A glucose biosensor, its preparation method and application

Abstract

The present invention discloses a glucose biosensor, a preparation method thereof and an application. Specifically, it includes coating an electrode paste on the surface of a flexible substrate and then preparing a three-dimensional graphene electrode body with a three-electrode structure by CO2 laser engraving; coating a conductive paste on the surface of any one of the electrodes in the three-dimensional graphene electrode body with a three-electrode structure to prepare a reference electrode; then immersing it in a sulfuric acid solution of pyrrole and preparing a working electrode by cyclic voltammetry to obtain a graphene-polypyrrole electrode; then soaking it in a sulfuric acid-acidified chloroauric acid solution for electrodeposition to obtain a graphene-polypyrrole-gold nanoparticle electrode, and finally loading glucose oxidase and connecting an analytical instrument to obtain a glucose biosensor. The glucose biosensor prepared by the present invention can significantly improve the electrochemical performance of the glucose biosensor through the synergistic enhancement effect of polypyrrole and gold nanoparticles, and is obtained by a non-physical binding method, having excellent structural stability and electrochemical performance.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and particularly relates to a glucose biosensor, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetes is a common chronic disease caused by insufficient insulin secretion in the human body, which reduces the ability of cells to absorb glucose from the blood, and has a serious impact on human society and economy. Although there is no way to cure or prevent diabetes, it is recommended that diabetic patients monitor their blood glucose levels every day and maintain stable blood glucose levels by taking hypoglycemic drugs or injecting insulin. Blood glucose is an important indicator for detecting diabetes and monitoring the condition changes of diabetic patients. Clinically, it mostly relies on blood sampling. However, the traditional fingertip blood glucose monitoring method still has some problems. First of all, when collecting blood, it is necessary to pierce the fingertip skin, which will not only damage the sensitive fingertip nerve endings, bring great pain to the patient, but also easily lead to wound infection. Secondly, the patient needs to collect blood multiple times a day, and repeated punctures will aggravate the pain and reduce the patient's compliance. Therefore, it is very important to develop a painless blood glucose monitoring method.

[0003] Scientists have found through research that human sweat provides valuable information about health status, mainly depending on the concentration levels of analytes such as lactate, ammonium, and glucose. Further research on the correlation between blood glucose concentration and sweat glucose concentration in diabetic patients found that the concentration range is mostly between 10 μM and 0.7 mM, determined the common sweat glucose concentration intervals for hypoglycemic and hyperglycemic patients as well as healthy people, and then determined the connection between glucose in sweat and blood glucose content. Based on this, a series of glucose biosensors for obtaining human blood glucose concentration by analyzing the glucose concentration in sweat have been developed.

[0004] The existing patent discloses a flexible glucose sensor and a preparation method thereof. This method prepares a multi-level glucose biosensor by in-situ growth of nanocomposites. However, most of the existing methods use physical methods for hierarchical stacking preparation. Therefore, the obtained biosensor has an unstable structure, and there are problems with unstable performance of each functional layer. Not only is its electrochemical performance poor, but it is also possible for each functional layer to fall off, seriously affecting the service life of the glucose biosensor. Therefore, there is an urgent need in the art for a glucose biosensor with stable electrochemical performance. Summary of the Invention

[0005] In view of the defects in the prior art, the present invention provides a glucose biosensor, a preparation method thereof and an application thereof. As an important category of wearable electronic products, flexible electrochemical sensors have attracted wide attention due to their great application potential in medical treatment, diagnosis and monitoring. Carbon-based nanomaterials have excellent physical properties and good biocompatibility, and are considered ideal materials for manufacturing wearable sensors. However, existing wearable flexible glucose biosensors often have problems such as poor electrochemical performance or short service life.

[0006] Based on this, the present invention provides a preparation method of a glucose biosensor, comprising the following steps:

[0007] S1: Coating electrode paste on the surface of a flexible substrate, and subjecting the flexible substrate to CO2 laser engraving to obtain a three-dimensional graphene electrode body with a three-electrode structure;

[0008] S2: Coating the surface of electrode 1 in the three-dimensional graphene electrode body with conductive paste to obtain a modified reference electrode, immersing the surface of electrode 2 in the three-dimensional graphene electrode body in a sulfuric acid solution of pyrrole and performing cyclic voltammetry to obtain a modified working electrode, and the unmodified electrode 3 is the counter electrode, thereby preparing a graphene-polypyrrole electrode;

[0009] S3: Electrochemically depositing and modifying the graphene-polypyrrole electrode obtained in S3 with a sulfuric acid-acidified chloroauric acid solution to obtain a graphene-polypyrrole-gold nanoparticle electrode;

[0010] S4: Loading glucose oxidase on the surface of the working electrode of the graphene-polypyrrole-gold nanoparticle electrode to obtain the glucose biosensor;

[0011] The electrode paste described in step S1 is a combination of carboxymethyl chitosan paste and nanocellulose paste;

[0012] The fibril diameter of the nanocellulose is 100-130 nm; through a large number of experiments, the present invention finds that the fibril diameter of the nanocellulose is crucial for the conductivity, mechanical properties and stability of the glucose biosensor. When the fibril diameter is small, it is not conducive to the overall structural stability of the electrode material during coating on the flexible substrate, the anti-deformation and anti-fracture abilities of the electrode are weakened, and the current response degree is small; when the fibril diameter is too large, it will change the molecular environment on the electrode surface, resulting in a decrease in recognition sensitivity, and the current response degree is also small. By selecting nanocellulose with a fibril diameter of 100-120 nm, the present invention ensures that the electrode can improve the current response degree while ensuring the overall structural stability of the electrode material, and maximally improves the sensitivity of the biosensor to glucose recognition.

[0013] The nanocellulose described in the present invention consists of 30 to 40 cellulose molecules to form a bundle-like extended chain structure. Its chemical composition is β-glycan condensed glucose units, and its macroscopic state is a transparent gel with pseudoplasticity.

[0014] In the present invention, a three-dimensional graphene layer is formed on the surface of the flexible substrate. Compared with conventional screen-printed electrodes, the use of a three-dimensional structure can significantly increase the specific surface area of the electrode and improve the electrocatalytic performance. The finally formed three-dimensional porous graphene structure has excellent hydrophilicity and N / O co-doped chemical properties, which can significantly promote the penetration and transport of solution ions.

[0015] In the present invention, in step S3, by modifying the graphene-polypyrrole electrode, gold nanoparticles are in-situ grown in the three-dimensional graphene-polypyrrole layer in the graphene-polypyrrole electrode. Therefore, the binding density is high, and the finally prepared glucose biosensor is not likely to have the problem of functional layer shedding. Moreover, when the in-situ grown gold nanoparticles are combined with polypyrrole with a high specific surface area, the synergistic enhancement effect of the two can further improve the electrocatalytic performance of the electrode.

[0016] In some embodiments, the flexible substrate in step S1 is any one of Nomex paper, polyester, polydimethylsiloxane, and silk protein; preferably, the flexible substrate is Nomex paper. The surface of the Nomex paper can be transformed into a carbon-based material suitable for the glucose biosensor by CO2 laser engraving. Its excellent mechanical strength and appropriate roughness can further improve the wearability of the glucose biosensor.

[0017] In some embodiments, the electrode paste in step S1 is a combination of carboxymethyl chitosan paste and nanocellulose paste. Specifically, carboxymethyl chitosan and nanofibers are respectively mixed with water to obtain carboxymethyl chitosan paste and nanocellulose paste, and the prepared carboxymethyl chitosan paste and nanocellulose paste are then mixed to obtain a mixed paste.

[0018] In some embodiments, the mass ratio of the carboxymethyl chitosan paste to the nanocellulose paste is 1:(0.5 to 1.5). As examples, the mass ratio of the carboxymethyl chitosan paste to the nanocellulose paste can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, as long as the mass ratio of the carboxymethyl chitosan paste to the nanocellulose paste is within this range.

[0019] The carboxymethyl chitosan syrup is obtained by mixing carboxymethyl chitosan (CMC) with water; wherein, the mass-volume ratio of carboxymethyl chitosan to water is (1.5 - 2.0) g : 40 mL. As an example, the mass-volume ratio of carboxymethyl chitosan to water can be 1.5 g : 40 mL, 1.6 g : 40 mL, 1.7 g : 40 mL, 1.8 g : 40 mL, 1.9 g : 40 mL, 2.0 g : 40 mL, as long as the mass-volume ratio of carboxymethyl chitosan to water is within this range.

[0020] The nanofibrillated cellulose slurry is obtained by mixing nanofibrillated cellulose with water; wherein, the mass-volume ratio of nanofibrillated cellulose to water is (15 - 20) g : 8 mL. As an example, the mass-volume ratio of nanofibrillated cellulose to water can be 15 g : 8 mL, 16 g : 8 mL, 17 g : 8 mL, 18 g : 8 mL, 19 g : 8 mL, 20 g : 8 mL, as long as the mass-volume ratio of nanofibrillated cellulose to water is within this range.

[0021] In some embodiments, the power of the CO2 laser engraving in step S1 is 10 - 15% of the full power; in some specific embodiments, the power of the CO2 laser engraving in step S10 is 10 - 15% of the full power. As an example, the power of the CO2 laser engraving can be 10%, 11%, 12%, 12.5%, 13%, 14%, 15%, as long as the power of the CO2 laser engraving is within this range.

[0022] In some embodiments, the scanning rate of the CO2 laser engraving in step S1 is 60 - 100 mm / s; as an example, the scanning rate of the CO2 laser engraving can be 60 mm / s, 65 mm / s, 70 mm / s, 75 mm / s, 80 mm / s, 85 mm / s, 90 mm / s, 95 mm / s, 100 mm / s, as long as the scanning rate of the CO2 laser engraving is within this range.

[0023] In some embodiments, the line spacing of the CO2 laser engraving in step S1 is 0.05 - 0.15 mm. As an example, the line spacing of the CO2 laser engraving can be 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.10 mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, 0.15 mm, as long as the line spacing of the CO2 laser engraving is within this range.

[0024] The present invention controls the depth and interval of laser engraving to obtain a three-dimensional structure with a micron scale, form an electrode with a high specific surface area, and enhance the conductivity of the electrode.

[0025] In some embodiments, the conductive paste described in step S2 is a mixed paste of silver and silver chloride. The silver-silver chloride mixed paste is coated on the surface of one of the electrodes in the three-dimensional graphene electrode body, and a reference electrode is obtained after the paste is dried.

[0026] In some embodiments, the concentration of sulfuric acid in the sulfuric acid solution of pyrrole in step S2 is 0.05 - 0.15 M; for example, the concentration of sulfuric acid in the sulfuric acid solution of pyrrole can be 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.10 M, 0.11 M, 0.12 M, 0.13 M, 0.14 M, 0.15 M, as long as the concentration of sulfuric acid in the sulfuric acid solution of pyrrole is within this range.

[0027] In some specific embodiments, the volume of sulfuric acid in the sulfuric acid solution of pyrrole in step S30 is 2.5 mL - 7.5 mL; for example, the volume of sulfuric acid in the sulfuric acid solution of pyrrole can be 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, 5.0 mL, 5.5 mL, 6.0 mL, 6.5 mL, 7.0 mL, 7.5 mL, as long as the volume of sulfuric acid in the sulfuric acid solution of pyrrole is within this range.

[0028] In some specific embodiments, the volume of pyrrole in the sulfuric acid solution of pyrrole in step S30 is 25 - 75 μL, for example, the volume of pyrrole can be 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, as long as the volume of pyrrole in the sulfuric acid solution of pyrrole is within this range.

[0029] In some embodiments, the scanning rate of the voltammetric cycle in step S2 is 75 - 125 mV / s; for example, the scanning rate of the voltammetric cycle is 75 mV / s, 80 mV / s, 85 mV / s, 90 mV / s, 95 mV / s, 100 mV / s, 105 mV / s, 110 mV / s, 115 mV / s, 120 mV / s, 125 mV / s, as long as the scanning rate of the voltammetric cycle is within this range.

[0030] The potential of cyclic voltammetry is +0.4 to +1.1 V; the number of cycles is 10 to 14 times. For example, the number of voltammetric cycles can be 10 times, 11 times, 12 times, 13 times, or 14 times, as long as the number of voltammetric cycles is within this range. As the voltammetric cycle progresses, pyrrole monomers are continuously oxidized to form radical cations or positive ions, and these intermediates then combine with counter anions and further polymerize to form polypyrrole. The sulfuric acid solution of polypyrrole is also an acidic environment, which is more conducive to the formation of polypyrrole, can increase the specific surface area of the graphene layer, and ultimately improve the electrochemical performance of the glucose biosensor.

[0031] In some embodiments, the potential of the electrochemical modification in step S3 is -0.8 to -1.0 V, a constant potential; the time of electrochemical modification is 180 to 300 s. As an example, the time of electrochemical modification can be 180 s, 190 s, 200 s, 210 s, 220 s, 230 s, 240 s, 250 s, 260 s, 270 s, 280 s, 290 s, or 300 s, as long as the time of electrochemical modification in step S40 is within this range.

[0032] In some embodiments, the concentration of sulfuric acid in the chloroauric acid solution acidified with sulfuric acid in step S3 is 0.25 to 0.75 M. As an example, the concentration of sulfuric acid in the chloroauric acid solution acidified with sulfuric acid is 0.25 M, 0.30 M, 0.35 M, 0.40 M, 0.45 M, 0.50 M, 0.55 M, 0.60 M, 0.65 M, 0.70 M, or 0.75 M, as long as the concentration of sulfuric acid in the chloroauric acid solution acidified with sulfuric acid is within this range.

[0033] In some embodiments, the concentration of chloroauric acid in the chloroauric acid solution acidified with sulfuric acid in step S3 is 25 mM to 75 mM. As an example, the concentration of chloroauric acid in the chloroauric acid solution acidified with sulfuric acid is 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, or 75 mM, as long as the concentration of chloroauric acid in the chloroauric acid solution acidified with sulfuric acid is within this range.

[0034] In some embodiments, the mass-volume ratio of glucose oxidase to chitosan solution in the glucose oxidase-chitosan solution used in step S4 is (8~12) mg: 1 mL. As an example, the mass-volume ratio of glucose oxidase to chitosan solution in the glucose oxidase-chitosan solution can be 8 mg: 1 mL, 9 mg: 1 mL, 10 mg: 1 mL, 11 mg: 1 mL, 12 mg: 1 mL, as long as the mass-volume ratio of glucose oxidase to chitosan solution is within this range.

[0035] In some embodiments, the volume ratio of Nafion to ethanol in the Nafion-ethanol solution used in step S4 is 1: (300~500). As an example, the volume ratio of Nafion to ethanol can be 1:300, 1:325, 1:350, 1:375, 1:400, 1:425, 1:450, 1:475, 1:500, as long as the volume ratio of Nafion to ethanol in the Nafion-ethanol solution is within this range.

[0036] In some embodiments, the specific steps of loading glucose oxidase include:

[0037] (1) Modify the surface of the working electrode using a crosslinking agent;

[0038] (2) Prepare a glucose oxidase-chitosan solution and coat it on the surface of the working electrode in (1) to obtain a glucose biosensor;

[0039] (3) Coat a Nafion-ethanol solution on the surface of the quasi-glucose biosensor in (2) to form a selective permeable membrane and then dry it to complete the loading of glucose oxidase.

[0040] The present invention provides a glucose biosensor obtained by the above preparation method.

[0041] The present invention also provides the application of the glucose biosensor in the preparation of products for detecting human blood glucose.

[0042] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0043] 1. The glucose biosensor prepared by the present invention can significantly improve the electrochemical performance of the glucose biosensor through the synergistic enhancement effect of polypyrrole and gold nanoparticles, and is obtained by a non-physical binding method, having excellent structural stability and electrocatalytic performance.

[0044] 2. The glucose biosensor prepared by the present invention can reflect the blood glucose concentration in the human body by analyzing the glucose concentration in human skin sweat, thereby obtaining the trend of blood glucose changes, and can realize the real-time monitoring of the blood glucose concentration in the human body.

[0045] 3. The glucose biosensor prepared by the present invention has a good linear range and a low detection limit for glucose, high sensitivity, excellent selectivity, and strong practicability.

[0046] 4. The substrate and carbon-based material used in the present invention not only solve the problems of green and environmental protection, but also effectively reduce the manufacturing cost of the substrate. Most importantly, its low-cost and rapid preparation strategy has broad potential in large-scale production and commercial development. Brief Description of the Drawings

[0047] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0048] Figure 1 SEM image of the flexible substrate Nomex paper used in Example 1 of the present invention.

[0049] Figure 2 SEM image of the three-dimensional graphene electrode body with a graphene three-electrode structure embedded on the surface of the flexible substrate obtained after drying the CMC-NFC slurry and then laser engraving with CO2 in Example 1 of the present invention, denoted as CMC-NFC / Nomex.

[0050] Figure 3 SEM image of the graphene-polypyrrole electrode obtained by modifying the three-dimensional graphene electrode body in Example 1 of the present invention with polypyrrole, denoted as PPy / CMC-NFC / Nomex.

[0051] Figure 4 SEM image of the graphene-polypyrrole-gold nanoparticle electrode obtained by electrochemically depositing and modifying the graphene-polypyrrole electrode in Example 1 of the present invention, denoted as Au / PPy / CMC-NFC / Nomex.

[0052] Figure 5 Current responses of Examples 1 to 3 of the present invention in three different substrates of Nomex paper, silk fibroin, and polydimethylsiloxane in a potassium ferricyanide redox probe.

[0053] Figure 6Current responses of nanocelluloses with diameters of 80 nm, 120 nm, and 160 nm in potassium ferricyanide redox probe for Example 1, Comparative Examples 1-2 of the present invention on flexible Nomex substrate paper.

[0054] Figure 7 Cyclic voltammetry performance characterization diagrams of four electrodes of CMC-NFC / Nomex, PPy / CMC-NFC / Nomex, Au / PPy / CMC-NFC / Nomex of the present invention, and Au / CMC-NFC / Nomex obtained in Test Example 1.

[0055] Figure 8 EIS spectra diagrams of four electrodes of CMC-NFC / Nomex, PPy / CMC-NFC / Nomex, Au / PPy / CMC-NFC / Nomex obtained in Example 1, and Au / CMC-NFC / Nomex obtained in Test Example 1.

[0056] Figure 9 Circuit diagrams of Randle equivalent circuit models of four electrodes of CMC-NFC / Nomex, PPy / CMC-NFC / Nomex, Au / PPy / CMC-NFC / Nomex obtained in Example 1, and Au / CMC-NFC / Nomex obtained in Test Example 1.

[0057] Figure 10 Test diagrams of current-time (i-t) curves corresponding to glucose sensors obtained in Example 1 at glucose concentrations of 5 μM to 1000 μM at room temperature.

[0058] Figure 11 Linear fitting curve diagrams of glucose sensors obtained in Example 1 in a current stable state.

[0059] Figure 12 Selectivity test diagrams of glucose sensors obtained in Example 1.

[0060] Figure 13 Test wear diagrams of the skin-adhesiveness of the electrode part of the glucose biosensor obtained in Example 1. Detailed implementation manners

[0061] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0063] A CO2 laser cutting system (Cixi Jinyue Laser Technology Co., Ltd., E5030) was used to prepare the sensing electrode. All electrochemical experiments (Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS), and Chronoamperometry (i-t)) were carried out at room temperature on a Shanghai Chenhua (CHI 660E) workstation. The workstation adopted a three-electrode system, with Au / PPy / CMC-NFC / Nomex as the working electrode (WE), CMC-NFC / Nomex and Ag / AgCl as the counter electrode (CE) and reference electrode (RE) respectively, which were used for the fabrication of all sensors.

[0064] The flexible electrode was characterized by environmental scanning electron microscopy (ESEM, ThermoFisher Quattro).

[0065] Example 1

[0066] (1) 1.6 g of carboxymethyl chitosan (abbreviated as CMC) was mixed with 40 mL of deionized water and stirred at 50 °C for 2 h to obtain a uniform CMC slurry. Then, 16 g of nanocellulose (NFC, fibril diameter of 120 nm) was added to 8 mL of deionized water and stirred at 55 °C for 1 h to obtain a uniform NFC slurry. Then, the CMC slurry and the NFC slurry were mixed in a mass ratio of 1:1 to obtain an electrode slurry. 1.5 g of the electrode slurry was coated on Nomex paper (coating area: 6×2.5 cm 2 ) and dried overnight at room temperature. Then, under the conditions of a fixed laser power of 12.5% of the full power, a scanning rate of 80 mm / s, and a line spacing of 0.1 mm, the Nomex paper coated with the electrode slurry was directly engraved with a CO2 laser device to obtain O / N co-doped laser-engraved graphene, denoted as CMC-NFC / Nomex. At this time, a three-electrode-structured three-dimensional graphene electrode body was formed on the surface of the Nomex paper after laser engraving.

[0067] In this example, Figure 1 Figure 19 is the SEM image of the unmodified Nomex paper in Example 1. Figure 2SEM image of the three-dimensional graphene electrode body with a graphene three-electrode structure embedded on the surface of the flexible substrate obtained after CO2 laser engraving in Example 1. After comparison, it can be found that after coating the electrode paste on the Nomex paper and then laser engraving, a graphene layer with a more complex and three-dimensional structure is formed.

[0068] (2)Drop approximately 20 μL of Ag / AgCl paste on the surface of any one of the three bare electrodes in the three-dimensional graphene electrode body, and dry it at 70 °C for 30 min to complete the preparation of the reference electrode in the three-dimensional graphene electrode body. In order to connect the prepared electrode to the electrochemical analyzer, the bottom end of each electrode is connected to a copper wire with silver paste.

[0069] (3)Coat a mixed solution of pyrrole and sulfuric acid on the surface of any one of the electrodes in the three-electrode structure of the three-dimensional graphene electrode body except the reference electrode and perform cyclic voltammetry. The concentration of sulfuric acid is 0.01 M, the volume is 5 mL, the volume of pyrrole is 50 μL, the scanning rate of cyclic voltammetry is 100 mV / s; the potential is from +0.4 V to +1.1 V; the electrode for cycling is the reference electrode in the three-dimensional graphene electrode body, and the number of cycles is 12 times to complete the preparation of the working electrode. The remaining unmodified electrode is the counter electrode. Thus, the preparation of the graphene-polypyrrole electrode (abbreviated as PPy / CMC-NFC / Nomex) is completed.

[0070] Figure 3 SEM picture of the PPy / CMC-NFC / Nomex obtained in Example 1. From Figure 3 It can be found that compared with the graphene electrode without polypyrrole modification, the PPy / CMC-NFC / Nomex obtained after cyclic voltammetry modification with polypyrrole has a more three-dimensional and rough internal structure, and the specific surface area is further improved.

[0071] (4)First, carefully wash the prepared PPy / CMC-NFC / Nomex with distilled water and keep it dry at room temperature for 10 min, then immerse it in a sulfuric acid-acidified chloroauric acid solution for electrodeposition. The concentration of sulfuric acid in the sulfuric acid-acidified chloroauric acid solution is 0.5 M, the concentration of chloroauric acid is 50 mM, set the potential to a constant potential of -0.9 V, and the deposition time is 240 s to complete the preparation of the graphene-polypyrrole-gold nanoparticle electrode (abbreviated as Au / PPy / CMC-NFC / Nomex).

[0072] Figure 4 SEM image of the Au / PPy / CMC-NFC / Nomex obtained in Example 1. Through Figure 4It can be seen that many gold nanoparticles have grown in-situ in the graphene-polypyrrole framework of the modified graphene-polypyrrole electrode. The complex three-dimensional structure of the graphene-polypyrrole electrode provides more in-situ growth sites for the gold nanoparticles, which can further improve the electrochemical performance of the electrode. At the same time, since the gold nanoparticles in the graphene-polypyrrole-gold nanoparticle electrode are prepared by the in-situ growth method of electrodeposition, their binding with the overall electrode is stronger and the structure is also very stable, and it is very difficult for the electrode functional layer to fall off.

[0073] (5)Coat 10 μL of cross-linking agent on the working electrode surface of the graphene-polypyrrole-gold nanoparticle electrode. The cross-linking agent is an aqueous solution with a volume ratio of EDC to NHS of 1:1, and let it stand overnight at 4 °C. Then coat 10 μL of glucose oxidase-chitosan solution (10 mg of glucose oxidase is contained in 1 mL of chitosan solution) on the working electrode surface modified by the cross-linking agent and let it stand overnight in an incubator at 4 °C. Finally, coat 5 μL of ethanol solution of Nafion (the volume ratio of Nafion to ethanol is 1:400) on the working electrode surface loaded with glucose oxidase to form a selective permeable membrane. After the Nafion-ethanol solution dries, the loading of glucose oxidase is completed. After the loading is completed, connect the analytical instrument to obtain a glucose biosensor.

[0074] Example 2

[0075] (1)Mix 1.5 g of CMC with 40 mL of deionized water and stir at 50 °C for 2 h to obtain a uniform CMC slurry. Then, add 15 g of NFC (fibril diameter of 120 nm) to 8 mL of deionized water and stir at 55 °C for 1 h to obtain a uniform NFC slurry. Then, mix the CMC slurry and the NFC slurry according to a mass ratio of 1:0.5 to obtain an electrode slurry. Take 1.5 g of the electrode slurry and coat it on polydimethylsiloxane (coating area is 6×2.5 cm 2 ), and dry it overnight at room temperature. Then, use a CO2 laser device to directly engrave the polydimethylsiloxane coated with the electrode slurry under the conditions of a fixed laser power of 10% of the full power, a scanning rate of 60 mm / s, and a line spacing of 0.05 mm to obtain O / N co-doped laser-engraved graphene.

[0076] (2)Drop about 20 μL of Ag / AgCl slurry on the surface of any one of the three bare electrodes in the three-dimensional graphene electrode body, and dry it at 70 °C for 30 min to complete the preparation of the reference electrode in the three-dimensional graphene electrode body.

[0077] (3) Coat a mixed solution of pyrrole and sulfuric acid on the surface of any one of the electrodes other than the reference electrode in the three-electrode structure of the three-dimensional graphene electrode body and perform voltammetric cycling, where the concentration of sulfuric acid is 0.05 M, the volume is 2.5 mL, the volume of pyrrole is 25 μL, and the scanning rate of voltammetric cycling is 75 mV / s; the potential is from +0.4 V to +1.1 V; the electrode for cycling is the reference electrode in the three-dimensional graphene electrode body, and the number of cycles is 10 times to complete the preparation of the working electrode. The remaining unmodified electrode is the counter electrode, and thus the preparation of the graphene-polypyrrole electrode is completed.

[0078] (4) First, carefully wash the prepared graphene-polypyrrole electrode with distilled water and keep it dry at room temperature for 10 min, and then immerse it in a sulfuric acid-acidified chloroauric acid solution for electrodeposition modification, where the concentration of sulfuric acid in the sulfuric acid-acidified chloroauric acid solution is 0.25 M, the concentration of chloroauric acid is 25 mM, the modification potential is a constant potential of -0.9 V, and the modification time is 180 s to complete the preparation of the graphene-polypyrrole-gold nanoparticle electrode.

[0079] (5) Coat 10 μL of a crosslinking agent on the working electrode surface of the graphene-polypyrrole-gold nanoparticle electrode. The crosslinking agent is an aqueous solution with a volume ratio of EDC to NHS of 1:1, and let it stand overnight at 4 °C. Then, coat 10 μL of a glucose oxidase-chitosan solution (8 mg of glucose oxidase is contained in 1 mL of chitosan solution) on the working electrode surface modified by the crosslinking agent and let it stand overnight in an incubator at 4 °C. Finally, coat 5 μL of an ethanol solution of Nafion (the volume ratio of Nafion to ethanol is 1:300) on the working electrode surface loaded with glucose oxidase to form a selective permeable membrane. After the Nafion-ethanol solution dries, the loading of glucose oxidase is completed. After the loading is completed, connect the analytical instrument to obtain a glucose biosensor.

[0080] Example 3

[0081] (1) Mix 2.0 g of CMC with 40 mL of deionized water and stir at 50 °C for 2 h to obtain a uniform CMC slurry. Then, add 20 g of NFC (fibril diameter of 120 nm) to 8 mL of deionized water and stir at 55 °C for 1 h to obtain a uniform NFC slurry. Then, mix the CMC slurry and the NFC slurry according to a mass ratio of 1:1.5 to obtain an electrode slurry. Take 1.5 g of the electrode slurry and coat it on silk fibroin (the coating area is 6×2.5 cm 2 ), and dry it overnight at room temperature. Then, directly engrave the silk fibroin coated with the electrode slurry with a CO2 laser device under the conditions of a fixed laser power of 15% of the full power, a scanning rate of 100 mm / s, and a line spacing of 0.15 mm to obtain an O / N co-doped laser-engraved graphene electrode.

[0082] (2) Drop approximately 20 μL of Ag / AgCl paste on the surface of any one of the three bare electrodes in the three-dimensional graphene electrode body, and dry it at 70 °C for 30 minutes to complete the preparation of the reference electrode in the three-dimensional graphene electrode body.

[0083] (3) Coat a mixed solution of pyrrole and sulfuric acid on the surface of any electrode other than the reference electrode in the three-electrode structure three-dimensional graphene electrode body and perform voltammetric cycling, where the concentration of sulfuric acid is 0.15 M, the volume is 7.5 mL, the volume of pyrrole is 75 μL, the scanning rate of voltammetric cycling is 125 mV / s; the potential is from +0.4 V to +1.1 V; the cycling electrode is the reference electrode in the three-dimensional graphene electrode body, and the number of cycles is 14 times to complete the preparation of the working electrode. The remaining unmodified electrode is the counter electrode. Thus, the preparation of the graphene-polypyrrole electrode is completed.

[0084] (4) First, carefully wash the prepared graphene-polypyrrole electrode with distilled water and keep it dry at room temperature for 10 min, then soak it in a sulfuric acid-acidified chloroauric acid solution for electrochemical modification, where the concentration of sulfuric acid in the sulfuric acid-acidified chloroauric acid solution is 0.75 M, the concentration of chloroauric acid is 75 mM, the modification potential is a constant potential of -0.9 V, and the modification time is 300 s to complete the preparation of the graphene-polypyrrole-gold nanoparticle electrode.

[0085] (5) Coat 10 μL of a cross-linking agent on the working electrode surface of the graphene-polypyrrole-gold nanoparticle electrode. The cross-linking agent is an aqueous solution with a volume ratio of EDC to NHS of 1:1, and let it stand overnight at 4 °C. Then coat 10 μL of a glucose oxidase-chitosan solution (12 mg of glucose oxidase is contained in 1 mL of chitosan solution) on the working electrode surface modified by the cross-linking agent and let it stand overnight in an incubator at 4 °C. Finally, coat 5 μL of an ethanol solution of Nafion (the volume ratio of Nafion to ethanol is 1:500) on the working electrode surface loaded with glucose oxidase to form a selective permeable membrane. After the Nafion-ethanol solution dries, the loading of glucose oxidase is completed. After the loading is completed, connect the analytical instrument to obtain a glucose biosensor.

[0086] Comparative Example 1

[0087] Compared with the preparation method of Example 1, the difference is that the diameter of the nanocellulose used is 80 nm.

[0088] Comparative Example 2

[0089] Compared with the preparation method of Example 1, the difference is that the diameter of the nanocellulose used is 160 nm.

[0090] To further prove the reliability of the glucose biosensor provided by the present invention, a comparative test is now carried out.

[0091] Test Example 1 Current Response Test of Different Substrates and Different Fiber Diameters

[0092] This test example is a CV test. The electrodes obtained in Examples 1 to 3 are subjected to CV test in 0.1 M KCl containing 5 mM [Fe(CN)6] 3- / 4- redox probe.

[0093] The test results are as Figure 5 shown. Among the three substrates, the Nomex substrate has the largest response current.

[0094] Subsequently, the electrodes obtained in Example 1 and Comparative Examples 1 to 2 are subjected to CV test in 0.1 M KCl containing 5 mM [Fe(CN)6] 3- / 4- redox probe.

[0095] The test results are as Figure 6 shown, which is a test comparison chart of different diameters of nanocellulose. The current magnitude in the figure shows that the response currents of the electrodes with 80 nm and 160 nm fiber diameters are less than that of 120 nm.

[0096] Test Example 2 Current Response Test of Different Electrodes

[0097] Now an additional electrode is provided. The difference between this electrode and the electrode obtained in Example 1 is that it directly undergoes electrochemical modification without using polypyrrole to modify O / N co-doped laser-engraved graphene, resulting in a graphene-gold nanoparticle electrode (abbreviated as Au / CMC-NFC / Nomex).

[0098] Now, the laser-engraved graphene obtained in Example 1 (abbreviated as Nomex), the O / N co-doped laser-engraved graphene electrode obtained in the comparative example (abbreviated as CMC-NFC / Nomex), the graphene-polypyrrole electrode obtained in Example 1 (abbreviated as PPy / CMC-NFC / Nomex), and the gold nanoparticle-graphene-polypyrrole electrode finally obtained in Example 1 (abbreviated as Au-PPy / CMC-NFC / Nomex) are respectively subjected to CV test in 0.1 M KCl containing 5 mM [Fe(CN)6] 3- / 4- redox probe, and the test results are as Figure 7 shown.

[0099] Figure 7 shows the CV curves of the bare electrode CMC-NFC / Nomex, Au / CMC-NFC / Nomex, PPy / CMC-NFC / Nomex, and Au / PPy / CMC-NFC / Nomex. ThroughFigure 7 It can be seen that in the CV curve of the graphene electrode (CMC-NFC / Nomex) without further modification, a pair of redox peaks is observed. The values of the redox current are not ideal, only showing a slow electron transfer rate at the interface, and its electrochemical performance is the worst. The electrochemical performance of the graphene electrode modified only with gold nanoparticles (Au / CMC-NFC / Nomex) is only slightly improved compared with the unmodified graphene electrode, with little change. The electrochemical performance of the graphene electrode modified with polypyrrole (PPy / CMC-NFC / Nomex) has been improved to a certain extent. The redox current of the gold nanoparticle-polypyrrole-graphene electrode (Au-PPy / CMC-NFC / Nomex) obtained after loading gold nanoparticles on the graphene electrode modified with polypyrrole increases significantly, indicating that the electron transfer rate at the electrode / electrolyte interface is significantly improved. The electrochemical performance has been significantly improved compared with the graphene electrode modified with polypyrrole or gold nanoparticles alone, and this improvement exceeds the linear expectation. It can be seen that the modification of polypyrrole and the modification of gold nanoparticles play a synergistic enhancement effect on the electrochemical performance of the graphene electrode.

[0100] Test Example 3 EIS Test of Four Electrodes

[0101] In this test example, the four electrodes used in Test Example 1 were subjected to EIS testing, and the interfacial performance differences of these electrodes were compared. Figure 8 are the EIS spectra of the bare electrodes CMC-NFC / Nomex, Au / CMC-NFC / Nomex, PPy / CMC-NFC / Nomex, and Au / PPy / CMC-NFC / Nomex. Figure 9 is the Randle equivalent circuit model for fitting the experimental results, where Cdl, Zw, Rct, and Rs represent the double-layer capacitance, Warburg element impedance, electron transfer resistance, and solution resistance, respectively. Figure 9 The semicircle diameter in corresponds to Rct.

[0102] Through Figure 8 and Figure 9 It can be known that the significant reduction of Rct is mainly attributed to the synergistic effect of polypyrrole and gold nanoparticles, which promotes the transfer of electrons from the electroactive marker to the electrode. These EIS results are consistent with the results of the CV test, indicating that the synergistic effect of polypyrrole and gold nanoparticles improves the electrochemical performance of CMC-NFC / Nomex.

[0103] Next, various performance tests were carried out on the glucose biosensor finally obtained in Example 1, as follows:

[0104] Test Example 4 Response Test of Glucose Biosensor to Glucose

[0105] This test example examines the response of the glucose biosensor obtained in Example 1 to glucose, as follows:

[0106] At room temperature, the glucose biosensor obtained in Example 1 was used to test glucose solutions with concentrations ranging from 5 μM to 1000 μM, and the response current curves of glucose at different concentrations at room temperature were obtained.

[0107] The results are shown in Figure 10 and Figure 11 respectively, where Figure 10 is the chronoamperometric response curve of the sensor in PBS (0.1 M, pH = 7.0) containing glucose concentrations in the range of 0 - 1000 μM. Figure 11 is the linear calibration curve obtained from Figure 10 the test data: y = 0.1093x + 14.90113 (n = 3), and the correlation coefficient (R 2 ) of the anodic current response is 0.997. From Figure 10 and Figure 11 , it can be seen that the glucose biosensor obtained in Example 1 has a good linear range of 0 - 1000 μM and a low detection limit of 0.041 μM, indicating its broad prospects in clinical applications.

[0108] Test Example 5 Selectivity Test of Glucose Biosensor

[0109] This test example tests the selectivity of the glucose biosensor obtained in Example 1. By adding interfering substances, the selectivity of the glucose biosensor is determined. The interfering substances are 10 mM lactic acid, 10 mM urea, 20 mM lysine, 20 mM valine, 20 mM serine, 10 mM uric acid, 10 mM paracetamol, 40 mM sodium chloride, and 40 mM potassium chloride. All interfering solutions are prepared with PBS.

[0110] The results are shown in Figure 12 . The glucose sensor prepared in this application shows no obvious current change for any interfering substance and does not respond to any interfering compound, indicating its high selectivity for glucose.

[0111] Test Example 6 Wearability Test

[0112] Figure 13 is the wearable graph for testing the skin adhesion of the graphene - polypyrrole - gold nanoparticle electrode obtained in Example 1. From Figure 13 , it can be seen that the polypyrrole - gold nanoparticle electrode obtained in this example has good skin adhesion, and subsequent connection to an electrochemical analysis device results in a relatively comfortable wearing experience for the glucose biosensor.

[0113] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a glucose biosensor, characterized in that, It includes the following steps: S1: Coat the electrode paste on the surface of the flexible substrate, and engrave the flexible substrate by CO2 laser to obtain a three-dimensional graphene electrode body with a three-electrode structure; S2: Coat the surface of electrode 1 in the three-dimensional graphene electrode body with a conductive paste to obtain a modified reference electrode. Immerse the surface of electrode 2 in the three-dimensional graphene electrode body in a sulfuric acid solution of pyrrole and perform cyclic voltammetry to obtain a modified working electrode. The unmodified electrode 3 is the counter electrode, and a graphene-polypyrrole electrode is prepared; S3: Electrochemically modify the graphene-polypyrrole electrode described in S2 with a sulfuric acid-acidified chloroauric acid solution to obtain a graphene-polypyrrole-gold nanoparticle electrode; S4: Load glucose oxidase on the surface of the working electrode of the graphene-polypyrrole-gold nanoparticle electrode to obtain the glucose biosensor; The electrode paste described in step S1 is a combination of carboxymethyl chitosan paste and nanocellulose paste; The fibril diameter of the nanocellulose is 100-130 nm; The flexible substrate described in step S1 is Nomex paper.

2. The preparation method according to claim 1, characterized in that, In the electrode paste described in step S1, the mass ratio of carboxymethyl chitosan paste to nanocellulose paste is 1:(0.5-1.5).

3. The preparation method according to claim 1, characterized in that, The power of the CO2 laser engraving described in step S1 is 10-15% of the full power; the scanning rate is 60-100 mm / s; the line spacing is 0.05-0.15 mm.

4. The preparation method according to claim 1, characterized in that, The conductive paste described in step S2 is a mixed paste of silver and silver chloride.

5. The preparation method according to claim 1, characterized in that, In step S2, the scanning rate of the voltammetric cycle is 75-125 mV / s; the potential is +0.4 to +1.1 V; the electrode for cycling is the reference electrode, and the number of cycles is 10-14 times.

6. The preparation method according to claim 1, characterized in that, In the sulfuric acid-acidified chloroauric acid solution in step S3, the concentration of sulfuric acid is 0.25-0.75 M; the concentration of the chloroauric acid solution is 25 mM-75 mM.

7. The preparation method according to claim 1, wherein The specific steps of loading glucose oxidase in step S4 include: (1) Modify the surface of the working electrode with a crosslinking agent; (2) Prepare a glucose oxidase-chitosan solution and coat it on the working electrode described in (1) to obtain a quasi-glucose biosensor; (3) Coat a Nafion-ethanol solution on the surface of the quasi-glucose biosensor described in (2) to form a selective permeable membrane and then dry it to complete the loading of glucose oxidase.

8. A glucose biosensor, characterized in that, Obtained by the preparation method according to any one of claims 1-7.

9. Application of the glucose biosensor according to claim 8 in the preparation of a product for detecting human blood glucose.

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