Preparation method of lactic acid and alpha fetoprotein detection biosensor

Abstract

The invention belongs to the technical field of biosensor preparation, and relates to a preparation method of a lactic acid and alpha fetoprotein detection biosensor. The preparation method comprises the following steps: adding single-layer graphene oxide into N, N-dimethylformamide, uniformly mixing, adding polyacrylonitrile, uniformly mixing, and adding dopamine hydrochloride to obtain a spinning solution; and after spinning with the spinning solution, drying in a vacuum drying oven, and then fixing on a gold electrode to obtain an electrode P. And uniformly mixing lactate oxidase and chitosan, dropwise adding the mixture to the surface of the electrode P, dropwise adding an antibody solution after drying, and performing secondary drying to obtain the sensing electrode. The nanofiber membrane with proper pore diameter and fiber diameter is regulated and controlled, is used for preparing a sensor for simultaneously detecting lactic acid and alpha fetoprotein, and has higher performance in detection of a real sample. The detection range of lactic acid and alpha fetoprotein is wide, the preparation process is simple, the cost is low, and the method can be used for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of biosensor fabrication technology, and relates to a method for fabricating a biosensor for detecting lactic acid and alpha-fetoprotein. Background Technology

[0002] Liver diseases have complex pathological mechanisms and unpredictable progression. Globally, over 844 million people suffer from chronic liver disease, and more than 2 million die from it annually. In many regions, the incidence of viral hepatitis and non-alcoholic fatty liver disease continues to rise. Currently, there are no specific drugs to completely cure these diseases, making early diagnosis and control crucial. However, clinical methods for immediate detection of liver diseases suffer from long processing times, complex instrument operation, and high costs, reducing the efficiency of early disease screening. Furthermore, current clinical diagnostic methods for liver diseases often lack clear etiological or pathological information and have low sensitivity in detecting early fibrosis. Invasive examinations require biopsies, which carry risks of bleeding and infection. Currently, no ideal methods for detecting early biomarkers of liver disease have been reported. Summary of the Invention

[0003] This invention addresses the problems existing in the detection of traditional liver disease biomarkers by proposing a method for preparing a biosensor for detecting lactate and alpha-fetoprotein.

[0004] To achieve the above objectives, the present invention is implemented using the following technical solution: A method for preparing a biosensor for detecting lactic acid and alpha-fetoprotein includes the following steps: (1) Add monolayer graphene oxide to N,N dimethylformamide and mix evenly. Add polyacrylonitrile and mix evenly. Then add dopamine hydrochloride to obtain the spinning solution.

[0005] (2) Nanofiber membranes are obtained after spinning the spinning solution.

[0006] (3) The nanofiber membrane was dried in a vacuum drying oven and then fixed on a gold electrode to obtain electrode P.

[0007] (4) Mix lactate oxidase and chitosan evenly to obtain an enzyme solution. Add the enzyme solution to the surface of electrode P. After drying, add antibody solution and dry again to obtain a sensing electrode. The antibody solution includes alpha-fetoprotein antibody, N-hydroxysuccinimide, and 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0008] Preferably, in step (1), monolayer graphene oxide and N,N dimethylformamide are pulverized and mixed using a cell pulverizer; the mass ratio of monolayer graphene oxide to N,N dimethylformamide to polyacrylonitrile is 1:(800-1000):(70-120), and the molecular weight of polyacrylonitrile is 200,000-300,000.

[0009] Preferably, the positive voltage of spinning in step (2) is 17-19 kV and the negative voltage is -(3-5) kV.

[0010] Preferably, the concentration of lactate oxidase in the enzyme solution is 1000-3000 U·mL. -1 The concentration of alpha-fetoprotein antibody in the antibody solution is 0.5-1.5 mg / mL; the amount of lactate oxidase added to the working electrode is 10-100 μL•cm-2, and the amount of AFP antibody added is 0.3-0.7 ng•cm-2.

[0011] 5. The application of the biosensor prepared by any one of claims 1-4 in the detection of lactic acid and alpha-fetoprotein.

[0012] This invention utilizes polyacrylonitrile (PAN) and graphene oxide (GO) to construct nanofiber membranes, achieving a balance between electrocatalysis and membrane separation functions. PAN is easily spun into nanofibers, forming a three-dimensional network structure with high porosity, suitable for separating blood cells. Simultaneously, the spun PAN possesses a large specific surface area, providing ample space for loading lactate oxidase and AFP antibodies. The addition of GO increases conductivity, delays the coagulation time of real blood samples on the membrane surface, and improves the membrane's mechanical strength and negative charge, preventing the adsorption of large protein molecules from the blood onto the membrane surface. Dopamine (DA) exhibits good biocompatibility and low toxicity in cell experiments. The biosensor prepared using PAN / GO-DA nanofibers for the simultaneous detection of lactate and alpha-fetoprotein demonstrates excellent biocompatibility and high accuracy in the detection of real blood samples.

[0013] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. This invention uses PAN / GO-DA nanofiber membranes as electrode materials. By controlling the amount of dopamine added, the pore size and fiber diameter of the nanofiber membrane are adjusted to obtain a nanofiber membrane with suitable pore size and fiber diameter. This membrane is then used to prepare a sensor for the simultaneous detection of lactic acid and alpha-fetoprotein, and it exhibits high performance in the detection of real samples.

[0014] 2. It exhibits a wide detection range for both lactic acid and alpha-fetoprotein, and the preparation process is simple and low-cost, making it suitable for large-scale production to achieve accurate real-time detection of lactic acid and alpha-fetoprotein in the human body. Attached Figure Description

[0015] Figure 1 is a scanning electron microscope image of PAN / GO-DA in Example 1.

[0016] Figure 2 is a TEM image of PAN / GO-DA in Example 1. Detailed Implementation

[0017] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0018] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0019] Example 1 9g of N,N-dimethylaminoamide was placed in a beaker, and 10mg of monolayer graphene oxide was added. The mixture was then placed in a cell disruptor and disrupted for 20 minutes. 1g of polyacrylonitrile (molecular weight 250,000) was added, and the mixture was stirred at 200 rpm for 24 hours at room temperature. 20mg of dopamine hydrochloride was then added, and the mixture was stirred at 200 rpm for 24 hours at room temperature to obtain a spinning solution. The spinning solution was then subjected to electrospinning under the following conditions: positive voltage 17kV, negative voltage -4kV, and spinning time 5 hours to obtain nanofibers. These nanofibers were then dried in a vacuum drying oven at 60℃ for 24 hours to obtain a PAN / GO-DA nanofiber membrane. 30 U of lactate oxidase (purchased from Sigma Biotechnology Co., Ltd.) was dissolved in 30 μL of PBS solution (containing 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, and 0.26 M dipotassium hydrogen phosphate, with water as the solvent). 15 μL of this solution was then added to a 0.2 mL centrifuge tube. 15 μL of a 5 mg / mL chitosan solution was added to dilute the lactate oxidase solution. After mixing thoroughly, 5 μL of this solution was added dropwise to the working electrode and dried at 0 °C for 24 h. 200 μL of AFP... Antibody (purchased from Sigma Biotechnology Co., Ltd.) was added to a 0.2 mL centrifuge tube, followed by 300 μL of deionized water. Then, 1 μL of this solution was added to the same 0.2 mL centrifuge tube, along with 1 μL of EDC (purchased from Sigma Biotechnology Co., Ltd.) and 1 μL of NHS (purchased from Sigma Biotechnology Co., Ltd.). After thorough mixing, 0.5 μL of this solution was added dropwise to the working electrode and dried at 0 °C for 24 h to obtain a biosensor for the simultaneous detection of lactate and alpha-fetoprotein. SEM characterization images of the PAN / GO-DA nanofibers are shown below. Figure 1 As shown, the TEM characterization image is as follows: Figure 2As shown in the figure, the fibers are of uniform thickness and have a smooth, defect-free surface.

[0020] Example 2 9g of N,N-dimethylaminoamide was placed in a beaker, and 10mg of monolayer graphene oxide was added. The mixture was then placed in a cell disruptor and disrupted for 20 minutes. 1g of polyacrylonitrile (molecular weight 250,000) was added, and the mixture was stirred at 200 rpm for 24 hours at room temperature. 50mg of dopamine hydrochloride was added, and the mixture was stirred at 200 rpm for 24 hours at room temperature to obtain a spinning solution. The spinning solution was then subjected to electrospinning under the following conditions: positive voltage 18kV, negative voltage -4kV, and spinning time 5 hours to obtain a nanofiber membrane. The membrane was then placed in a vacuum drying oven and dried at 60℃ for 24 hours to obtain a PAN / GO-DA nanofiber membrane. 30 U of lactate oxidase was dissolved in 30 μL of PBS solution (containing 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, and 0.26 M dipotassium hydrogen phosphate, with water as the solvent). 5 μL of this solution was then added to a 0.2 mL centrifuge tube. 20 μL of a 5 mg / mL chitosan solution was added to dilute the lactate oxidase solution. After mixing thoroughly, 5 μL of this solution was added to the working electrode and dried at 0 °C for 24 h. 200 μL of AFP antibody was added to a 0.2 mL centrifuge tube. 300 μL of deionized water was added, and 1 μL of this solution was added to the centrifuge tube. 1 μL of EDC and 1 μL of NHS were added, and after mixing thoroughly, 1 μL of this solution was added to the working electrode and dried at 20 °C for 24 h. This yielded a biosensor for the simultaneous detection of lactate and alpha-fetoprotein.

[0021] Example 3 9g of N,N-dimethylaminoamide was placed in a beaker, and 10mg of monolayer graphene oxide was added. The mixture was then placed in a cell disruptor and disrupted for 20 minutes. 1g of polyacrylonitrile (molecular weight 250,000) was added, and the mixture was stirred at 200 rpm for 24 hours at room temperature. 80mg of dopamine hydrochloride was added, and the mixture was stirred at 200 rpm for 24 hours at room temperature to obtain a spinning solution. The spinning solution was then subjected to electrospinning under the following conditions: positive voltage 18kV, negative voltage -4kV, and spinning time 5 hours to obtain a nanofiber membrane. The membrane was then placed in a vacuum drying oven and dried at 60℃ for 24 hours to obtain a PAN / GO-DA nanofiber membrane. 30 U of lactate oxidase was dissolved in 30 μL of PBS solution (containing 0.5 M potassium chloride, 0.3 M potassium dihydrogen phosphate, and 0.26 M dipotassium hydrogen phosphate, with water as the solvent). 10 μL of this solution was then added to a 0.2 mL centrifuge tube. 20 μL of chitosan solution (5 mg / mL) was added to dilute the lactate oxidase solution. After mixing thoroughly, 5 μL of the solution was added to the working electrode and dried at 0 °C for 24 h. 200 μL of AFP antibody was added to a 0.2 mL centrifuge tube. 300 μL of deionized water was added, and 1 μL of this solution was added to the centrifuge tube. 1 μL of EDC and 1 μL of NHS were added to the mixture. After mixing thoroughly, 1.5 μL of this solution was added to the working electrode and dried at 20 °C for 24 h to obtain a biosensor for the simultaneous detection of lactate and alpha-fetoprotein.

[0022] The performance of the sensors prepared in each embodiment was tested as follows.

[0023] 1. Linear range test of lactic acid and alpha-fetoprotein concentrations.

[0024] Using the biosensor for detecting lactic acid and alpha-fetoprotein (AFP) prepared in Example 1, the changes in lactic acid concentration and AFP levels were tested using a constant current (IT) and cyclic voltammetry (CV) method with a Shanghai Chenhua CHI660E electrochemical workstation. PBS electrolyte (pH 7.0) was used in the electrolytic cell. After each sampling, 40 μL of sample was added to 40 mL of electrolyte for detection. The concentration of the sample was calculated using a standard curve after detection. The sensor prepared in Example 1 showed a linear range of 0.5-25 mM for lactic acid and 10 fg-100 ng for AFP. After storing the sensor in a refrigerator at 4°C for 30 days, its steady-state response current still maintained more than 85% of the initial sensitivity, indicating that the prepared biosensor for detecting lactic acid and AFP has good stability.

[0025] 2. Accuracy Verification Using the standard addition method, samples were added to the electrolyte to obtain electrolytes of different concentrations of lactic acid (sample AD) and alpha-fetoprotein (sample EH) for testing. The sensors prepared in Example 1 were used for detection (test values). The detected values ​​were compared with the actual concentrations to verify the accuracy of the biosensor prepared in Example 1. The results are shown in Tables 1 and 2 below.

[0026] Table 1 Lactic acid test results Table 2. Results of Alpha-fetoprotein (AFP) Detection The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a biosensor for detecting lactic acid and alpha-fetoprotein, characterized in that, The steps are as follows: (1) Add monolayer graphene oxide to N,N dimethylformamide and mix evenly. Add polyacrylonitrile and mix evenly. Then add dopamine hydrochloride to obtain the spinning solution. (2) Nanofiber membranes are obtained after spinning the spinning solution; (3) The nanofiber membrane was dried in a vacuum drying oven and then fixed on a gold electrode to obtain electrode P; (4) Mix lactate oxidase and chitosan evenly to obtain an enzyme solution. Add the enzyme solution to the surface of electrode P. After drying, add antibody solution and dry again to obtain a sensing electrode. The antibody solution includes alpha-fetoprotein antibody, N-hydroxysuccinimide, and 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride.

2. The method for preparing the biosensor for detecting lactic acid and alpha-fetoprotein according to claim 1, characterized in that, In step (1), monolayer graphene oxide and N,N-dimethylformamide are pulverized and mixed using a cell disruptor; the mass ratio of monolayer graphene oxide to N,N-dimethylformamide to polyacrylonitrile is 1:(800-1000):(70-120), and the molecular weight of polyacrylonitrile is 200,000-300,000.

3. The method for preparing the lactic acid and alpha-fetoprotein detection biosensor according to claim 1, characterized in that, In step (2), the positive voltage for spinning is 17-19 kV and the negative voltage is -(3-5) kV.

4. The method for preparing the biosensor for detecting lactic acid and alpha-fetoprotein according to claim 1, characterized in that, The concentration of lactate oxidase in the enzyme solution is 1000-3000 U·mL. -1 The concentration of alpha-fetoprotein antibody in the antibody solution was 0.5-1.5 mg / mL; the amount of lactate oxidase added to the working electrode was 10-100 μL·cm⁻¹. -2 The dosage of AFP antibody added is 0.3-0.7 ng / cm³. -2 .

5. The application of the biosensor prepared by any one of claims 1-4 in the detection of lactic acid and alpha-fetoprotein.

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