Spiral carbon nanotube chitosan loaded with gold nanoparticles on surface as well as preparation and application of spiral carbon nanotube chitosan

Abstract

The invention provides helical carbon nanotube chitosan with gold nanoparticles loaded on the surface as well as preparation and application thereof, and the preparation method comprises the following steps: (1) pretreating helical carbon nanotubes, adding the helical carbon nanotubes into a hydrochloric acid solution, and carrying out ultrasonic mixing, washing and drying, (2) adding the helical carbon nanotubes into a sodium citrate solution, ultrasonically adding deionized water, heating until boiling, adding a chloroauric acid solution, continuously heating and stirring, filtering, washing and drying to obtain the helical carbon nanotube chitosan with gold nanoparticles loaded on the surface. Cooling, washing and drying; (3) dispersing in deionized water, performing ultrasonic treatment to obtain a mixed solution, adding a chitosan solution into the mixed solution, and performing stirring and ultrasonic mixing to obtain a helical carbon nanotube chitosan composite material mixed solution with gold nanoparticles loaded on the surface. According to the nanogold-modified helical carbon nanotube, due to the high aspect ratio, the helical carbon nanotube enhances an electronic signal in electrochemical measurement by providing a surface area which is much larger than that of a bare electrode at the same site, and the nanogold-modified helical carbon nanotube also serves as a connecting layer between biomolecules of an immunosensor and AuNPs, so that signal amplification is caused.

Description

Technical Field

[0001] The invention belongs to the technical field of nanocomposite materials and biological immune sensor detection, and specifically relates to the field of improving sensor performance, especially a preparation method and application of a signal amplification surface-loaded gold nanoparticle spiral carbon nanotube and chitosan electrochemical immunosensor. Background Art

[0002] Carcinoembryonic antigen (CEA) is an acidic glycoprotein with a molecular weight of about 170 kDa and is known as one of the most important clinical tumor biomarkers. An increase in CEA concentration in adult plasma may be an early sign of many cancerous diseases. The average level of CEA in the serum of healthy adults is usually less than 5 ng / mL; although some diseases can cause a slight increase in the CEA index, if the CEA level in the serum is higher than 20 ng / mL, it means the possibility of tumors, including colon tumors, breast tumors, ovarian cancer, colorectal cancer and cystadenocarcinoma. Therefore, early detection of CEA is an important step in controlling, effectively treating, monitoring and screening for disease recurrence.

[0003] Therefore, it is very important to prepare an electrochemical immunosensor that can significantly improve the sensitivity, stability, repeatability and low detection limit to achieve early detection of carcinoembryonic antigen. Summary of the invention

[0004] The first object of the present invention is to provide a method for preparing a spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface, in view of the problems existing in the background technology. The technical scheme is as follows:

[0005] A method for preparing a spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface comprises the following steps:

[0006] Step 1: pre-treating the helical carbon nanotubes, adding them into a hydrochloric acid solution, ultrasonically mixing for 2 hours, washing, and drying to obtain purified helical carbon nanotubes;

[0007] Step 2: Add the purified helical carbon nanotube powder obtained in step 1 to a sodium citrate solution, perform ultrasonication for 10 minutes, add deionized water, heat to boiling, add chloroauric acid solution, continue heating and stirring for 5 minutes, cool, wash, and dry to obtain a helical carbon nanotube with gold nanoparticles loaded on the surface;

[0008] Step 3: Disperse the surface-loaded gold nanoparticles spiral carbon nanotubes obtained in step 2 in deionized water, ultrasonicate for 5 minutes to obtain a mixed solution, add chitosan solution to the mixed solution, and stir and ultrasonicate at 25°C to obtain a surface-loaded gold nanoparticles spiral carbon nanotube chitosan composite material mixed solution.

[0009] As a preferred embodiment, in the step 1, the outer diameter of the spiral carbon nanotube is 200-700 nm, and the length is between 1-10 μm; and the mass fraction of hydrochloric acid is 37%.

[0010] As a preferred embodiment, in the step 2, the volume ratio of the spiral carbon nanotube mass to the sodium citrate solution is 3:4; the mass fraction of the chloroauric acid solution is 1%, the mass fraction of the sodium citrate solution is 1%, and the volume ratio of the chloroauric acid solution to the sodium citrate solution is 1:8; the size of the obtained loaded gold nanoparticles is between 10-40nm.

[0011] As a preferred embodiment, in step three, the concentration of the spiral carbon nanotube ion aqueous solution loaded with gold nanoparticles on the surface is 1.5 mg / mL; the concentration of the chitosan solution is 200 μg / mL, wherein the solvent is a glacial acetic acid solution with a volume fraction of 1%; and the volume ratio of the spiral carbon nanotube mixed solution loaded with gold nanoparticles on the surface to the chitosan solution is 1:1.

[0012] The second object of the present invention is to provide a spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface obtained by the preparation method.

[0013] The third object of the present invention is to provide a method for preparing an electrochemical immunosensor, wherein the mixed droplets of the spiral carbon nanotube chitosan loaded with gold nanoparticles on the surface are applied to the surface of a glassy carbon electrode, and then dried and sequentially combined with antibodies, blocking agents and antigens to obtain the electrochemical immunosensor.

[0014] As a preferred embodiment, the blocking agent in the preparation method is bovine serum albumin, the mass fraction of the bovine serum albumin is 1%, and the blocking agent binding time is 1 hour; the antibody is the antibody corresponding to the carcinoembryonic antigen, the antibody concentration is 100 μg / mL, and the antibody incubation time is 4 hours; the antigen incubation time is 30 minutes.

[0015] The fourth object of the present invention is to provide an electrochemical immunosensor prepared by the preparation method.

[0016] A fifth object of the present invention is to provide an application of an electrochemical immunosensor in the detection of biomarkers in human serum samples.

[0017] The beneficial effects of the present invention are:

[0018] (1) Gold nanoparticles modified helical carbon nanotubes, due to the high aspect ratio, helical carbon nanotubes enhance the electronic signal in electrochemical measurements by providing a much larger surface area than bare electrodes at the same site, and are also used as a connecting layer between biomolecules and AuNPs in immunosensors, leading to signal amplification. Chitosan is a polysaccharide derived from the deacetylation of chitin, which has excellent film-forming ability, high water permeability, good adhesion, biocompatibility and high mechanical strength, and has been widely used as an immobilization matrix for bioprocessing.

[0019] (2) The electrochemical immunosensor constructed based on the surface-loaded gold nanoparticles, spiral carbon nanotubes and chitosan composite material has the advantages of good biocompatibility, high conductivity, low detection limit and high sensitivity, and can be further applied to the detection of various disease biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the preparation process of electrochemical immunosensor based on surface-loaded gold nanoparticles helical carbon nanotube and chitosan nanocomposite;

[0021] FIG2 is a scanning electron microscope and mapping image of a surface-loaded gold nanoparticle-coiled carbon nanotube and a surface-loaded gold nanoparticle-coiled carbon nanotube and chitosan nanocomposite material;

[0022] FIG. 2(A) is a scanning electron microscope and mapping image of a surface-loaded gold nanoparticle spiral carbon nanotube; FIG. 2(B) is a scanning electron microscope image of a surface-loaded gold nanoparticle spiral carbon nanotube and chitosan nanocomposite material;

[0023] Figure 3 is a comparison of cyclic voltammograms before and after electrode modification;

[0024] Among them, Figure 3 (A) shows a blank glassy carbon electrode, and Figure 3 (B) shows a surface-loaded gold nanoparticle-loaded spiral carbon nanotube and chitosan-modified electrode;

[0025] FIG4(A) is a square wave voltammetry curve of gold nanoparticles / helical carbon nanotubes / chitosan / carcinoembryonic antigen antibody / bovine serum albumin / carcinoembryonic antigen electrochemical immunosensor, and FIG4(B) is a standard curve established between the logarithm of carcinoembryonic antigen concentration and the percentage of current reduction;

[0026] Figure 5 The bar graph shows the current response of serum at different dilution multiples. DETAILED DESCRIPTION

[0027] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0028] 1. A method for preparing helical carbon nanotube chitosan with gold nanoparticles loaded on the surface

[0029] The embodiment provides a method for preparing a spiral carbon nanotube and chitosan loaded with gold nanoparticles on the surface, comprising the following steps:

[0030] Step 1: pre-treating the helical carbon nanotubes, adding them into a hydrochloric acid solution, ultrasonically mixing for 2 hours, washing, and drying to obtain purified helical carbon nanotubes;

[0031] Step 2: Add the purified helical carbon nanotube powder obtained in step 1 to a sodium citrate solution, perform ultrasonication for 10 minutes, add deionized water, heat to boiling, add chloroauric acid solution, continue heating and stirring for 5 minutes, cool, wash, and dry to obtain a helical carbon nanotube with gold nanoparticles loaded on the surface;

[0032] Step 3: Disperse the surface-loaded gold nanoparticles spiral carbon nanotubes obtained in step 2 in deionized water, ultrasonicate for 5 minutes to obtain a mixed solution, add chitosan solution to the mixed solution, and stir and ultrasonicate at 25°C to obtain a surface-loaded gold nanoparticles spiral carbon nanotube chitosan composite material mixed solution.

[0033] In the step 1, the outer diameter of the spiral carbon nanotube is 200-700 nm, and the length is between 1-10 μm; the mass fraction of hydrochloric acid is 37%;

[0034] In the step 2, the volume ratio of the helical carbon nanotube mass to the sodium citrate solution is 3:4; the mass fraction of the chloroauric acid solution is 2%, the mass fraction of the sodium citrate solution is 1%, and the volume ratio of the chloroauric acid solution to the sodium citrate solution is 1:8; the size of the obtained loaded gold nanoparticles is between 10-40nm.

[0035] In the step three, the concentration of the surface-loaded gold nanoparticles spiral carbon nanotube solution is 1.5 mg / mL, and the concentration of the chitosan solution is 200 μg / mL, wherein the solvent is 1% glacial acetic acid solution; and the volume ratio of the surface-loaded gold nanoparticles spiral carbon nanotube mixed solution to the chitosan solution is 1:1.

[0036] 2. A method for preparing an electrochemical immunosensor

[0037] The embodiment provides a method for preparing an electrochemical immunosensor: a mixed solution comprising the above-mentioned surface-loaded gold nanoparticles, spiral carbon nanotubes and chitosan is droplet-coated on the surface of a glassy carbon electrode, and then dried and sequentially combined with an antibody, a blocking agent and an antigen to obtain the electrochemical immunosensor.

[0038] The blocking agent is bovine serum albumin, the mass concentration of the bovine serum albumin is 1%, and the blocking agent binding time is 1 hour; the antibody is the antibody corresponding to the carcinoembryonic antigen, the antibody concentration is 100 μg / mL, and the antibody incubation time is 4 hours; the antigen incubation time is 30 minutes.

[0039] Specifically, in the following embodiments, the preparation method of the electrochemical immunosensor with signal amplification function includes the following steps:

[0040] (1) 10 μL of gold nanoparticle / helical carbon nanotube / chitosan mixed solution was dropped onto the treated glassy carbon electrode surface. After natural drying, the gold nanoparticle / helical carbon nanotube / chitosan / glassy carbon electrode was immersed in 100 μg / mL carcinoembryonic antigen antibody solution. After incubation for 4 hours, it was washed with a PBS buffer solution with a pH of 7.4 to remove the unfixed antibody. 6 μL of 1% bovine serum albumin was dropped onto the gold nanoparticle / helical carbon nanotube / chitosan / carcinoembryonic antigen antibody electrode surface, incubated for 1 hour to block nonspecific active sites, and dried naturally to prepare a gold nanoparticle / helical carbon nanotube / chitosan / carcinoembryonic antigen antibody / bovine serum albumin electrochemical sensor with signal amplification.

[0041] (2) The immunosensor uses a Shanghai Chenhua CHI660E electrochemical workstation with gold nanoparticles / helical carbon nanotubes / chitosan / glassy carbon electrodes as working electrodes, saturated Ag / AgCl electrodes as reference electrodes, and platinum wire electrodes as counter electrodes in an aqueous solution containing 5 mM [Fe(CN)6] 3- / 4- Cyclic voltammetry and electrochemical impedance spectroscopy were carried out in PBS buffer solution to test the electron transfer ability of the prepared sensor.

[0042] Based on the above preparation method, further, the optional standard antigen incubation time in step (1) is 15 min, 30 min, 45 min, and the optimal incubation temperature is 30 min.

[0043] 3. Application of the electrochemical immunosensor in the detection of biomarkers in human serum samples

[0044] The present invention also provides an application of the electrochemical immunosensor prepared by the above preparation method in the detection of biomarkers in human serum samples.

[0045] Specifically, the method for detecting biomarkers in human serum samples using an electrochemical immunosensor with a signal amplification function comprises the following steps:

[0046] The working electrode modified with gold nanoparticles / helical carbon nanotubes / chitosan / CEA antibody / bovine serum albumin was immersed in CEA solutions of different concentrations. The prepared CEA electrochemical immunosensor was then placed in a solution containing 5 mM [Fe(CN)6] 3- / 4- The current response was detected by square wave voltammetry in PBS buffer solution. The current response signal changed with the change of antigen concentration, and the linear regression equation was fitted using the logarithmic relationship between the percentage of current response signal reduction and the antigen concentration; the constructed electrochemical immunosensor was used to detect the current response of carcinoembryonic antigen in human serum, and the carcinoembryonic antigen concentration in human serum was obtained according to the fitted linear regression equation.

[0047] In the following examples, the electrochemical performance of the electrodes before and after modification was evaluated, comprising the following steps:

[0048] (1) The glassy carbon electrode was polished with 0.3 μm and 0.05 μm Al2O3 powders to remove the surface oxide layer. After polishing, it was ultrasonically cleaned with ultrapure water and ethanol to remove any other physically adsorbed substances. The cleaned electrode was subjected to cyclic voltammetry in PBS buffer solution with a pH of 7.4 and 5 mM [Fe(CN)6] 3- / 4- The electrode was activated in PBS buffer solution until the cyclic voltammetry current was stable. The activated electrode was washed with ultrapure water and dried for later use.

[0049] (2) 10 μL of the gold nanoparticle / helical carbon nanotube / chitosan mixture was added to the pretreated glassy carbon electrode and dried naturally to prepare a gold nanoparticle / helical carbon nanotube / chitosan modified electrode. Cyclic voltammetry was performed using an electrochemical workstation.

[0050] The present invention is further illustrated by the following examples.

[0051] Embodiment 1:

[0052] The method for preparing spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface comprises the following steps:

[0053] (1) Purification of helical carbon nanotubes

[0054] 1 g of helical carbon nanotubes was pretreated, added into 100 mL of 37% hydrochloric acid solution, ultrasonically mixed at 50° C. for 2 h for purification, filtered and washed until neutral, and vacuum dried at 50° C. to obtain purified helical carbon nanotubes.

[0055] (2) Preparation of helical carbon nanotubes loaded with gold nanoparticles

[0056] Take 12 mg of purified helical carbon nanotubes and ultrasonically disperse them in 16 mL of 1% sodium citrate solution, then add 84 mL of deionized water, stir and heat to boiling, then add 2 mL of 1% chloroauric acid solution, boil for 5 minutes until the solution turns wine red, cool to room temperature, wash and dry to obtain helical carbon nanotubes with gold nanoparticles loaded on the surface.

[0057] (3) Preparation of helical carbon nanotube and chitosan composites loaded with gold nanoparticles

[0058] 20 mg of chitosan was dissolved in 100 mL of 1% acetic acid solution, and ultrasonic mixing was performed to obtain a 200 μg / mL chitosan solution. 15 mg of surface-loaded gold nanoparticles spiral carbon nanotubes were dispersed in 10 mL of deionized water, and ultrasonic mixing was performed for 5 min to obtain a 100 μg / mL mixed solution. The same volume of 200 μg / mL chitosan solution was added to the mixed solution, and ultrasonic mixing was performed at 25°C for 1 h to obtain a surface-loaded gold nanoparticles spiral carbon nanotube and chitosan composite material mixed solution.

[0059] Embodiment 2:

[0060] The electrochemical performance evaluation of the electrode before and after modification includes the following steps:

[0061] 10 μL of the mixed solution of the spiral carbon nanotube chitosan composite material loaded with gold nanoparticles on the surface was dropped onto the glassy carbon electrode and dried naturally at room temperature to prepare the gold nanoparticle / spiral carbon nanotube / chitosan modified electrode. The cyclic voltammetry curve of the modified electrode was detected by an electrochemical workstation. The cyclic voltammetry curves and current response bar graphs of the blank glassy carbon electrode, gold nanoparticle / spiral carbon nanotube / chitosan modified electrode are shown in Figures 3 (A) and 3 (B). Combined with Figures 3 (A) and 3 (B), the experimental results show that the redox peak current of the gold nanoparticle / spiral carbon nanotube / chitosan modified electrode is significantly increased compared with the blank glassy carbon electrode. According to the Randles-Sevcik equation i p =2.69×10 5 n 3 / 2 AD 1 / 2 V 1 / 2 C0, the surface area A of the glassy carbon electrode and the modified glassy carbon electrode can be determined. Calculations show that the surface area of ​​the modified electrode (gold nanoparticles / helical carbon nanotubes / chitosan / glassy carbon electrode) is about 200% higher than that of the glassy carbon electrode, and the electronic conductivity is about 200% higher. The increase in specific surface area and the continuous increase in electronic conductivity are due to the synergistic electronic conductive behavior of gold nanoparticles and helical carbon nanotubes and the high surface area of ​​nanomaterials.

[0062] Example 3

[0063] A preparation method and application of an electrochemical immunosensor with signal amplification function, comprising the following steps:

[0064] (1) 10 μL of the gold nanoparticle / helical carbon nanotube / chitosan mixture was dropped onto the treated glassy carbon electrode surface. After natural drying, the gold nanoparticle / helical carbon nanotube / chitosan / glassy carbon electrode was immersed in a 100 μg / mL carcinoembryonic antigen antibody solution, incubated at 25°C for 4 h, and then washed with a PBS buffer solution with a pH of 7.4 to remove the unfixed antibody. 6 μL of 1% bovine serum albumin was dropped onto the gold nanoparticle / helical carbon nanotube / chitosan / carcinoembryonic antigen antibody electrode surface, incubated at 25°C for 1 h to block nonspecific active sites, and dried naturally to construct the gold nanoparticle / helical carbon nanotube / chitosan / anti-carcinoembryonic antigen / bovine serum albumin electrochemical immunosensor.

[0065] (2) An electrochemical immunosensor with a signal amplification function for determining the content of carcinoembryonic antigen (CEA), comprising the following steps:

[0066] Drawing of standard curve:

[0067] The prepared electrochemical immunosensor was used to detect different concentrations (1pg / mL, 10pg / mL, 100pg / mL, 10 3 pg / mL, 10 4 pg / mL, 10 5 pg / mL, 2×10 5 pg / mL and 4×10 5 pg / mL) of carcinoembryonic antigen was detected by square wave voltammetry, with a scanning voltage of -0.5 to 0.8V, a step height of 2mV, and a pulse height of 50mV. The standard curve established between the percentage reduction of the current response signal corresponding to different concentrations of carcinoembryonic antigen and the logarithmic change of its concentration is shown in Figures 4(A) and 4(B). Figure 4(A) is a square wave voltammetric current response diagram of the gold nanoparticle / helical carbon nanotube / chitosan / carcinoembryonic antigen antibody / bovine serum albumin electrochemical immunosensor, and Figure 4(B) is a standard curve established between the logarithm of the carcinoembryonic antigen concentration and the percentage reduction of the response current. Combined with Figures 4(A) and 4(B), the linear range of the electrochemical immunosensor is 1pg / mL-400ng / mL, and the linear regression equation is y=9.58245x+6.43328, (R 2 =0.99379), and the detection limit was 0.98581 pg / mL.

[0068] Actual sample analysis application

[0069] In order to evaluate the application of the electrochemical immunosensor with signal amplification function based on gold nanoparticles / helical carbon nanotubes / chitosan prepared in Example 3 in human serum detection, the spike method in serum was used for further exploration. The serum sample was diluted 1X, 10X, 100X, and 1000X with buffer, and 1ng / mL carcinoembryonic antigen solution was added to the blank serum. The square wave voltammetry detection was performed using a traditional three-electrode system, and the current response was compared with that of the pure 1ng / mL carcinoembryonic antigen solution. The experimental results are shown in Figure 2. Figure 5 As shown, the results show that human serum is closer to the response of pure carcinoembryonic antigen when diluted at 100X, so the serum was subsequently diluted to 100X for testing at different concentrations. By adding 1ng / mL, 10ng / mL, and 100ng / mL serum samples to the blank serum in sequence. The results in Table 1 show that the recovery rate of this method is between 95% and 111%, and the relative standard deviation of each sensor after three repeated measurements is less than 9.5%. Therefore, the prepared electrochemical immunosensor is expected to be used in the clinical determination of carcinoembryonic antigen.

[0070] Table 1 Experimental results of spike recovery of carcinoembryonic antigen

[0071]

[0072] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing a spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface, characterized in that: The following steps are involved: Step 1: pre-treating the helical carbon nanotubes, adding them into a hydrochloric acid solution, ultrasonically mixing for 2 hours, washing, and drying to obtain purified helical carbon nanotubes; Step 2: Add the purified helical carbon nanotube powder obtained in step 1 to a sodium citrate solution, perform ultrasonication for 10 minutes, add deionized water, heat to boiling, add chloroauric acid solution, continue heating and stirring for 5 minutes, cool, wash, and dry to obtain a helical carbon nanotube with gold nanoparticles loaded on the surface; Step 3: Disperse the surface-loaded gold nanoparticles spiral carbon nanotubes obtained in step 2 in deionized water, ultrasonicate for 5 minutes to obtain a mixed solution, add chitosan solution to the mixed solution, and stir and ultrasonicate at 25°C to obtain a surface-loaded gold nanoparticles spiral carbon nanotube chitosan composite material mixed solution.

2. The method for preparing the spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface according to claim 1, characterized in that: In the step 1, the outer diameter of the spiral carbon nanotube is 200-700 nm, and the length is between 1-10 μm; the mass fraction of hydrochloric acid is 37%.

3. The method for preparing the spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface according to claim 1, characterized in that: In the step 2, the volume ratio of the helical carbon nanotube mass to the sodium citrate solution is 3:4; the mass fraction of the chloroauric acid solution is 1%, the mass fraction of the sodium citrate solution is 1%, and the volume ratio of the chloroauric acid solution to the sodium citrate solution is 1:8; the size of the obtained loaded gold nanoparticles is between 10-40nm.

4. The method for preparing the spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface according to claim 1, characterized in that: In the step three, the concentration of the spiral carbon nanotube ion aqueous solution loaded with gold nanoparticles on the surface is 1.5 mg / mL; the concentration of the chitosan solution is 200 μg / mL, wherein the solvent is a glacial acetic acid solution with a volume fraction of 1%; and the volume ratio of the spiral carbon nanotube mixed solution loaded with gold nanoparticles on the surface to the chitosan solution is 1:

1.

5. A spiral carbon nanotube chitosan with gold nanoparticles loaded on the surface obtained by the preparation method according to any one of claims 1 to 4.

6. A method for preparing an electrochemical immunosensor, characterized in that: The mixed liquid drop of the spiral carbon nanotube chitosan loaded with gold nanoparticles on the surface of claim 5 is applied to the surface of the glassy carbon electrode, and then dried and combined with antibodies, blocking agents and antigens in sequence to obtain the electrochemical immunosensor.

7. The method for preparing the electrochemical immunosensor according to claim 6, characterized in that: The blocking agent is bovine serum albumin, the mass fraction of the bovine serum albumin is 1%, and the blocking agent binding time is 1 hour; the antibody is the antibody corresponding to the carcinoembryonic antigen, the antibody concentration is 100 μg / mL, and the antibody incubation time is 4 hours; the antigen incubation time is 30 minutes.

8. An electrochemical immunosensor, characterized in that Prepared by the preparation method of claim 6 or 7.

9. Use of the electrochemical immunosensor according to claim 8 in the detection of biomarkers in human serum samples.

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