An electrochemical biosensor and a preparation method and application thereof

Abstract

The application discloses an electrochemical biosensor and a preparation method and application thereof. The electrochemical biosensor comprises a sensor body, two pieces of nickel foam electrode sheets and two copper wires, the two copper wires are respectively inserted into the two pieces of nickel foam electrode sheets, and then are fixed on a natural birch of the sensor body; the natural birch is modified with CYFRA21-1 antibodies and a stabilizer. First, the natural birch is cut into a block with a proper size; the birch block is immersed into a mixed solution containing the CYFRA21-1 antibodies and the stabilizer for incubation, and after the incubation, the birch block is used as the body of the electrochemical biosensor; two pieces of nickel foam sheets are cut out and are perforated at edges; the two copper wires are respectively inserted into the holes of the two pieces of the cut-out nickel foam sheets, and are fixed to the birch block of the electrochemical biosensor body through two rubber bands, so that the electrochemical biosensor is obtained. The electrochemical biosensor has the advantages of portability, sensitivity, high stability, miniaturization, low cost, no need of pretreatment, rapid detection and the like.

Description

I. TECHNICAL FIELD

[0001] The present application relates to the field of electrochemical analysis and detection, in particular to an electrochemical biosensor and a preparation method and application thereof. II. BACKGROUND

[0002] Biosensors belong to the emerging field of bioelectronics, which is an interdisciplinary field involving biology, chemistry, materials, medicine, and nanotechnology, microelectronics, information technology, and other multidisciplinary technologies. Whether it is the mechanism of bioelectronic systems or the development of practical applications, biosensors have aroused widespread and lasting interest among researchers around the world. The application fields of biosensors mainly include medical detection, environmental monitoring, food safety and national defense applications.

[0003] At present, the application technology of biosensors generally uses fluorescence labeling to detect signals, and magnetic beads are used to extract samples by adsorption. Magnetic beads are magnetic microspheres coated with specific biomolecules, which specifically bind to the corresponding target substances to form new complexes. When a magnetic field is applied, the complex is retained and separated from other components for the detection of biological samples. The magnetic bead process is very time-consuming, complex and costly to produce due to the use of a large number of reagents.

[0004] As a marker for non-small cell lung cancer, CYFRA21-1 antigen will appear abnormal concentration increase in patients. In clinical practice, the concentration of CYFRA21-1 antigen is often used as one of the main standards for diagnosing whether a patient is ill or not. In the application technology of biosensors, fluorescence labeling is generally used to detect signals, and magnetic beads are used to extract samples by adsorption. Magnetic beads are magnetic microspheres coated with specific biomolecules, which can specifically bind to the corresponding target substances to form new complexes. When a magnetic field is applied, the complex is retained and separated from other components for the detection of biological samples. The magnetic bead process uses a large number of reagents, so it takes a lot of time, and the operation process is complex and costly to produce.

[0005] An electrochemical biosensor is an analytical device that can convert biological immune signals into electrical signals, involving the concentration of a substance to be analyzed or a group of substances to be analyzed and the detectable electrical signal response. The main feature of an electrochemical biosensor for real-time detection is fast detection, simple operation, small size, portability, stable results and comparability. III. SUMMARY

[0006] The technical problem solved by the present application is: according to the development and existing problems of the existing electrochemical biosensor, the present application provides an electrochemical biosensor and a preparation method and application thereof.

[0007] In order to solve the above problems, the technical scheme adopted by the present application is:

[0008] The present application provides an electrochemical biosensor, comprising a sensor body of natural birch, two pieces of nickel foam electrode sheet and two copper wires, the two copper wires are respectively inserted into the two pieces of nickel foam electrode sheet, and the two pieces of nickel foam electrode sheet with copper wires are fixed on the sensor body of natural birch; the sensor body of natural birch is modified with CYFRA21-1 antibody and stabilizer.

[0009] According to the above-mentioned electrochemical biosensor, the stabilizer is Tween-20.

[0010] In addition, a preparation method of an electrochemical biosensor is provided, the preparation method comprises the following steps:

[0011] a. cutting natural birch into wood blocks with a size of 1.5*1.5*1cm 3 ;

[0012] b. adding CYFRA21-1 antibody and stabilizer into saturated sodium chloride solution for dissolution, and obtaining a mixed solution after sufficient dissolution;

[0013] c. immersing the birch blocks obtained in step a into a container containing the mixed solution obtained in step b for incubation; taking out the birch blocks after incubation, and the birch blocks obtained after incubation are used as the body of the electrochemical biosensor;

[0014] d. cutting two pieces of nickel foam sheet with a size of 1.5*1.5cm 2 , and perforating at the edge;

[0015] e. inserting two ordinary copper wires with a length of 15cm into the holes of the two pieces of nickel foam sheet obtained by cutting;

[0016] f. fixing the two pieces of nickel foam sheet with copper wires obtained in step e to the birch block of the electrochemical biosensor body obtained in step c by two rubber bands, to obtain an electrochemical biosensor.

[0017] According to the above-mentioned preparation method of the electrochemical biosensor, the stabilizer in step b is Tween-20.

[0018] According to the preparation method of the electrochemical biosensor, the concentration of the CYFRA21-1 antibody in the mixed solution obtained in step b is 5-20 ng·mL -1 , and the concentration of the stabilizer is 0.01-0.1 wt%.

[0019] According to the preparation method of the electrochemical biosensor, the incubation temperature of the antibody is 4 DEG C, and the incubation time of the antibody is 24-36 h in step c; and the antibody is stored in the dark after incubation.

[0020] The application of the electrochemical biosensor in the detection of non-small cell lung cancer.

[0021] In the technical scheme of the application, the birch used is natural birch without any chemical treatment.

[0022] The positive beneficial effects of the application are as follows:

[0023] 1. In the technical scheme of the application, the natural birch loaded with CYFRA21-1 antibody is used as the sensor body, and the capillary phenomenon of the closely arranged 10-20 mu m channels of the natural birch is used to transport the solution upward to generate a microelectric potential, thereby realizing detection. Tween-20 plays the role of a stabilizer in the incubation process. The assembled electrochemical biosensor is placed in an antigen solution with different concentrations, the antigen and the antibody in the upward solution transport are combined, and the relationship between the electric response signal (current, impedance) and the antigen concentration is detected.

[0024] 2. The electrochemical biosensor prepared by the application has the advantages of portability, sensitivity, high stability, miniaturization, low cost, no need for pretreatment, rapid detection, etc., and can realize early diagnosis and early treatment of non-small cell lung cancer patients, and simultaneously provides a brand-new idea for designing a clinical rapid diagnosis of non-small cell lung cancer. IV. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The structure diagram of the electrochemical biosensor.

[0026] Figure 1 In the middle: 1 is a foam nickel electrode layer, 2 is CYFRA21-1 antibody loaded after incubation, 3 is an antigen transported upward from a liquid, 4 is a birch block of the sensor body, 5 is a copper wire for transmitting an electric signal, 6 is an electrochemical workstation, 7 is a solution containing anions and cations and CYFRA21-1 antibody, 8 is an anion in the solution, and 9 is a cation.

[0027] Figure 2 The principle diagram of the microelectric potential generated by the electrochemical sensor.

[0028] Emerging forms of osmotic power generation have attracted increasing attention from researchers. This electrodynamic phenomenon, based on osmotic induction, occurs when an electrolyte solution passes through a capillary or capillary-like tube. The liquid regulates the electrical double layer, causing ion dissociation and generating a potential difference across the capillary-like tube, thus producing a so-called flow or induced potential and current. Previous studies have reported combining this effect with porous, hydrophilic natural wood materials to generate continuous microcurrents. By utilizing the current generated by its own potential, precise detection of disease biomarkers can be achieved without an external voltage.

[0029] Figure 3 The current-concentration calibration curve of the CYFRA21-1 antigen electrochemical biosensor of this invention.

[0030] Depend on Figure 3 It can be seen that the current measured by the electrochemical biosensor gradually decreases with increasing antigen concentration. Furthermore, the correlation coefficient of the linear fit is greater than 0.96, indicating a good fit.

[0031] Figure 4 The impedance-concentration calibration curve of the CYFRA21-1 antigen electrochemical biosensor of this invention.

[0032] Depend on Figure 4 It can be seen that the impedance measured by the electrochemical biosensor gradually decreases with increasing antigen concentration. Furthermore, the correlation coefficient of the linear fit is greater than 0.98, indicating a good fit. V. Detailed Implementation Methods:

[0033] The present invention is further illustrated by the following embodiments, but these embodiments do not limit the scope of protection of the technical solutions of the present invention.

[0034] Example 1:

[0035] The present invention relates to an electrochemical biosensor comprising a sensor body made of natural birch wood, two nickel foam electrode plates, and two copper wires. The two copper wires are respectively threaded into the two nickel foam electrode plates, and the two nickel foam electrode plates with copper wires are fixed on the sensor body made of natural birch wood. The sensor body made of natural birch wood is modified with CYFRA21-1 antibody and the stabilizer Tween-20.

[0036] See appendix Figure 1The specific structure of the electrochemical biosensor for non-small cell lung cancer in the application is as follows: 1 is a foamed nickel electrode layer, the foamed nickel electrode layer 1 includes two pieces of foamed nickel, which are respectively arranged on the upper and lower ends of a birch block, and the lower end has been immersed in water; CYFRA21-1 antibody 2 loaded after incubation, antigen 3 transmitted upward from liquid, the birch block 4 as a sensor body and copper wire 5 for transmitting an electric signal, electrochemical workstation 6, solution 7 containing anions and cations and CYFRA21-1 antibody, anions 8 and cations 9 in the solution.

[0037] The foamed nickel electrode located between the electrode layer 1 and the foamed nickel not shown as immersed in water transmits the signal to the electrochemical workstation 6 through the copper wire 5 as an electrode for transmitting an electric signal.

[0038] The generation of the electric signal of the electrochemical biosensor in the application is as shown in the accompanying drawings. Figure 2 During the upward transmission of the bottom solution due to capillary phenomenon, a large number of hydroxyl groups (-OH) and carboxyl groups (-COOH) contained in the birch itself are hydrolyzed, and in addition, the anions and cations rub against the inner wall of the pore during the transmission of the solution, resulting in a potential difference between the upper and lower ends of the sensor body, that is, the birch block. The micro-potential generated by the solution containing different concentrations of antigens is also different, and the electrochemical biosensor in the application utilizes this principle to realize the detection of tumor markers.

[0039] Example 2:

[0040] The preparation method of the electrochemical biosensor in the application includes the following detailed steps:

[0041] a. Cut a natural birch into a wood block with a size of 1.5*1.5*1 cm 3 (the cutting process needs to pay attention to the upper and lower surfaces of the birch block, that is, the surfaces with a size of 1.5*1.5 cm 2 , as the cross section of the wood);

[0042] b. Add CYFRA21-1 antibody and stabilizer Tween-20 into saturated sodium chloride solution for dissolution, and obtain a mixed solution after sufficient dissolution; in the obtained mixed solution, the concentration of CYFRA21-1 antibody is 20 ng·mL -1 , and the concentration of the stabilizer is 0.01 wt.%;

[0043] c. immerse the birch block obtained by cutting in step a into a weighing bottle containing 5 mL of the mixed solution obtained in step b for incubation, the incubation temperature of the antibody is 4 DEG C, and the incubation time of the antibody is 30 h; after incubation, store in the dark; the birch block obtained after incubation is used as the body of the electrochemical biosensor;

[0044] d. cut two pieces of foamed nickel with a size of 1.5*1.5 cm 2 , and perforate the edges;

[0045] e、 two 15cm long copper wires are respectively inserted into the holes of the two pieces of nickel foam;

[0046] f、 the two pieces of nickel foam with copper wires inserted in step e are fixed to the birch block of the electrochemical biosensor body obtained in step c by two rubber bands, to obtain the electrochemical biosensor of the application.

[0047] Application example of the electrochemical biosensor prepared in Example 2 of the application in non-small cell lung cancer detection:

[0048] The electrochemical biosensor prepared in Example 2 is placed in a substrate solution containing different concentrations of antigens, incubated at room temperature for 10 min, and the relationship between the current response value and the AC impedance response value and the antigen concentration is measured by an electrochemical workstation.

[0049] The antibody used in this example is CYFRA21-1 Antibody, and the antibody stock solution concentration is 675ng·mL -1 ; the antigen used is CYFRA21-1 Antigen, and the antigen stock solution concentration is 2mg·mL -1 , and the molecular weight is 44KD.

[0050] The change in current of the electrochemical biosensor prepared in Example 2 of the application is measured; the relationship between the current response value and the concentration in the CYFRA21-1 antigen concentration range of 2-7ng·mL -1 is shown in the following figure: Figure 3 Each concentration is tested for 15 groups of data, and the relationship between the average test current and the antigen concentration is known by statistical fitting, that is, the current measured by the electrochemical biosensor gradually decreases with the increase of the antigen concentration. The correlation coefficient of linear fitting is greater than 0.96, and the fitting degree is good.

[0051] The change in AC impedance of the electrochemical biosensor prepared in Example 2 of the application is measured; the relationship between the impedance response value and the concentration in the CYFRA21-1 antigen concentration range of 2-7ng·mL -1 is shown in the following figure: Figure 4 Each concentration is tested for 15 groups of data, and the relationship between the average test impedance and the antigen concentration is known by statistical fitting, that is, the impedance measured by the electrochemical biosensor gradually decreases with the increase of the antigen concentration. The correlation coefficient of linear fitting is greater than 0.98, and the fitting degree is also good.

[0052] In summary, the electrochemical biosensor is a high-sensitivity immunological diagnosis method without sample pretreatment or labeling, the sensor can be connected with an electrochemical workstation by software in a computer, without large detection equipment, and has considerable advantages in reagent cost, detection cost, professional skill requirement of operating personnel, detection time and the like, and has the advantages of short detection time, no need of professional personnel for assisting in measurement, and convenience for popularization. The prepared electrochemical biosensor has the advantages of portability, sensitivity, high stability, miniaturization, low cost, no need of pretreatment, rapid detection and the like, can realize early diagnosis and early treatment of non-small cell lung cancer patients, and provides a brand-new idea for designing on-site rapid diagnosis equipment.

Claims

1. An electrochemical biosensor, characterized in that: The sensor body is made of natural birch wood, two nickel foam electrode plates, and two copper wires. The two copper wires are respectively threaded into the two nickel foam electrode plates, and the two nickel foam electrode plates with copper wires are fixed to the natural birch wood sensor body. The natural birch wood sensor body is modified with CYFRA21-1 antibody and stabilizer. The electrochemical biosensor is prepared by the following method: a. Cut the natural birch wood into pieces measuring 1.5 × 1.5 × 1 cm. 3 The birch wood blocks were obtained; the top and bottom surfaces of the resulting birch wood blocks were 1.5 × 1.5 cm. 2 The surface is the cross-section of the wood; b. Dissolve the CYFRA21-1 antibody and stabilizer in a saturated sodium chloride solution until fully dissolved to obtain a mixed solution; c. Immerse the birch block obtained in step a into a container containing the mixed solution obtained in step b for incubation; after incubation, remove the birch block, and use the birch block obtained after incubation as the body of the electrochemical biosensor; d. Cut out two pieces, each 1.5 x 1.5 cm in size. 2 The foamed nickel sheet is perforated at the edges; e. Thread two ordinary copper wires, each 15cm long, through the holes in the two pieces of cut nickel foam sheets; f. Fix the two pieces of foam nickel sheets with copper wires obtained in step e to the birch block of the electrochemical biosensor body obtained in step c using two rubber bands to obtain the electrochemical biosensor.

2. The electrochemical biosensor according to claim 1, characterized in that: The stabilizer mentioned in step b is Tween-20.

3. The electrochemical biosensor according to claim 1, characterized in that: The concentration of CYFRA21-1 antibody in the mixed solution obtained in step b is 5–20 ng / mL. -1 The concentration of the stabilizer is 0.01 to 0.1 wt%.

4. The electrochemical biosensor according to claim 1, characterized in that: During the incubation process described in step c, the antibody is incubated at a temperature of 4°C for 24–36 hours; after incubation, it is stored in the dark.

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