed solution (SEM image as shown in Figure 2 Take 1 mL of the dispersed mixed solution and evenly coat it on the surface of the aforementioned cut working electrode substrate layer area (d = 0.15 cm), then place it in a drying oven to dry, obtaining a working electrode.

[0082] Modification of counter electrode: Put the aforementioned cut counter electrode substrate layer area into a magnetron sputtering machine and use magnetron sputtering technology (sputtering conditions: vacuum degree less than 1.8 x 10-3 The gold-coated surface of the working electrode was used as the counter electrode.

[0083] Modification of the reference electrode: A mixture of silver and silver chloride (weight ratio of silver to silver chloride was 2.0:1) was coated on the area of the aforementioned cut reference electrode substrate layer, dried in an oven at 90°C for 15 min, and then a polyvinyl butyral mixture solution (78.0 mg of polyvinyl butyral and 50.0 mg of sodium chloride were dissolved in 1 mL of methanol, and then ultrasonically treated for 20 min to obtain) was added dropwise, and dried at room temperature to obtain the reference electrode.

[0084] (7) Assembly of the glucose sensor: The modified working electrode, counter electrode, and reference electrode were assembled to form a sensor layer 20 (as shown in Figure 3 ) on the outer surface of the flexible material layer 10, and a layer of perfluorosulfonic acid-based polymer layer 30 was provided on the outer surface of the sensor layer 20 away from the flexible material layer 10 to obtain the glucose sensor. The thickness of the flexible material layer 10 was 0.104 mm; the thickness of the sensor layer 20 was 0.05 mm; and the thickness of the perfluorosulfonic acid-based polymer layer 30 was 0.02 mm.

[0085] Comparative Example 1

[0086] The difference between Example 1 and Comparative Example 1 is only in the modification of the working electrode: 1 mg of CuO nanofiber was dispersed in 1.0 mL of 0.5% perfluorosulfonic acid-based polymer aqueous solution under ultrasonic conditions to form a mixed solution. The mixed solution was stirred for 15 min, and then the dispersion was ultrasonically treated for 20 min until the dispersion was uniform. 1 mL of the dispersed mixed solution was uniformly coated on the surface of the aforementioned cut working electrode substrate layer area (d = 0.15 cm), and then it was placed in a drying oven for drying to obtain the working electrode.

[0087] The CuO nanofiber was prepared by electrospinning: 0.4 g of copper acetate was slowly added to 7.6 g of PVA aqueous solution. The solution was kept under vigorous magnetic stirring for 12 h to obtain a viscous gel. The viscous gel was continuously loaded into a syringe and connected to a high-voltage power supply for electrospinning, and an electric potential of 15 kV was applied, and the obtained spinning was CuO nanofiber. For details, see: Wang, W.; Zhang, L.; Tong, S.; Li, X.; Song, W. Three-dimensional network films of electrospun copper oxide nanofibers for glucose determination. Biosens. Bioelectron. 2009, 25, 708-714.

[0088] Comparative Example 2

[0089] The difference between Example 1 and Comparative Example 2 is only in the modification of the working electrode: 1 mg of Cu / CuO / ZnO sheet was dispersed in 1.0 mL of aqueous solution of perfluorosulfonic acid-based polymer with a concentration of 0.5% under ultrasonic conditions to form a mixed solution. The mixed solution was stirred for 15 min, and then the dispersion was ultrasonically treated for 20 min until the dispersion was uniform. 1 mL of the dispersed mixed solution was uniformly coated on the surface of the aforementioned cut working electrode base layer area (d = 0.15 cm), and then it was placed in a drying oven for drying to obtain the working electrode.

[0090] The Cu / CuO / ZnO sheet was synthesized as follows: first, copper oxide nanosheets were arranged on the outer surface of the copper sheet by chemical oxidation method, and then a zinc oxide layer was arranged on the outer surface of the copper oxide nanosheets away from the copper sheet by spin-coating sol-gel precursor method to obtain the Cu / CuO / ZnO sheet. For details, see: S. So Yoon, A. Ramadoss, B. Saravanakumar, S. J. Kim, Novel Cu / CuO / ZnO hybrid hierarchical nanostructures for non-enzymatic glucose sensor application, J. Electroanal. Chem. 717 (2014) 90-95.

[0091] Comparative Example 3

[0092] The difference between Example 1 and Comparative Example 3 is only in the modification of the working electrode: 1 mg of NPG / CuO was dispersed in 1.0 mL of aqueous solution of perfluorosulfonic acid-based polymer with a concentration of 0.5% under ultrasonic conditions to form a mixed solution. The mixed solution was stirred for 15 min, and then the dispersion was ultrasonically treated for 20 min until the dispersion was uniform. 1 mL of the dispersed mixed solution was uniformly coated on the surface of the aforementioned cut working electrode base layer area (d = 0.15 cm), and then it was placed in a drying oven for drying to obtain the working electrode.

[0093] The NPG / CuO was synthesized as follows: NPG / CuO was synthesized by electrodeposition of CuO on the surface of NPG electrode by plating potential cycling method. Here, NPG is dealloyed nanoporous gold. For details, see: Xiao X, Li H, Pan Y, et al. Non-enzymatic glucose sensors based on controllable nanoporous gold / copper oxide nanohybrids [J]. Talanta, 2014, 125: 366-371.

[0094] Comparative Example 4

[0095] The difference between Example 1 and this example is only in the modification of the working electrode: 1 mg of CuO nanorods was dispersed in 1.0 mL of aqueous solution of perfluorosulfonic acid-based polymer with a volume concentration of 0.5% to form a mixed solution under ultrasonic conditions. The mixed solution was stirred for 15 min, and then the dispersion was ultrasonically treated for 20 min until the dispersion was uniform. 1 mL of the dispersed mixed solution was uniformly coated on the surface of the aforementioned cut working electrode base layer area (d = 0.15 cm), and then it was placed in a drying oven to dry, obtaining the working electrode.

[0096] wherein the CuO nanorods were synthesized: 9 g of a mixture of sodium hydroxide and potassium hydroxide (weight ratio of NaOH and KOH was 51.5:48.5) was placed in a 25 mL Teflon container. Then 2 mM of copper chloride and 0.1 g of sodium sulfide were added to the container. The container was sealed and placed in a furnace preheated to 200°C. After 24 h of reaction, the container was removed and allowed to cool naturally to room temperature. Finally, the black product was washed several times with deionized water and ethanol to obtain the product, which was CuO nanorods. For details, see: Wang X, Hu C, Liu H, et al. Synthesis of CuO nanostructures and their application for nonenzymatic glucose sensing [J]. Sensors and Actuators B: Chemical, 2010, 144(1): 220-225.

[0097] Comparative Example 5

[0098] The difference between Example 1 and this example is only in the modification of the working electrode: 1 mg of CuO microparticles was dispersed in 1.0 mL of aqueous solution of perfluorosulfonic acid-based polymer with a volume concentration of 0.5% to form a mixed solution under ultrasonic conditions. The mixed solution was stirred for 15 min, and then the dispersion was ultrasonically treated for 20 min until the dispersion was uniform. 1 mL of the dispersed mixed solution was uniformly coated on the surface of the aforementioned cut working electrode base layer area (d = 0.15 cm), and then it was placed in a drying oven to dry, obtaining the working electrode.

[0099] Wherein, CuO microparticles synthesis: 0.5 g of copper chloride was dissolved in 5 mL of water, sonicated for 10 min, and 0.8 M oxalic acid solution was added to the copper chloride solution. The solution color changed to dark blue to blue-green, which corresponds to the conversion of copper chloride to copper oxalate. The copper oxalate was then filtered out and transferred to a quartz beaker and kept in the furnace. The temperature was kept at 450 °C for 2 h. Subsequently, the furnace was allowed to reduce the temperature to 30 °C. The sample was collected and allowed to granulate well. The obtained compound was washed with double distilled water to remove the impurities. The sample was dried in an air oven at 45 °C for 24 h, and the obtained sample was CuO microparticles. For more details, see: Yang Z, Fan M, Liu J, et al. Intelligent sensor of glucose based on CuO nanomaterials [J]. Int. J. Electrochem. Sci, 2019, 14: 11531-11540.

[0100] Comparative Example 6

[0101] The difference between Example 1 and this example is only in the modification of the working electrode: 1 mg of rose-like nanostructured copper oxide was dispersed in 1.0 mL of a 0.5% volume concentration of a perfluorosulfonic acid-based polymer aqueous solution under ultrasonic conditions to form a mixed solution. The mixed solution was taken and stirred for 15 min, and then the dispersion was ultrasonically treated for 20 min until the dispersion was uniform. 1 mL of the dispersed mixed solution was uniformly coated on the surface of the aforementioned cut working electrode base layer area (d = 0.15 cm), and then it was placed in a drying box to dry, obtaining the working electrode.

[0102] Wherein, rose-like nanostructured copper oxide synthesis: 0.1 M aqueous solution of copper nitrate and 0.1 M aqueous solution of hexamethylene tetramine were continuously stirred and mixed for 30 min, both in 50 mL of deionized water. A few drops of ammonium hydroxide were added to the resulting solution to maintain pH = 10, and stirred again for 20 minutes. After that, the resulting solution was transferred to an autoclave and heated to 120 ± 10 °C for 6 h. After 6 h of reaction, the autoclave was cooled to room temperature, obtaining a green-black precipitate, which was washed with deionized water and ethanol in turn, and dried at 65 °C for 3 hours, and the obtained sample was rose-like nanostructured copper oxide. For more details, see: Kim S H, Umar A, Hwang S W. Rose-like CuO nanostructures for highly sensitive glucose chemical sensor application [J]. Ceramics International, 2015, 41(8): 9468-9475.

[0103] Performance test:

[0104] (I) Sensitivity, linear range, and detection limit testing

[0105] Sensitivity: Test Example 1

[0106] Standard glucose solutions with concentration gradients of 0.01 mM to 0.05 mM were prepared. A 0.1 M sodium hydroxide solution with pH 13 was used as a buffer solution. The sodium hydroxide buffer solution was first drop-coated until it completely covered the electrode detection area. Then, glucose solutions of different concentrations were sequentially drop-coated onto the electrode detection area. An electrochemical workstation was used to perform I / O tests on the sensor under different glucose concentrations, obtaining the response curves for glucose detection by the flexible sensor. A calibration curve for glucose detection was also plotted. Based on the calibration curves, the corresponding linear equations and correlation coefficients for low glucose concentrations from 0.01 mM to 0.05 mM were derived, and the sensitivity of the flexible glucose sensor was calculated. For example, the response curve for glucose detection by the flexible sensor in Example 1 is shown below. Figure 4 As shown, the calibration curve for glucose detection is as follows: Figure 5 As shown, based on the calibration curve, the corresponding linear equation for low concentrations of glucose from 0.01 mM to 0.05 mM is Ipc(μA) = 34.43Cglucose(mM) + 7.592, with a correlation coefficient R. 2 =0.993, the calculated sensitivity of this flexible glucose sensor is 487.3 μAmM. -1 cm -2 Sensitivity calculations were performed for Comparative Examples 1 through 6, and the results are shown in Table 1.

[0107] Linear range: Test Case 2

[0108] Standard glucose solutions with concentration gradients ranging from 0.01 mM to 2 mM were prepared. A 0.1 M sodium hydroxide solution with a pH of 13 was used as a buffer solution. The sodium hydroxide buffer solution was first drop-coated until it completely covered the electrode detection area. Then, glucose solutions of different concentrations were sequentially drop-coated onto the electrode detection area. The sensor under different concentrations of glucose solutions was tested using an electrochemical workstation. The response curve of the flexible sensor for glucose detection in Example 1 is shown below. Figure 6 As shown in the table. The results indicate that the flexible glucose biosensor of Example 1 can produce efficient detection results for glucose concentration in the range of 0.01 mM to 2 mM, and the linear range for glucose detection by the flexible sensor of Example 1 is 0.01 mM to 2 mM. Linear range tests were conducted similarly for Comparative Examples 1 to 6, and the results are shown in Table 1.

[0109] Detection limit: The concentration value corresponding to three times the instrument noise. The estimated detection limits for Example 1, Comparative Examples 1-6 are shown in Table 1.

[0110] Table 1

[0111]

[0112] Compared with Comparative Examples 1-6, the copper oxide nanoparticles, calcium titanate nanoparticles and perfluorosulfonic acid-based polymer used in Example 1 of the application as the material of the working electrode modification layer can make the device have more excellent sensitivity, linear range and detection limit performance. Among them, the addition of calcium titanate nanoparticles increases the specific surface area of copper oxide nanoparticles and increases the conductivity of the entire working electrode; at the same time, the dispersion of calcium titanate nanoparticles on the surface of copper oxide nanoparticles can improve the electron transfer rate in the detection process and broaden the linear range of detection; the perfluorosulfonic acid-based polymer can increase the conductivity and reduce the interference of other substances in the sweat.

[0113] (ii) Biocompatibility test

[0114] Test Example 3

[0115] (a) Figure 7 H&E images of the tissue around the flexible sensor of Example 1 at the time point of day 14 are shown (scale bar: 20 μm).

[0116] (b) The toxicity of the leachate of the flexible sensor of Example 1 on L929 cells was determined by a calcein-AM / propidium iodide staining test. As shown in Figure 8 , live cells-green; dead cells-red; scale bar: 1 cm: 100 μm.

[0117] (c) The effect of the leachate of the flexible sensor of Example 1 on the migration of L929 cells was detected by a scratch test. As shown in Figure 9 , scale bar: 1 cm: 100 μm.

[0118] In the above-mentioned (a), (b), (c) three tests, the control group did not paste the flexible sensor, and the experimental group pasted the flexible sensor of Example 1. The results show that the flexible glucose biosensor of the application has good biocompatibility and can be safely pasted on the human skin for sweat glucose testing.

[0119] (iii) Real-time human sweat glucose test

[0120] Test Example 4

[0121] In this test, the volunteer is a 26-year-old healthy male with no history of diabetes. Before installing the flexible sensor of Example 1 on his arm, the skin where the sensor is placed will be carefully disinfected and cleaned to prevent other substances from affecting the experimental results. A layer of PU film will be used to secure the sensor to prevent it from loosening during movement. During this process, the volunteer will be asked to ride a stationary bike and climb a hill for 30 minutes, respectively, to secrete a certain amount of sweat.

[0122] This test records the initial state of the volunteer before exercise and the late state after 30 minutes of exercise. Figure 10 The initial state of the volunteer before riding a stationary bike and the late state after 30 minutes of exercise are shown. Figure 10 The initial state of the volunteer before climbing a hill and the late state after 30 minutes of exercise are shown. Figure 10 and Figure 11 It can be found that the current changes significantly before and after exercise. Compared with the initial state of the volunteer, the current decreases after cycling and climbing, which proves that the glucose concentration in sweat decreases with the consumption of energy and the passage of time during exercise. The more intense the exercise, the more energy consumed and the more sweat secreted, which will lead to the reduction of glucose in the human body. Figure 11 The current change during climbing is more obvious than that during cycling, because cycling is more tiring and consumes more energy than climbing. Although there are fluctuations in the current signal in this test, the results still show that when the glucose concentration in the volunteer's sweat changes, the signal of the sensor will also change, which indicates that the above-mentioned glucose sensor of the present application is wearable and can be applied to monitor the real-time change of glucose concentration in human sweat.

[0123] (IV) Effect of pressing temperature and time on the appearance of the finished product of the flexible sensor manufactured by heat transfer printing technology

[0124] Test group (1): sensor layer of Example 1.

[0125] Test group (2): the difference between the sensor layer and Example 1 is only that the processing temperature of heat transfer printing is 100°C and the processing time is 5 minutes.

[0126] Test group (3): the difference between the sensor layer and Example 1 is only that the processing temperature of heat transfer printing is 100°C and the processing time is 10 minutes.

[0127] Test group (4): the difference between the sensor layer and Example 1 is only that the processing temperature of heat transfer printing is 150°C and the processing time is 10 minutes.

[0128] Test group (5): the difference between the sensor layer and Example 1 is only that the processing temperature of heat transfer printing is 200°C and the processing time is 15 minutes.

[0129] The photos of the sensing layer of the test groups (1)-(5) are shown in Figure 12 The test group (1) sensor image is clear and complete, and the corresponding temperature and pressing time can be accurately transferred to the designed sensor pattern. The test group (2) has poor transfer completeness. The test group (3) has better pattern completeness, but also has the disadvantage of unclearness. The test group (4) has good completeness, but slightly poor accuracy. The test group (5) has some slight adhesion of the pattern due to the long time.

[0130] (V) Stability test

[0131] Test Example 6

[0132] The stability was studied by monitoring the current response in the presence of 0.01 mM glucose for five weeks. The flexible sensor of Example 1 was stored in dry air at room temperature without use for five weeks. The initial current was 5.882 μA, and the current after storage for five weeks was 5.187 μA, as shown in Figure 13 , showing good stability, and the current intensity after five weeks was 88% of the initial current intensity.

[0133] (VI) Anti-interference test

[0134] Test Example 7

[0135] The anti-interference test was performed by current response in the presence of some common interference substances in human sweat. As shown in Figure 14 , after the continuous addition of 0.1 mM glucose (Glu), 1 mM uric acid (UA), 1 mM NaCl, 1 mM ascorbic acid (AA), 1 mM lactic acid (LA), and 0.1 mM glucose (Glu), there was no obvious current response when various interference substances were added, and the current significantly increased after the addition of glucose, which can indicate that the present application has good anti-interference ability.

Claims

1. An enzyme-free glucose biosensor, wherein, The sensor includes a flexible material layer, a sensor layer, and a perfluorosulfonic acid-based polymer layer stacked sequentially; the sensor layer includes a metal sensing material substrate layer and at least one set of three-electrode modification layers disposed on the outer surface of the substrate layer away from the perfluorosulfonic acid-based polymer layer; each set of three-electrode modification layers includes a working electrode modification layer, a counter electrode modification layer, and a reference electrode modification layer. The material of the working electrode modification layer includes copper oxide nanoparticles, calcium titanate nanoparticles, and perfluorosulfonic acid-based polymers, and the weight ratio of the copper oxide nanoparticles, the calcium titanate nanoparticles, and the perfluorosulfonic acid-based polymers is 0.5-1.5:0.5-1.5:1.5-2.

5.

2. The enzyme-free glucose biosensor according to claim 1, wherein, The metal sensing material substrate is made of copper; the flexible material layer is made of polyimide.

3. The enzyme-free glucose biosensor according to claim 1 or 2, wherein, The material of the electrode modification layer is selected from one or more combinations of gold, silver and platinum.

4. The enzyme-free glucose biosensor according to claim 1, wherein, The material of the reference electrode modification layer is silver and silver chloride.

5. The enzyme-free glucose biosensor according to claim 4, wherein, A solid electrolyte layer is also disposed on the outer surface of the reference electrode modification layer, and the material of the solid electrolyte layer includes polyvinyl butyral and sodium chloride.

6. The enzyme-free glucose biosensor according to claim 1 or 2, wherein, The thickness of the flexible material layer is 0.102–0.106 mm; The thickness of the sensor layer is 0.03–0.07 mm; The thickness of the perfluorosulfonic acid-based polymer layer is 0.01 to 0.03 mm.

7. A method for preparing an enzyme-free glucose biosensor according to any one of claims 1 to 6, wherein, Includes the following steps: A sample preparation substrate is provided, the sample preparation substrate comprising a flexible material layer and a metal sensing material layer disposed on the outer surface of the flexible material layer; Provide paper pre-made products printed with images of carbon sensors; The carbon sensor image is transferred from the paper preform to the outer surface of the metal sensing material layer in the sample preparation substrate using a thermal transfer method. Remove the metallic sensing material on the sample substrate that is not covered by the carbonaceous sensor image; Remove the carbonaceous sensor image from the sample substrate to obtain a flexible material layer with a metallic sensing material substrate layer on its outer surface. At least one set of three-electrode modification layers is disposed on the outer surface of the metal sensing material substrate to form a sensor layer; A perfluorosulfonic acid-based polymer layer is disposed on the outer surface of the sensor layer away from the flexible material layer to obtain the enzyme-free glucose biosensor.

8. The preparation method according to claim 7, wherein, The heat transfer process is carried out at a temperature of 180–220°C.

9. The preparation method according to claim 7, wherein, The three-electrode modification layer is formed on the outer surface of the metal sensing material substrate by the following steps: Working electrode modification step: A dispersion containing copper oxide nanoparticles, calcium titanate nanoparticles and perfluorosulfonic acid polymer is coated on the outer surface of the working electrode corresponding to the metal sensing material substrate layer, and the working electrode modification layer is obtained after drying. Electrode modification step: The electrode modification layer is deposited on the outer surface of the metal sensing material substrate corresponding to the working electrode by magnetron sputtering; Reference electrode modification steps: The reference electrode material is coated onto the outer surface of the reference electrode corresponding to the metal sensing material substrate layer, and after drying, the reference electrode modification layer is obtained; a solid electrolyte material is coated onto the outer surface of the reference electrode modification layer, and after drying, a solid electrolyte layer is obtained. The working electrode modification layer, the counter electrode modification layer, and the reference electrode modification layer are assembled to form the three-electrode modification layer.

10. The preparation method according to claim 7, wherein, The copper oxide nanoparticles were prepared by the following steps: a mixture of copper sulfate aqueous solution and urea was subjected to a hydrothermal reaction at a temperature of 130-150°C for 18-22 hours to obtain the copper oxide nanoparticles. The molar ratio of copper sulfate to urea in the copper sulfate aqueous solution is 1:8.0 to 9.

0.

11. The preparation method according to claim 7, wherein, The calcium titanate nanoparticles were prepared by the following steps: calcium nitrate tetrahydrate and titanium dioxide P25 were heat-treated at 550-650℃ for 9-11 hours to obtain the calcium titanate nanoparticles. The weight ratio of the calcium nitrate tetrahydrate to the titanium dioxide P25 is 1:1 to 1.

3.

12. The application of an enzyme-free glucose biosensor according to any one of claims 1 to 6 in a smart wearable electronic device.

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