CN102507693A - Functional-material-based glucose biosensor and manufacturing method thereof - Google Patents

Abstract

Glucose biosensor based on functionalized materials, its working electrode is made of graphene nanomaterials, which is made by using the working electrode made of graphene nanomaterials for the assembly of glucose biosensors. It is a quantitative testing technology. It shows good linearity and resolution, and the detection sensitivity, detection range, and detection speed are improved; especially in the range of human blood glucose concentration, the response current amplitude can be increased by 50%, and the resolution is increased by more than two times. Graphene nano-materials form nano-functional membranes, and its immobilized enzymes can be analyzed continuously for more than 1,000 times. The cost of determination is low, and the analysis accuracy is higher than other methods. Greatly extended, it can accurately measure glucose concentration at regular intervals, high specificity, short-term, low-cost analysis, no special requirements for analyte substances, safe and simple operation, convenient for on-site determination, etc., and can be used for daily blood glucose measurement of diabetic patients.

Description

Glucose biosensor based on functionalized material and preparation method thereof

technical field

The invention relates to biotechnology analysis and detection, in particular to a biosensor, more specifically to a glucose biosensor based on a functional material and a preparation method thereof.

Background technique

A biosensor is an instrument that is sensitive to biological substances and converts its concentration into an electrical signal for detection. It is made of immobilized biosensitive materials as recognition elements (including enzymes, antibodies, antigens, microorganisms, cells, tissues, nucleic acids and other biologically active substances) and appropriate physical and chemical transducers (such as oxygen electrodes, photosensitive tubes, field effect tubes, Piezoelectric crystals, etc.) and an analysis tool or system composed of a signal amplification device. Biosensors have the functions of receptors and converters.

Since the advent of the enzyme electrode in the 1960s, biosensors have undergone tremendous development and have become an increasingly important component of enzymatic assays. The production of biosensors is the product of the cross-infiltration of biology, medicine, electrochemistry, thermal science, optics and electronic technology. It has the characteristics of high selectivity, fast analysis speed, simple operation and low price. It is widely used in industrial and agricultural production. , environmental protection, food industry, medical diagnosis and other fields have been widely used.

The advantages of biosensors are: low cost and cost, using immobilized enzymes as catalysts, which can be used repeatedly; good specificity, only reacting to specific substrates, so generally no need for sample pretreatment, less interference ; The analysis speed is fast, and the results can usually be obtained within 1 minute; the accuracy is high, and the relative error is generally less than 1%; the operating system is simple, and it is easy to realize automatic analysis.

Biosensors include the following types: (1) Enzyme sensors are the earliest and most mature type of biosensors. Under the catalysis of immobilized enzymes, after the biomolecules undergo chemical changes, the changes are recorded by the transducer to indirectly measure the concentration of the analyte. At present, there are more than 20 kinds of enzyme sensors that have been successfully developed in the world, the most mature of which is the glucose sensor. When in use, the enzyme electrode is immersed in the sample solution, the glucose in the solution diffuses to the enzyme membrane, and gluconic acid is generated under the action of glucose oxidase fixed on the enzyme membrane, and oxygen is consumed at the same time, and the oxygen concentration in the solution is measured by the oxygen electrode The concentration of glucose in the sample can be inferred; (2) Tissue sensor uses the catalysis of multi-enzyme system in animal and plant tissues to detect the analyte. Since the enzymes in the tissue are used, there is no need for artificial purification process, so it is relatively stable and has a long service life; (3) The microbial sensor immobilizes the microorganisms on the biosensitive film, and uses the respiration of the microorganisms or the enzymes contained in them to detect Determination of the concentration of the substance to be tested, especially during the fermentation process; (4) The immunosensor uses the high specificity between the antigen and the antibody to bind the antigen (or antibody) to the biosensitive membrane to measure the corresponding antibody (or antibody) in the sample (5) The field effect transistor biosensor combines the transistor process, requires a small amount of enzyme or antibody, and is considered a third-generation biosensor. At present, there are not many practical applications, but the development potential is huge.

In recent years, remarkable progress has been made in the research and development of biosensors, which have potential applications in many industries. In the future, in the process of combining with cutting-edge disciplines such as molecular electronics and bioelectronics, more sensitive and novel biosensors will be created, and biosensors will be miniaturized, portable and practical.

Chinese patent 99249332.2 proposes a method for immobilizing glucose oxidase, which uses titanium dioxide paste to immobilize glucose oxidase as an electronic intermediate. This invention is relatively novel and can be used many times, but the electron transfer effect of titanium dioxide is not as good as that of potassium ferricyanide, and the sensitivity and accuracy are also limited, and it is easy to encounter problems such as enzyme activity maintenance and enzyme body shedding after repeated use. Chinese patent CN1219676A uses a disposable dual-electron dual-enzyme biosensor to detect cholesterol and glucose in blood, but this type of sensor requires the participation of oxygen and is easily disturbed by changes in oxygen content in blood.

Contents of the invention

The object of the present invention is to provide a glucose biosensor based on functionalized materials.

Another object of the present invention is to provide a method for preparing a glucose biosensor based on functionalized materials.

The present invention is based on the national standard GB7665-87's definition of sensor as: "a device or device that can sense a specified measured value and convert it into a usable signal according to a certain rule, usually composed of a sensitive element and a conversion element". The development principle is based on the action of glucose oxidase (GOD) membrane, the oxidation reaction of glucose occurs, and oxygen is consumed to generate gluconolactone and hydrogen peroxide. The change in the amount of the substance can be converted into an electrical signal, and the signal change can be detected by the above-mentioned biosensor.

In the functionalized material-based glucose biosensor of the present invention, its working electrode is made of graphene nanomaterials.

Wherein, the graphene nanomaterial is selected from: palladium nanoparticle/graphene composite material, platinum nanoparticle/graphene composite material, gold nanoparticle/graphene composite material, silver nanoparticle/graphene composite material, copper nanoparticle/graphene composite material ene composite material, germanium nanoparticle/graphene composite material, ferric oxide nanoparticle/graphene composite material, ferroferric oxide nanoparticle/graphene composite material, indium tin oxide nanoparticle/graphene composite material and indium oxide Tin-platinum binary composite nanoparticles/graphene nanocomposites.

In the present invention, the preparation method of its graphene is selected from: hydrazine hydrate reduction method, tape method, epitaxial growth method on SiC substrate, sodium borohydride and hydrazine hydrate two-step reduction method, alkali reduction method, acid reduction method, water Thermal method, solvothermal method, chemical vapor deposition method, graphite direct exfoliation method, microwave-assisted reduction method, carbon tube shearing method, electrochemical reduction method, flame method and free radical reduction method.

Graphene nano material preparation method of the present invention comprises the steps:

a. Dispersing graphene oxide into a mixed medium of thionyl chloride and N,N-dimethylformamide, sonicating at room temperature, and then refluxing to prepare a brown acid chloride compound;

b. At the same time, mix phthaloyl chitosan, LiCl and N, N-dimethylacetamide, and react at 120-150 ^o C under the protection of nitrogen; after cooling the reaction system,

c. Add the product of a and pyridine into the reaction system, reflux under nitrogen protection, filter, wash, and vacuum-dry;

d. The dried solid was stirred in distilled water, filtered, and the retained solid was redispersed in distilled water, sonicated, filtered, washed, and dried in vacuum to obtain the intermediate PHCS-GO;

e. The intermediate obtained in d is dispersed in hydrazine hydrate and reacted at 60-90 ^o C, filtered, washed, and vacuum-dried to obtain an intermediate CSGR-NH ₂ containing an active amine group;

f. Add the intermediate CSGR-NH ₂ containing active amine groups to water, then add ethylene glycol, add the ethylene glycol solution or aqueous solution of the metal nanoparticle precursor dropwise under ultrasonic state, and react under acidic conditions under the protection of nitrogen, Then react under alkaline conditions at 120-140 ^o C;

g. After cooling to room temperature, filter, wash, and vacuum-dry to obtain a graphene nanomaterial nanoparticle/graphene composite material.

The most suitable graphene nanomaterial preparation method comprises the following steps:

a. Dispersing graphene oxide into a mixed medium of thionyl chloride and N,N-dimethylformamide, ultrasonicating at room temperature for 20 to 40 minutes, and then refluxing for 40 to 60 hours to prepare a brown acid chloride compound;

b. At the same time, mix phthaloyl chitosan, LiCl and N, N-dimethylacetamide, and react at 120-150 ^o C for 2-4 hours under the protection of nitrogen; after cooling the reaction system,

c. Add the product of a and pyridine into the reaction system, reflux for 40-60 hours under the protection of nitrogen, after cooling, filter, wash and dry in vacuum;

d. After drying, the solid was stirred in distilled water for 3-9 hours, filtered, and the retained solid was redispersed in distilled water, sonicated for 0.5-2 hours, filtered, washed, and the obtained solid was dried under vacuum to obtain the intermediate PHCS-GO;

e. Disperse the intermediate obtained in d in hydrazine hydrate, react at 60-90 ^o C for 10-24 hours, filter, wash, and vacuum-dry to obtain the intermediate CSGR-NH ₂ containing active amine groups;

f. The intermediate CSGR-NH ₂ containing active amine groups was added to water. Then add ethylene glycol, after ultrasonication for 20-30 minutes, add the ethylene glycol solution or aqueous solution of the metal nanoparticle precursor drop by drop, and react under the acidic condition of pH 5-6 for 12-24 hours under the protection of nitrogen, and then Reaction at pH 12-13 alkaline condition 120-140 ^o C;

g. After cooling to room temperature, filter, wash with water, wash with ethanol, and dry under vacuum to obtain a graphene nanomaterial nanoparticle/graphene composite material.

The glucose biosensor based on functionalized materials of the present invention is made by using the working electrode made of graphene nanomaterial for the assembly of glucose biosensor, and the preparation method of the working electrode made of graphene nanomaterial comprises the following steps :

a. The glassy carbon electrode is ground and then polished;

b. After cleaning, dry at room temperature for later use;

c. Graphene nanomaterials are ultrasonically dispersed in 3% to 10% acetic acid;

d. Take the above-mentioned dispersed liquid and drop it on the glassy carbon electrode after b, and dry it; then,

e. Soak the electrode with 0.1%-1% glutaraldehyde solution at room temperature, then wash with deionized water and dry;

f. Drop the glucose oxidase solution on the electrode after e, and dry it at 1-4 ^o C; after that,

g. Soak in phosphate buffer solution with pH = 7.5, and store at 1-4 ^o C for later use.

The most suitable preparation method of the working electrode made of graphene nanomaterials comprises the following steps:

The glassy carbon electrode was first polished with 1200# metallographic sandpaper, and then polished with 1 μm, 0.3 μm, and 0.05 μm Al ₂ O ₃ in sequence;

a. After cleaning with distilled water, ultrasonically clean in 1:1 HNO ₃ aqueous solution, absolute ethanol, and distilled water for 5-10 min, and dry at room temperature for later use;

b. Get the graphene nanomaterials and ultrasonically disperse them in 5% acetic acid;

c. Take 5-10 μL of the above-mentioned dispersion liquid and drop it on the surface of the above-mentioned glassy carbon electrode, and after drying, the nano-particle functionalized graphene material-modified electrode is obtained;

d. The electrode was soaked with 0.5% glutaraldehyde solution at room temperature, then washed with deionized water and dried at room temperature;

e. Drop 5-10 μL of 4 mg/ml glucose oxidase solution on the nanoparticle-functionalized graphene material-modified electrode, and dry at 1-4 ^o C to obtain the nanoparticle-functionalized graphene material-glucose oxidase-modified electrode. electrode;

f. The above nanoparticle functionalized graphene material glucose oxidase modified electrode was soaked in 0.05 M pH = 7.5 phosphate buffer solution for 10-30 minutes, and then soaked in 0.05 M pH = 7.5 phosphate buffer solution after taking it out. Store in 4 ^o C refrigerator for later use.

On the basis of many years of research, the present invention uses graphene nanomaterials in the assembly of glucose biosensors to form nano-functional membranes. Compared with traditional enzyme electrodes, it has high specificity, short-term and low-cost analysis. , Safe operation, convenient on-site measurement and other significant advantages, it is the first choice for daily blood glucose measurement of diabetic patients, and has important economic and social significance. Nano-functional membrane-immobilized enzymes can be analyzed continuously for more than 1,000 times, and can achieve the same current response height as one-time consumption of enzymes. The cost of determination is only a few cents, and the analysis accuracy is higher than other methods, with a relative error of about 1%. And the response time is shortened to 20 seconds, and the service life is greatly extended. This current-type glucose sensor can accurately measure the glucose concentration at regular intervals. It has high specificity, short-term, low-cost analysis, no special requirements for analytical substances, and safe operation. , Simple, convenient for on-site determination, etc., can be used for daily blood glucose measurement of diabetic patients. It also provides favorable conditions for the popularization of national standards.

The glucose biosensor based on functionalized graphene nanomaterials of the present invention is a quantitative testing technology with accurate data and small errors, and can be used for efficient, simple and rapid determination of blood glucose concentration of patients, so it has broad clinical application prospects , is the development trend of clinical and household biosensors.

Description of drawings

Figure 1a is a scanning electron microscope image of palladium nanoparticles/graphene composites;

Figure 1b is a high-resolution transmission electron microscope image of palladium nanoparticles/graphene composites;

Figure 1c is an electron diffraction pattern of palladium nanoparticles/graphene composite;

Figure 1d is the X-ray energy spectrum (EDS) of the palladium nanoparticles/graphene composite;

Figure 2 is a typical Raman spectrum of graphene nanocomposites;

Fig. 3 is the infrared spectrogram of palladium nanoparticle/graphene composite material and related materials;

Figure 4 is the X-ray diffraction of palladium nanoparticles/graphene composites and related materials.

Detailed ways

Preparation Example 1 Graphene Preparation

Improved Hummers method combined with hydrazine hydrate reduction method: First, graphite (1.5 g, 325 mesh) was added to a mixture of 12 ml concentrated H ₂ SO ₄ , 2.5 g K ₂ S ₂ O ₈ and 2.5 g P ₂ O ₅ , heated The above mixed system was brought to 80 ^o C, and the temperature was maintained with magnetic stirring for 5 hours. Then the reaction system was cooled to room temperature, and the mixture was poured into 500 ml of deionized water and left to stand overnight. Filter the above standing matter through a 0.2 micron filter membrane, wash and dry naturally to obtain pre-oxidized graphite. Add the pre-oxidized graphite to 120 ml of concentrated H ₂ SO ₄ at 0 ^o C, then slowly add 15 g of KMnO ₄ , and control the reaction temperature at 20 ^o C to stir. After the addition of potassium permanganate, the reaction system was controlled to stir at 35 ^o C for 4 hours, then 250 ml of deionized water was added, and the temperature was controlled below 50 ^o C by a peripheral ice bath. After stirring for 1.5 hours, 700 ml of deionized water was added, and after half an hour, 20 ml of 30% H ₂ O ₂ was added dropwise, and the reaction system quickly changed from brown to brown yellow. Remove the stirring device, filter the brown-yellow mixture, wash with 1 L of 1: 10 HCl to remove metal ions, and then wash repeatedly with 1 L of deionized water to obtain a brown solid. After drying at room temperature, the above-mentioned brown solid is made into a weight ratio 0.5% w/w water dispersion, continuous dialysis for one week, finally filtered, washed, re-dispersed by ultrasonication for 1 hour, filtered, and vacuum-dried at 60 ^o C for 24 hours to prepare graphene oxide. Prepare 100 ml of the above graphene oxide dispersion with a concentration of 1 mg/ml, add 1 ml of hydrazine hydrate, reflux at 100 ^o C for 24 hours after ultrasonication for 30 minutes, filter, wash, and vacuum dry at 60 ^o C for 24 hours, that is Graphene can be prepared;

Preparation Example 2 Preparation of Graphene Nanomaterials (Palladium Nanoparticles/Graphene Composite Pd NPs/CSGR)

First, disperse 200 mg of graphene oxide into a mixed medium of 40 ml thionyl chloride and 1 ml N,N-dimethylformamide, sonicate at room temperature for 0.5 hours, and then reflux for 52 hours to prepare 219.2 mg of brown acid chloride compound . At the same time, 1.753 g of phthaloyl chitosan), 1.201 g of LiCl and 120 ml of N,N-dimethylacetamide were mixed and reacted at 140 ^o C for 2 hours under the protection of nitrogen. After cooling the reaction system, 219.2 mg of the brown acid chloride compound prepared in the previous step and 14 ml of pyridine were added to the reaction system, and refluxed for 48 hours under nitrogen protection. After cooling, it was filtered, washed, and vacuum-dried. After drying, the solid was stirred in 120 ml of distilled water for 6 hours, filtered, and the retained solid was redispersed in 200 ml of water, ultrasonicated for 1 hour, filtered, and washed. The obtained solid was dried under vacuum at 65 ^o C for 24 hours to obtain the intermediate PHCS-GO (205 g). The intermediate was dispersed in 15 ml of hydrazine hydrate and reacted at 80 ^o C for 16 hours to remove the protection of phthaloyl. Filter, wash, and vacuum-dry to obtain an intermediate CSGR-NH ₂ (175 mg) containing an active amine group. Intermediate CSGR- _NH2 (100 mg) containing reactive amine groups was added to 1.5 ml of water. Subsequently, 10 ml of ethylene glycol was added, ultrasonicated for 20 minutes, 10 ml of palladium chloride ethylene glycol solution with a concentration of 2.03 mg Pd/ml was added dropwise, the pH value was adjusted to 5-6, and the reaction system was vigorously ultrasonicated for 5 minutes, followed by stirring 20 hours. Subsequently, the pH value was adjusted to 13 with 2.5 M sodium hydroxide solution, and the reaction was carried out at 140 ^o C for 3 hours, and the whole reaction process was protected by nitrogen gas. After cooling to room temperature, filter, wash with water and ethanol three times each, and dry under vacuum at 65 ^o C for 24 hours to obtain palladium nanoparticles/graphene composite (Pd NPs/CSGR);

Material Characterization

The characterization of product size and morphology was performed on JEM-2010F Transmission Electron Microscope (TEM), JEOL-2010F High Resolution Transmission Electron Microscope (HRTEM) and JSM-7401F Field Emission Scanning Electron Microscope (FESEM), and the operating voltage of the electron microscope was 200 kV . X-ray Energy Spectroscopy (EDS) and Selected Area Diffraction (SAED) experiments were completed under JEOL-2010F high-resolution transmission electron microscope. The powder X-ray diffraction (XRD) characterization of the product was carried out on a Bruker D8-advance X-ray diffractometer in Germany. The X-ray is monochromatic CuKα radiation (λ=1.5418 Å), and the 2θ scanning angle is from 10 to 70°. The step size is 0.02°. Fourier transform infrared spectroscopy (FTIR) experiments were carried out on a FTIR-8201 (PerkinElmer) infrared spectrometer. Infrared spectrum analysis was tested after the sample was pressed with KBr. The Raman spectrum was selected from Renishaw, a British company. Microprobe RM1000 Raman spectrometer, excitation wavelength 633 nm (He/Ne laser). Atomic force test (Atomic force Microscopic, AFM) adopts the Nanoscope of Veeco Precision Instrument Co., Ltd. III MultiMode SPM (digital display) scanning probe atomic force microscope. Elemental analysis was performed using a CE-440 elemental analyzer from EAI, USA.

The characterization results of palladium nanoparticles/graphene composites are as follows: the diameter range of palladium nanoparticles is between 2-8 nm, with smaller particle size and narrower particle size distribution (characterized according to SEM Figure 1a and TEM Figure 1b results); the electron diffraction pattern (see Figure 1c) can be observed from the inner ring to the outer ring from strong to weak four diffraction rings, corresponding to (111), (200), (220), (311 ) and (400) crystal planes. Raman spectrum (see Figure 2) shows that it has characteristic Raman peaks at 1331 cm ^-1 and 1590 cm ^-1 , which are assigned to the D band and G band of graphene respectively; infrared spectrum (see Figure 3, where a. chitosan sugar, b. graphene, c. chitosan-modified graphene, d. palladium nanoparticles/graphene composite) show that the electrode material (Fig. 3, d.) has 890 , 1150 cm ^-1 and 1545 cm ^{- 1} characteristic peaks, the first two peaks are the characteristic peaks of chitosan, and the third peak is attributed to the vibration of the graphene skeleton. X-ray diffraction pattern (XRD pattern, see Figure 4, where a. graphene, b. palladium nanoparticles/graphene composite material) observed palladium nanoparticles/graphene composite material at 24.7 ^° , 39.6°, 45.5° and 67.3° Wait for four Bragg reflection peaks, the first one corresponds to the (002) crystal plane of graphene, and the last three peaks correspond to the (111), (200), (220) crystal planes of palladium in turn;

Preparation Example 3 Sensor Working Electrode Preparation

The glassy carbon electrode was first polished with 1200# metallographic sandpaper, and then polished with 1 μm, 0.3 μm, and 0.05 μm Al ₂ O ₃ in sequence, cleaned with distilled water, and washed in 1:1 HNO ₃ aqueous solution, absolute ethanol , distilled water, ultrasonic cleaning for 5 min each, and dry at room temperature for later use. Take 2 mg of the nanoparticle-functionalized graphene material prepared above, and disperse it in 1 ml of 5% acetic acid. After ultrasonication, take 8 μL of the above-mentioned dispersion solution and drop it on the surface of the above-mentioned glassy carbon electrode, and obtain the nanoparticle-functionalized graphene material after drying. Graphene material modified electrodes. The electrode was soaked with 0.5% glutaraldehyde solution at room temperature, then washed with deionized water and dried at room temperature. 8 μL of 4 mg/ml glucose oxidase (GOD) solution was dropped on the nanoparticle-functionalized graphene material-modified electrode, and dried at 4 ^o C. The above glucose oxidase modified electrode was soaked in 0.05 M phosphate buffer solution with pH = 7.5 for 20 minutes to remove unbound GOD. Soak the above-prepared working electrode of the glucose biosensor in 0.05 M phosphate buffer solution with pH = 7.5 and store it in a 4 ^o C refrigerator for later use.

Application Example 1

Cyclic voltammetry and AC impedance tests were performed using a CHI830B electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.), with a three-electrode system: a saturated calomel electrode as the residual wall electrode, a platinum electrode as the auxiliary electrode, and an enzyme electrode as the working electrode. Dip the prepared glucose biosensor working electrode into 5 mL In the 0.05 M phosphate buffer solution with pH = 7.5, add different volumes of glucose standard solution or solution to be tested under uniform stirring, after the dissolved oxygen signal is captured by the oxygen electrode, the change of the electrode potential is input into the computer data processor, and calculated as Output in the form of changes in dissolved oxygen concentration, record the drop curve of dissolved oxygen concentration, and use the calibration curve method to detect the glucose content in the sample.

The results show that the glucose biosensor based on the functionalized material of the present invention has the characteristics of wide linear range, low detection limit, good reproducibility, long life, etc.: it presents a good linear relationship between the glucose concentration of 0.010-1.10 mmol/L , the linear regression equation is y(mg/L)=6.7471x(mmol/L)－0.00501(r=0.9989); the detection limit of the sensor is calculated by dividing the standard deviation of 3 times the blank by the slope of the standard working curve to be 0.2 μmol /L (S/N=3). The sensor made of the same enzyme membrane was repeatedly measured for 0.25 mmol/L glucose solution 10 times, and the response average RSD=2.5%. Four glucose biosensors were prepared by placing different immobilized enzyme membranes on the surface of the oxygen electrode, for 0.25 Measured with mmol/L glucose solution, RSD=4.7%; 200 consecutive tests (about 24 h) of this concentration of glucose, the response signal can still reach more than 98% of the initial value; store this glucose biosensor in a refrigerator at 4 °C , Repeat the test every 3 to 4 days. The response value of 0.25 mmol/L glucose solution. After 3 months, the response signal was 86.5% of the initial value.

Claims (6)

1. A glucose biosensor based on a functionalized material, comprising a working electrode, characterized in that the working electrode is made of graphene nanomaterials. 2. according to the described biosensor of claim 1, it is characterized in that: graphene nanomaterial is selected from: palladium nanoparticle/graphene composite material, platinum nanoparticle/graphene composite material, gold nanoparticle/graphene composite material, silver Nanoparticle/graphene composite material, copper nanoparticle/graphene composite material, germanium nanoparticle/graphene composite material, ferric oxide nanoparticle/graphene composite material, ferric oxide nanoparticle/graphene composite material, Indium tin oxide nanoparticle/graphene composite material and indium tin oxide-platinum binary composite nanoparticle/graphene nanocomposite material. 3. The biosensor according to claim 1 or 2, characterized in that: the graphene nanomaterial preparation method comprises the steps: a. Dispersing graphene oxide into a mixed medium of thionyl chloride and N,N-dimethylformamide, sonicating at room temperature, and then refluxing to prepare a brown acid chloride compound; b. At the same time, mix phthaloyl chitosan, LiCl and N, N-dimethylacetamide, and react at 120-150 ^o C under the protection of nitrogen; after cooling the reaction system, c. The product of a and pyridine are added to the reaction system, refluxed under nitrogen protection, filtered, washed, and vacuum-dried; d. The dried solid was stirred in distilled water, filtered, and the retained solid was redispersed in distilled water, sonicated, filtered, washed, and dried in vacuum to obtain the intermediate PHCS-GO; e. The intermediate obtained in d is dispersed in hydrazine hydrate and reacted at 60-90 ^o C, filtered, washed, and vacuum-dried to obtain an intermediate CSGR-NH ₂ containing an active amine group; f. Add the intermediate CSGR-NH ₂ containing active amine groups to water, then add ethylene glycol, add the ethylene glycol solution or aqueous solution of the metal nanoparticle precursor dropwise under ultrasonic state, and react under acidic conditions under the protection of nitrogen, Then react under alkaline conditions at 120-140 ^o C; g. After cooling to room temperature, filter, wash, and vacuum-dry to obtain a graphene nanomaterial nanoparticle/graphene composite material. 4. The biosensor according to claim 1 or 2, characterized in that: the graphene nanomaterial preparation method comprises the steps: a. Dispersing graphene oxide into a mixed medium of thionyl chloride and N,N-dimethylformamide, ultrasonicating at room temperature for 20 to 40 minutes, and then refluxing for 40 to 60 hours to prepare a brown acid chloride compound; b. At the same time, mix phthaloyl chitosan, LiCl and N, N-dimethylacetamide, and react at 120-150 ^o C for 2-4 hours under the protection of nitrogen; after cooling the reaction system, c. Add the product of a and pyridine into the reaction system, reflux for 40-60 hours under the protection of nitrogen, after cooling, filter, wash, and vacuum-dry; d. After drying, the solid was stirred in distilled water for 3-9 hours, filtered, and the retained solid was redispersed in distilled water, sonicated for 0.5-2 hours, filtered, washed, and the obtained solid was dried under vacuum to obtain the intermediate PHCS-GO; e. Disperse the intermediate obtained in d in hydrazine hydrate, react at 60-90 ^o C for 10-24 hours, filter, wash, and vacuum-dry to obtain the intermediate CSGR-NH ₂ containing active amine groups; f. Add the intermediate CSGR-NH ₂ containing active amine groups into water; then add ethylene glycol, and after ultrasonication for 20-30 minutes, add the ethylene glycol solution or aqueous solution of the metal nanoparticle precursor drop by drop, and firstly adjust the pH value under nitrogen protection. React under acidic conditions with a value of 5-6 for 12-24 hours, and then react under alkaline conditions with a pH value of 12-13 at 120-140 ^o C; g. After cooling to room temperature, filter, wash with water, wash with ethanol, and dry under vacuum to obtain a graphene nanomaterial nanoparticle/graphene composite material. 5. according to the preparation method of the described biosensor of claim 1, it is characterized in that: the working electrode that graphene nanomaterial is made is used for the assembly of glucose biosensor and is made, the working electrode that is made by graphene nanomaterial The preparation method comprises the following steps: a. The glassy carbon electrode is ground and then polished; b. After cleaning, dry at room temperature for later use; c. Graphene nanomaterials are ultrasonically dispersed in 3% to 10% acetic acid; d. Take the above-mentioned dispersed liquid and drop it on the glassy carbon electrode after b, and dry it; then, e. Soak the electrode with 0.1%-1% glutaraldehyde solution at room temperature, then wash with deionized water and dry; f. Drop the glucose oxidase solution on the electrode after e, and dry it at 1-4 ^o C; after that, g. Soak in phosphate buffer solution with pH = 7.5, and store at 1-4 ^o C for later use. 6. according to the preparation method of the described biosensor of claim 1 or 5, it is characterized in that: the working electrode that graphene nano material is made is used for the assembly of glucose biosensor and is made, the working electrode that is made of graphene nano material The preparation method of electrode comprises the steps: a. Grind the glassy carbon electrode with 1200# metallographic sandpaper, and then polish it with 1 μm, 0.3 μm, 0.05 μm Al ₂ O ₃ in sequence; b. After cleaning with distilled water, ultrasonically clean in 1:1 HNO ₃ aqueous solution, absolute ethanol, and distilled water for 5-10 minutes respectively, and dry at room temperature for later use; c. Take graphene nanomaterials and ultrasonically disperse them in 5% acetic acid; d. Take 5-10 μL of the above-mentioned dispersion liquid and drop it on the surface of the above-mentioned glassy carbon electrode, and after drying, the electrode modified by nano-particle functionalized graphene material is obtained; e. soak the electrode with 0.5% glutaraldehyde solution at room temperature, then wash with deionized water and dry at room temperature; f. Drop 5-10 μL of 4 mg/ml glucose oxidase solution on the nanoparticle-functionalized graphene material-modified electrode, and dry at 1-4 ^o C to obtain the nano-particle-functionalized graphene material glucose oxidation enzyme. Enzyme modified electrodes; g. The above nanoparticle functionalized graphene material glucose oxidase modified electrode was soaked in 0.05 M pH = 7.5 phosphate buffer solution for 10 to 30 minutes, and then soaked in 0.05 M pH = 7.5 phosphate buffer solution after taking it out. Store in the refrigerator at 1-4 ^o C for later use.

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