CN110376380B

CN110376380B - Electrochemical enzyme-linked immunosensor and preparation and application thereof to antigen detection

Info

Publication number: CN110376380B
Application number: CN201910678544.6A
Authority: CN
Inventors: 吴康兵; 郝俊兴
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2019-07-25
Filing date: 2019-07-25
Publication date: 2020-07-24
Anticipated expiration: 2039-07-25
Also published as: CN110376380A

Abstract

The invention relates to an electrochemical enzyme-linked immunosensor and preparation and application thereof for detecting antigen, belonging to the technical field of protein detection.A preparation method of the electrochemical enzyme-linked immunosensor comprises the steps of adding the antigen into a biological conjugate of a magnetic bead and a capture antibody for incubation, removing supernatant under the condition of an external magnetic field, adding nanogold, a detection antibody and a biological conjugate of enzyme for incubation, and obtaining the electrochemical enzyme-linked immunosensor after double-antibody sandwich reaction.A dispersion solution of the probe is modified on the surface of a carbon paste substrate electrode to obtain the electrochemical enzyme-linked immunosensor, wherein the linear range of the electrochemical enzyme-linked immunosensor (MB-eElisa) provided by the invention is 0.01-6.0ng m L^‑1The detection limit is 4.0pg m L^‑1(S/N ═ 3); compared with the existing electrochemical immunodetection method, the method has lower detection limit and higher sensitivity.

Description

Electrochemical enzyme-linked immunosensor and preparation and application thereof to antigen detection

Technical Field

The invention belongs to the technical field of protein detection, and particularly relates to an electrochemical enzyme-linked immunosensor, preparation thereof and application thereof in antigen detection.

Background

The antigen is capable of stimulating immune system of living body, inducing immune feedback, and generating humoral immunityIt has been found that there are many antigens associated with tumor diseases, such as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), prostate specific Protein (PSA), and carbohydrate antigen (CA19-9), among which, CEA is the most widely applicable test object for current tumor diseases, a cell surface glycoprotein with a molecular weight of about 200kDa, as a typical tumor marker for clinical diagnosis of diseases such as colon cancer, breast cancer, lung cancer, stomach cancer, and pancreatic cancer, however, CEA, an antigen, generally exhibits very low content (0-2.5ng L ng) in blood of healthy people^-1) (ii) a In addition, the content of antigen CEA in human blood changes only slightly during early tumor formation. Therefore, clinical assays for antigens should be fast, accurate, sensitive, and specific to detect small changes in antigen content in complex biological samples.

Electrochemical immunosensing technology has the advantages of portability, fast response, high sensitivity, low cost and the like, and is most commonly applied to the measurement of antigens. However, most of the previously reported antigen electrochemical immunosensors are generally constructed directly at the sensing electrode interface, which often faces the following problems when dealing with complex biological sample detection: tedious detection steps, low detection flux and electrode interfaces that are easily passivated by proteins. Research shows that the Magnetic Beads (MB) are introduced into an electrochemical immunosensing system, so that the passivation of an electrode interface can be effectively avoided, and an immunodetection process can be simplified. Compared with an immobilized electrode sensing interface, the MB has faster reaction kinetics, larger specific surface area and better stability in a solution; in addition, various functional groups, DNA, enzymes, antibodies, etc. can be easily modified on the MB surface by only a simple chemical reaction and an external magnetic field cleaning operation. Existing studies have shown that MB can bind to DNA probes or antibody molecules and has been widely used in the determination of DNA and biological proteins. However, current MB-based electrochemical immunosensors provide signal amplificationThe vast majority of strategies rely on horseradish peroxidase (HRP) to catalyze hydrogen peroxide (H)₂O₂) And (3) reduction reaction of (2). However, although HRP is capable of catalyzing H with a variety of redox mediators₂O₂Reduction but H₂O₂The direct reduction potential of (A) is usually very close to those of these mediators, resulting in relatively poor signal-to-noise ratio of the enzyme-type sensor, which is very disadvantageous for highly sensitive measurement of antigens. Therefore, how to rapidly, accurately, highly sensitively and specifically measure the content of antigen in a complex biological sample remains a popular topic of attention.

Disclosure of Invention

The invention solves the technical problems of low detection sensitivity and accuracy and low specificity of the electrochemical immunosensor in the prior art. The invention prepares an electrochemical enzyme-linked immunosorbent assay probe, which is characterized in that an antigen is added into a biological conjugate of a magnetic bead and a capture antibody for incubation, after supernatant liquid is removed under the condition of an external magnetic field, nanogold, a detection antibody and a biological conjugate of enzyme are added for incubation, and the electrochemical enzyme-linked immunosorbent assay probe is obtained after double-antibody sandwich reaction. The dispersion liquid of the probe is modified on the surface of a carbon paste substrate electrode to obtain the electrochemical enzyme-linked immunosensor, and the sensor has lower detection limit and higher sensitivity when being used for antigen detection.

According to the first aspect of the invention, the preparation method of the electrochemical enzyme-linked immunosorbent probe is provided, which comprises the following steps:

(1) preparation of bioconjugates of magnetic beads and capture antibodies: incubating magnetic beads and capture antibodies, enabling the capture antibodies to be covalently connected with the magnetic beads, and then adding blocking protein, wherein the blocking protein is used for blocking groups which are not bonded by the capture antibodies on the surfaces of the magnetic beads to obtain biological conjugates of the magnetic beads and the capture antibodies;

(2) preparing a biological conjugate of nanogold, a detection antibody and an enzyme: adding a detection antibody and at least one enzyme into a nanogold solution, enabling the detection antibody and the enzyme to be covalently connected to the surface of the nanogold, and then adding a blocking protein, wherein the blocking protein is used for blocking exposed sites on the surface of the nanogold, so as to obtain a biological conjugate of the nanogold, the detection antibody and the enzyme;

(3) preparing an electrochemical enzyme-linked immunosorbent probe: adding an antigen to the bioconjugate of the magnetic beads and the capture antibody obtained in the step (1) for incubation, wherein the antigen can be specifically combined with the capture antibody and the detection antibody; and (3) removing the supernatant under the condition of an external magnetic field, adding the nanogold obtained in the step (2), the detection antibody and the biological conjugate of the enzyme for incubation, and performing double-antibody sandwich reaction to obtain the electrochemical enzyme-linked immunosorbent assay probe.

Preferably, the blocking protein in the steps (1) and (2) is bovine serum albumin; the enzyme is alkaline phosphatase, DT-diaphorase, horseradish catalase or glucose oxidase.

According to another aspect of the invention, the electrochemical enzyme-linked immunosorbent probe prepared by any one of the methods is provided.

According to another aspect of the invention, the invention provides a preparation method of an electrochemical enzyme-linked immunosensor, which comprises the steps of dropwise coating the dispersion liquid of the electrochemical enzyme-linked immunosensor on the surface of a substrate electrode, wherein the substrate electrode is a carbon paste electrode, and after the dispersion liquid is dried in the air, modifying the electrochemical enzyme-linked immunosensor on the surface of the substrate electrode to obtain the electrochemical enzyme-linked immunosensor.

Preferably, the substrate electrode is a carbon paste electrode modified by a zinc oxide graphene nano composite.

According to another aspect of the invention, the electrochemical enzyme-linked immunosensor prepared by the method is provided.

According to another aspect of the invention, the use of the electrochemical enzyme-linked immunosensor for the detection of antigens is provided.

Preferably, the electrochemical enzyme-linked immunosensor is placed in electrolyte containing a substrate for incubation, the substrate and enzyme in a probe on the surface of the sensor are subjected to enzymolysis reaction, an electrochemical signal of an enzymolysis product is detected, and the concentration of the antigen is calculated.

Preferably, the electrolyte contains a surfactant;

preferably, the surfactant is cetyltrimethylammonium bromide, sodium dodecyl sulphate or sodium dodecyl benzene sulphonate.

Preferably, the antigen is carcinoembryonic antigen, alpha-fetoprotein or prostate specific protein; the enzyme is alkaline phosphatase, and the substrate is phenyl phosphate;

preferably, the substrate is 1-naphthyl phosphate or phenyl diphosphate.

Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:

(1) the invention solves the problems of long and tedious detection steps, low detection flux, easy passivation of an electrode interface by protein and the like existing in the prior art (an immobilized electrode sensing interface). the linear range of the electrochemical enzyme-linked immunosensor (MB-eElisa) provided by the invention is 0.01-6.0ngm L^-1The detection limit is 4.0pg m L^-1(S/N ═ 3); compared with the existing electrochemical immunodetection method, the method has lower detection limit and higher sensitivity. In addition, the sensor is applied to the measurement of the antigen (CEA) in the serum of healthy people and tumor patients, and the test result has good accuracy after being compared with a standard detection method (a commercial human CEA kit).

(2) According to the electrochemical enzyme-linked immunosensor provided by the invention, Magnetic Beads (MB) are introduced into the system, so that the passivation of an electrode interface can be effectively avoided, and an immunodetection process can be simplified. In addition, the high binding property of the nanogold and the protein is utilized, so that the nanogold can be efficiently bound with a detection antibody and an enzyme.

(3) The invention preferably utilizes a high signal-to-noise ratio/renewable zinc oxide graphene nano composite modified carbon paste electrode (ZnO @ rGO/CPE) to construct a magnetic bead electrochemical enzyme-linked immunosensor (MB-eElisa) for signal transduction of alkaline phosphatase (A L P), and the specific preparation method comprises the steps of introducing Magnetic Beads (MB), alkaline phosphatase (A L P), a surfactant (CTAB), high signal-to-noise ratio/renewable (ZnO @ rGO/CPE) and the like when detecting antigen (CEA) in electrochemical immunity, and greatly improving the detection process and sensitivity of MB-eElisa.

(4) The enzyme in the electrochemical enzyme-linked immunosensor is preferably alkaline phosphatase (A L P), the enzyme can catalyze and hydrolyze 1-naphthyl phosphate (1-NPP) into 1-naphthol (1-NP) with electrochemical activity, a signal transduction system close to zero background is constructed, specifically, a carbon paste electrode with low background current is adopted to combine with a substrate 1-NPP with zero background, and then zinc oxide graphene nano compound and CTAB are introduced to further enhance the signal sensitivity of the enzymolysis product 1-NP, so that a higher signal-to-noise ratio is obtained.

Drawings

FIG. 1 is a schematic diagram of a A L P based MB-eElisa construction of a ZnO @ rGO/CPE sensing interface.

Figure 2 is XRD of ZnO @ rGO nanocomposite.

FIG. 3 is an SEM of ZnO @ rGO nanocomposites.

FIG. 4 is XPS of ZnO @ rGO nanocomposites.

FIG. 5 is a ZnO @ rGO nanocomposite Zn_2pXPS。

FIG. 6 is a differential pulse plot of ZnO @ rGO/GCE (a, b, c) and ZnO @ rGO/CPE (d, e, f) in the absence of (a, d) and in the presence of (b, c, e, f) 1-NP; wherein the curves (c, f) are for further addition of CTAB.

FIG. 7 is a differential pulse plot of 6 ZnO @ rGO/CPE modified electrodes in 1-NP.

FIG. 8 shows AuNPs and Ab₂UV-visible absorption spectrum of AuNPs-A L P.

FIG. 9 is a cyclic voltammogram of different electrodes in a potassium ferricyanide probe solution, where curve (a) CPE; curve (b) is ZnO @ rGO/CPE; curve (c) is Ab₁-MB-ZnO @ rGO/CPE, curve (d) is A L P-AuNPs-Ab₂-CEA-Ab₁-MB-ZnO@rGO/CPE。

FIG. 10 is a differential pulse plot of CEA measured by this MB-eElisa method with CPE (a, b) and ZnO @ rGO/CPE (c, d, e) in an electrolyte solution containing 1-NPP; wherein (b, d) is in the presence of CTAB, and (e) is in the absence of CEA.

FIG. 11 is a diagram showing the mechanism of electrochemical sensitization of CTAB to 1-NP.

FIG. 12 is MB-eElisa Ab for CEA detection₂-dose optimization plot of AuNPs-a L P.

FIG. 13 is a diagram showing the optimization of the time for the enzymatic reaction for detecting CEA in MB-eElisa.

FIG. 14 is a pH-optimized graph of electrolyte solutions for detection of CEA from MB-eElisa.

FIG. 15 is a graph of the optimization of the amount of ZnO @ rGO added to modified electrodes for CEA detection from MB-eElisa.

FIG. 16 is a graph of differential impulse response for different concentrations of CEA.

Figure 17 is a linear plot of the corresponding response signals for different concentrations of CEA.

FIG. 18 is a stability test chart of MB-eElisa.

FIG. 19 is a selectivity test chart of MB-eElisa; wherein, (a) is CEA, (b-i) is adding different interferents to CEA: insulin (b), prostate specific antigen (c), human serum albumin (d), human immunoglobulin (e), glucose (f), lactic acid (g), uric acid (h), and dopamine (i).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The construction method of the electrochemical enzyme-linked immunosensor MB-eElisa comprises the following steps:

(1) magnetic bead MB and capture antibody Ab₁Obtaining MB-Ab after incubation reaction₁The bioconjugate of (1).

(2) Alkaline phosphatase A L P and detection antibody Ab₂Adding into nano gold (AuNPs) solution to obtain Ab after incubation reaction₂-AuNPs-a L P bioconjugate.

(3) The bioconjugates obtained in (1) and (2) are bothAdding into a solution containing antigen CEA to obtain MB-Ab after double-antibody sandwich immunoreaction₁-CEA-Ab₂AuNPs-A L P bioprobe.

(4) And (4) modifying the biological probe obtained in the step (3) to a prepared zinc oxide graphene nano composite modified carbon paste ZnO @ rGO/CPE electrode, and airing at room temperature for later use.

(5) And (3) placing the immune probe electrode constructed in the step (4) in an electrolyte solution containing 1-naphthyl phosphate (1-NPP) and CTAB for incubation for a certain time to perform enzymolysis reaction, and recording an electrochemical signal of an enzymolysis product 1-naphthol (1-NP) by using a Differential Pulse Voltammetry (DPV), wherein the signal size can indirectly reflect the content of the CEA to be measured.

When the enzyme in the step (2) is DT-diaphorase, nitroso or nitro can be catalyzed to generate amino; when the enzyme in the step (2) is horseradish catalase, hydrogen peroxide can be catalyzed to reduce; when the enzyme in the step (2) is glucose oxidase, the glucose oxidation can be catalyzed.

Example 1

The preparation of the electrochemical enzyme-linked immunosensor and the method for the high-sensitivity electrochemical immunodetection of the alkaline phosphatase-labeled magnetic beads for the antigen have the following processes and steps:

(1) preparation of ZnO @ rGO/CPE

Preparing high-yield graphene oxide gel (GO, 1.0-100mg m L) based on an improved Kovttyukhova two-step oxidation stripping method^-1) Ultrasonically dispersing 0.1-5.0m L GO in 9.0m L water, and then adding 10-80mg Zn powder and 0.1-5.0m L NH₄Aqueous Cl solution (0.2g m L)^-1) Magnetically stirring at proper rotation speed for 1-60min, standing, reacting for 30min, centrifuging and washing black product with ultrapure water for at least 6 times, and drying in an electric oven at 60 deg.C for 2-9h to obtain final product (ZnO @ rGO).

Accurately weighing 0.05g ZnO @ rGO and 0.95g graphite powder, uniformly mixing in platinum grinding, adding 200 mu L paraffin oil, mechanically grinding for 30min to obtain a pasty mixture, pressing the pasty mixture into a cavity (CPE, the diameter is 3mm) at the bottom of a carbon paste electrode, and grinding the surface of the electrode on carbon paste paper to be smooth for later use (5 percent, ZnO @ rGO/CPE) except for the condition that the graphite powder is not usedThe preparation method of the naked CPE is similar to that of the ZnO @ rGO/CPE except that the ZnO @ rGO nano composite material is contained; for comparison, ZnO @ rGO modified glassy carbon electrode (ZnO @ rGO/GCE) is also used for detecting 1-NP of enzymolysis product of the system, and 50nm Al is firstly added before GCE modification₂O₃Grinding and polishing, ultrasonically washing with ethanol and secondary water, and finally washing with 5 μ L ZnO @ rGO (DMF, 1mg m L)^-1) And modifying the dispersion liquid to a clean GCE surface, and drying under an infrared lamp for later use.

(2) Preparation of MB-Ab₁

Collecting 1-40 μ L MBs (10mg m L)^-1) To a 1.5m L PE tube, washed at least 2 times with PBST wash solution with the aid of an externally applied magnetic field, and EDC (30 μ L, 50mg m L)^-1) And NHS (30 μ L, 50mg m L^-1) Activating at room temperature for 30min on a multifunctional shaking table, and further adding Ab₁(100μL,4μg mL^-1) After overnight incubation at room temperature, excess Ab₁PBST was added under an applied magnetic field and washed 3 times to remove, and finally BSA (100. mu. L, 1 wt%) was added to block the non-Ab on MBs₁The bound carboxyl groups, excess BSA were removed with PBST wash. Finally obtained MB-Ab₁The bioconjugate was redispersed in 100. mu. L0.01.01 MPBS (pH7.4) and placed in a refrigerator for use (4 ℃).

(3) Preparation of Ab₂-AuNPs-ALP

First, use 0.2M K₂CO₃The pH of a 1.0-2.0m L AuNPs (20nm) solution was adjusted to 8.2, and Ab was added continuously₂(150μL,4μg mL^-1) And A L P (150 μ L, 8 μ g m L)^-1) Incubating at room temperature for 2h, adding BSA (200 μ L, 1 wt%) to block other non-specific sites on the AuNPs surface, centrifuging the mixture with a refrigerated high-speed centrifuge, washing the obtained precipitate with PBST for at least 3 times by the above-mentioned low-temperature centrifugation method₂The AuNPs-A L P bioconjugate was redispersed in 200. mu. L1% BSA and placed in a refrigerator for future use (4 ℃).

(4) Electrochemical immunoassay for CEA

Add 10. mu. L CEA to 20. mu. L of MB-Ab prepared above₁Incubating the bioconjugate for 1h at room temperature; removing under the condition of an external magnetic fieldAfter the supernatant, 40. mu. L Ab was added₂after-AuNPs-A L P, incubating for 1h at room temperature to obtain MB-Ab₁-CEA-Ab₂The AuNPs-A L P bioprobe was washed 3 times with PBST and then redispersed in 10. mu. L0.01M PBS (pH7.4), 10. mu. L MB-Ab was taken₁-CEA-Ab₂Modifying the dispersion of-AuNPs-A L P on the surface of ZnO @ rGO/CPE, airing for later use at room temperature, placing the constructed immune probe electrode in 0.5mM 1-NPP and 6 mu M0.1M Tris-HCl electrolyte solution for incubation for 15min for enzymolysis reaction, and recording the electrochemical signal of hydrolysate 1-NP by Differential Pulse Voltammetry (DPV).

(5) Determination of actual samples

The actual serum samples (healthy and cancer patients) used in the experiment were provided by Tongji medical college-Sunghe Hospital, university of science and technology in Huazhong, and the serum samples were centrifuged at 4000rpm for 5min before collecting the supernatant for later use; before the serum sample is measured by the MB-eElisa method, the serum sample is diluted by 100 times by PBST, and other detection procedures are consistent with those of the standard CEA measurement. In addition, the commercial method-human CEA kit (RAB0411-1KT, Sigma-Aldrich) was used to further determine the CEA content in the serum samples to verify the accuracy of the method.

FIG. 1 shows that the MB-eElisa system adopts a magnetic bead-based strategy to greatly simplify the washing and incubation time of the antigen and the antibody, and in addition, the A L P enzymolysis reaction and the CTAB sensitization effect are adopted to greatly improve the detection sensitivity of CEA.

Figure 2 shows that the ZnO @ rGO nanocomposite has been successfully prepared from the characteristic diffraction peaks of XRD of ZnO and rGO.

Fig. 3 shows that the SEM image of the ZnO @ rGO nanocomposite shows a typical graphene wrinkle shape, and in addition, a plurality of rod-shaped ZnO nanoparticles are loaded on the surface of the ZnO @ rGO nanocomposite, and the unique morphology structure can provide a large specific surface area and rich active sites for electrochemical sensing applications.

FIG. 4 shows that XPS total spectrum confirms that ZnO @ rGO contains characteristic peaks of Zn, O and C.

Fig. 5 shows that XPS high resolution spectroscopy can further confirm that the rod-like material loaded on the rGO surface is ZnO nanoparticles.

FIG. 6 is a graph of differential pulse curves for ZnO @ rGO/GCE and 1-NP of ZnO @ rGO/CPE, where a and d are differential pulse curves without 1-NP, b and e are differential pulse curves with 1-NP, and c and f are differential pulse curves with 1-NP further into CTAB. in contrast to commercial glassy carbon modified electrode ZnO @ rGO/GCE, the system was constructed to have better sensitivity and signal-to-noise ratio for the determination of this A L P labeled enzymatic product 1-NP, and from the DPV response signal, the response current of ZnO @ rGO/CPE was 12.5 times that of ZnO rGO/GCE, while the background current was only 0.05 times that of ZnO @ rGO/GCE, and further, the introduction of CTAB can improve the signal-to-noise ratio of ZnO @ rGO/CPE, which adversely affects ZnO @ rGO/GCE.

FIG. 7 shows that the standard deviation of the response signals of multiple ZnO @ rGO/CPE is 0.8%, and the reproducibility is better than that of the commercial glassy carbon modified electrode.

FIG. 8 shows, Ab₂The ultraviolet visible absorption of AuNPs-A L P has red shift phenomenon compared with AuNPs, Ab is known from nano gold surface plasma resonance theory₂And A L P successfully adsorbed on AuNPs, Ab₂Successful construction of AuNPs-A L P bioconjugates.

FIG. 9 shows that the redox peak current and peak potential difference of CPE are significantly improved after ZnO @ rGO modification, indicating that ZnO @ rGO/CPE has better electron transfer capability; through MB-Ab₁Or A L P-AuNPs-Ab₂-CEA-Ab₁After MB modification, the redox peak current of the modified MB is remarkably reduced, and A L P-AuNPs-Ab₂-CEA-Ab₁The most severe decrease after MB modification, which indicates the successful construction of various immunobioprobes and MB-Ab₁With A L P-AuNPs-Ab₂An effective double-antibody sandwich immune reaction is generated between the two.

FIG. 10 shows that the sensitivity of the constructed A L P labeled MB-eElisa on ZnO @ rGO/CPE is superior to that of CPE, on the basis, the induction of CTAB and the sensitivity of ZnO @ rGO/CPE are remarkably improved, and in addition, under the same other immunoreaction conditions, only CEA is not contained, and MB-Ab is known₁With A L P-AuNPs-Ab₂No immune response occurred, and no recognition was found on curve eThe other response signal, i.e., the MB-eElisa constructed by the method, is close to zero background for the CEA determination.

FIG. 11 shows that the CTAB on the surface of the hydrophobic carbon paste electrode and the hydrophobic 1-NP phenolic substance have synergistic adsorption and enrichment effects, so that the sensitization effect of the sensing signal is generated, as can be seen from the mechanism diagram of the sensitization of the surfactant CTAB.

FIGS. 12 to 15 show the optimization of the CEA detection conditions of MB-eElisa; ab₂The dosage of-AuNPs-A L P is 40 mu L, the enzymolysis reaction time is 15min, the pH value of the electrolyte solution is 8.5, and the addition of ZnO @ rGO in the modified electrode is 5%.

FIGS. 16 to 17 show that the linearity of the MB-eElisa for CEA detection is in the range of 0.01 to 6.0ng m L^-1The detection limit is 4.0pg m L^-1(S/N ═ 3); compared with the existing electrochemical immunodetection method, the method has lower detection limit and higher sensitivity. In addition, the ZnO @ rGO/CPE of the sensing electrode can realize the updating and regeneration of a sensing interface through simple polishing treatment, and then the quick and low-cost determination of a large amount of CEA samples can be realized by combining with simple operation and high-flux MB-eElisa.

FIG. 18 shows that the response signals of the constructed MB-eElisa sensor, which is tested after being placed for 7 days and 14 days, are only reduced by 4.66% and 9.68% compared with the initial state, indicating that the sensor has good stability.

FIG. 19 shows that the potential interferents (insulin, prostate specific antigen, human serum albumin, human immunoglobulin, glucose, lactate, uric acid, dopamine) in human serum were selected by this work, and even in the presence of higher concentrations of interferents, the response signal of the MB-eElisa sensor to CEA was not substantially affected, indicating that it has good selectivity.

TABLE 1

^aRef, is to adopt commercial method-human CEA kit to determine CEA content in serum sample.

Table 1 shows that the MB-eElisa method and the commercial E L ISA kit method are used for measuring the content of CEA in serum samples of healthy people and cancer patients, wherein the serum samples of the healthy people (A, B), colon cancer (C), breast cancer (D), lung cancer (E), stomach cancer (F) and pancreatic cancer (G) are obtained from the MB-eElisa sensor, the standard addition method is used for measuring the content of CEA in the serum samples of the healthy people and the cancer patients, and the addition mark rate is between 96.4% and 109.7%, which indicates that the method has good accuracy.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The preparation method of the electrochemical enzyme-linked immunosensor is characterized by comprising the following steps:

(2) preparing a biological conjugate of nanogold, a detection antibody and an enzyme: adding a detection antibody and at least one enzyme into a nanogold solution, enabling the detection antibody and the enzyme to be covalently connected to the surface of the nanogold, and then adding a blocking protein, wherein the blocking protein is used for blocking exposed sites on the surface of the nanogold, so as to obtain a biological conjugate of the nanogold, the detection antibody and the enzyme; the enzyme is alkaline phosphatase;

(3) preparing an electrochemical enzyme-linked immunosorbent probe: adding an antigen to the bioconjugate of the magnetic beads and the capture antibody obtained in the step (1) for incubation, wherein the antigen can be specifically combined with the capture antibody and the detection antibody; removing supernatant under the condition of an external magnetic field, adding the nanogold obtained in the step (2), the detection antibody and the biological conjugate of the enzyme for incubation, and obtaining the electrochemical enzyme-linked immunosorbent assay probe after double-antibody sandwich reaction;

(4) and dropwise coating the dispersion of the electrochemical enzyme-linked immunosorbent assay probe on the surface of a substrate electrode, wherein the substrate electrode is a carbon paste electrode, and after the dispersion is dried in the air, modifying the electrochemical enzyme-linked immunosorbent assay probe on the surface of the substrate electrode to obtain the electrochemical enzyme-linked immunosensor.

2. The method for preparing an electrochemical ELISA sensor of claim 1 wherein the blocking protein in step (1) and step (2) is bovine serum albumin.

3. The method for preparing an electrochemical enzyme-linked immunosensor of claim 1, wherein the substrate electrode is a carbon paste electrode modified by zinc oxide graphene nano-composites.

4. An electrochemical enzyme-linked immunosensor prepared according to any one of claims 1-3.

5. Use of the electrochemical enzyme-linked immunosensor of claim 4 for detecting an antigen.

6. The use of claim 5, wherein the electrochemical enzyme-linked immunosensor is incubated in an electrolyte containing a substrate, the substrate is subjected to enzymatic hydrolysis reaction with enzymes in the probe on the sensor surface, an electrochemical signal of an enzymatic hydrolysis product is detected, and the concentration of the antigen is calculated.

7. The use of claim 6, wherein the electrolyte comprises a surfactant.

8. Use according to claim 7, wherein the surfactant is cetyltrimethylammonium bromide, sodium dodecyl sulphate or sodium dodecylbenzene sulphonate.

9. The use of claim 6, wherein the antigen is carcinoembryonic antigen, alpha fetoprotein or prostate specific protein; the substrate is phenyl phosphate.

10. The use of claim 9, wherein the substrate is 1-naphthyl phosphate or phenyl-diyl phosphate.