Electrochemical sensor for glycoprotein detection and glycoprotein detection method

Abstract

The invention discloses an electrochemical sensor for glycoprotein detection and a glycoprotein detection method. The method comprises the following steps: anchoring an aptamer-protein conjugate on the surface of a gold electrode, and then assembling an anti-fouling peptide around the aptamer-protein conjugate to form an imprinted self-assembled monolayer film. And removing the combined protein through an acid solution to form a biocompatible imprinting cavity capable of being used for recombining a target object. The imprinted cavity can eliminate non-specific adsorption and enhance targeted binding efficiency. The sensor can be used for directly detecting carcino-embryonic antigens with the concentration as low as 0.1 ng / mL through an electrochemical impedance spectroscopy method. Besides, homodimer concanavalin A (ConA) is used as a recognition element and a cross-linking agent, homodimer glucose oxidase (GOx) is assembled on the surface of the electrode in situ, a protein network can be formed, and enzyme catalysis signal amplification detection is achieved. Through in-situ formation of a ConA-GOx assembly, the sensitivity is improved by 100 times.

Description

Technical Field

[0001] This invention specifically relates to an electrochemical sensor and a method for glycoprotein detection, belonging to the field of electrochemical detection technology for glycoproteins. Background Technology

[0002] Glycoproteins serve as diagnostic biomarkers and therapeutic targets for various diseases, and their quantitative detection is crucial for disease prevention and treatment. Electrochemical biosensors, with their advantages of high sensitivity, rapid response, cost-effectiveness, and miniaturization, have become a new tool for protein detection. The core components of electrochemical biosensors include recognition elements and signal labeling groups. Traditional electrochemical biosensors typically employ biological receptors and enzymes as recognition elements and signal labeling groups. Among numerous protein recognition elements, antibodies are highly favored due to their high specificity, strong affinity, and multifunctionality. However, the high production cost, limited shelf life, weak stability, and stringent storage conditions of antibodies have forced researchers to seek next-generation recognition elements.

[0003] As a potential alternative to antibodies, aptamers possess the potential for specific target recognition, offering advantages such as simple preparation processes, ease of modification, high stability, and relatively low production costs. However, aptamers are prone to inactivation and degradation by nucleases during detection. Molecularly imprinted polymers, acting as "artificial antibodies," also exhibit high specificity for target recognition. Given the significant advantages of both aptamers and molecularly imprinted polymers, researchers have combined them to prepare dual-recognition elements, achieving a synergistic effect of "1+1>2" to enhance recognition performance. For example, aptamers can promote the fixation of template proteins in a preferred orientation, while imprinted polymers can restrict conformational changes of aptamers hidden within the imprint cavity and resist nuclease-induced degradation through steric hindrance. Therefore, the combination of aptamers and imprinted polymers significantly increases the binding efficiency within the imprint cavity, effectively reducing the non-specific adsorption of interfering substances in complex samples, thereby greatly improving the accuracy of analytical methods. Despite some encouraging progress in this field, biosensors based on aptamer / imprinted polymer dual-recognition elements for detecting protein biomarkers still face numerous challenges, such as complex polymerization reaction operations and demanding template removal conditions. These issues can affect the three-dimensional structure of the aptamer and template protein, leading to deformation or collapse of the imprinted cavity, thus limiting the recombination of the target protein in its native conformation. Furthermore, proteins and other biomolecules in the biological medium may adsorb onto the sensor surface through non-specific interactions, thereby reducing specificity, contaminating the device, or passivating the electrodes. In addition, sensitivity and detection efficiency are two key parameters for evaluating the performance of electrochemical biosensors, and the sensitivity and detection efficiency of existing sensors still need improvement. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for preparing an electrochemical sensor for glycoprotein detection and its application, aiming to solve at least one of the many problems of existing glycoprotein detection sensors, such as complicated preparation operation, harsh template removal conditions, poor detection specificity and low sensitivity.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides an electrochemical sensor for glycoprotein detection, comprising: One gold electrode; Several aptamers are attached to the gold electrode as imprinting carriers, and these aptamers can form conjugates with the target glycoprotein. Several antifouling peptides are attached to the gold electrode and surround the aptamer to form an imprinted cavity for capturing the target glycoprotein.

[0006] Secondly, the present invention also provides a method for preparing an electrochemical sensor for glycoprotein detection, comprising the following steps: (1) Prepare clean gold electrodes; (2) An aptamer-glycoprotein conjugate was prepared by mixing an aptamer solution and a glycoprotein solution of a certain concentration. (3) Place the gold electrode in the coupling solution and incubate for t1, so that the aptamers in the coupling are fixed on the gold surface by chemical bonds; wherein t1 is 10-15h, preferably 12h; (4) Add the antifouling peptide solution to the solution in step (3) and continue incubation for time t2, so that the antifouling peptide is also fixed on the gold surface by chemical bonds and the antifouling peptide surrounds the conjugate; wherein t2 is 10-15h, preferably 12h; (5) Add cysteine ​​solution to the solution in step (4) and continue incubation for t3, so that cysteine ​​is also fixed on the gold surface by chemical bonds to further seal the unreacted gold surface; wherein t3 is 0.5-2h, preferably 1h; (6) The electrode from step (5) is washed with dilute hydrochloric acid under slight ultrasonication to remove glycoproteins from the coupling material, resulting in an aptamer-mediated antifouling peptide-imprinted self-assembled monolayer electrode, referred to as the imprinted electrode. This imprinted electrode is an electrochemical sensor that can be used for glycoprotein detection.

[0007] As one specific embodiment, the sequence of the aptamer described in the first and second aspects is: 5'-HS-C3-ATACCAGCTTATTCAATT-3', and the aptamer is fixed to the gold surface by a gold-sulfur bond.

[0008] In one specific embodiment, the antifouling peptide described in the first and second aspects is a C-terminal amidated polypeptide with the amino acid sequence EKEKEKEPPPPC, which is immobilized on a gold surface via a gold-sulfur bond.

[0009] In one specific embodiment, in the first and second aspects, the glycoprotein is carcinoembryonic antigen (CEA).

[0010] Thirdly, the present invention also provides an electrochemical detection method for glycoprotein CEA, comprising the following steps: (1) Prepare the electrochemical sensor electrode (imprinted electrode) in the first or second aspect. (2) The imprinted electrode was incubated in a CEA standard solution of known concentration for a certain time t4; after removal, it was thoroughly rinsed with phosphate buffer. Using the imprinted electrode as the working electrode, a platinum wire as the auxiliary electrode, and a silver / silver chloride electrode as the reference electrode, the three electrodes were placed in a 5 mM [Fe(CN)6] solution containing 0.1 M potassium chloride. 3− / [Fe(CN)6] 4− In a 1:1 solution, the other ends of the three electrodes are connected to an electrochemical workstation for EIS measurement. After the measurement is completed, the imprinted electrodes are washed with 10 mM hydrochloric acid under slight ultrasonic action to release the captured CEA on the imprinted surface and regenerate the imprinted electrodes. The t4 is 20-40 min, preferably 30 min. (3) Change the concentration of the CEA standard solution and repeat step (2) at least three times to establish R. et - [CEA] standard curve and obtain the fitting equation; (4) Using the CEA solution to be tested, repeat the EIS measurement in step (2), and calculate the R value based on the measured value. et代 The concentration of the CEA solution to be tested is obtained by fitting the equation.

[0011] Fourthly, the present invention also provides a method for amplifying and detecting the enzyme catalytic signal of glycoprotein CEA, comprising the following steps: (1) Prepare the electrochemical sensor (imprinted electrode) in the first or second aspect. (2) The imprinted electrode was incubated in a CEA standard solution of known concentration for a certain time t4; then the electrode was incubated in a 0.1 mg / mL ConA solution for a certain time t5, and then 0.1 mg / mL GOx was added to the solution and incubated for a certain time t6; after being taken out and thoroughly rinsed with ultrapure water, the imprinted electrode was used as the working electrode, a platinum wire as the auxiliary electrode, and a silver / silver chloride electrode as the reference electrode. The three electrodes were placed in a phosphate buffer containing 50 μM ferrocene methanol and 5 mM glucose for linear sweep voltammetry (LSV) measurement; after the measurement was completed, the imprinted electrode was washed with 10 mM hydrochloric acid under slight sonication to release the captured CEA on the imprint surface and regenerate the imprinted electrode; t4 was 20-40 min, preferably 30 min; t5 was 5-15 min, preferably 10 min; t6 was 20-40 min, preferably 30 min; (3) Change the concentration of the CEA standard solution and repeat step (2) at least three times to establish I. pa - [CEA] standard curve and obtain the fitting equation; (4) Using the CEA solution to be tested, repeat the LSV measurement in step (2), and calculate the peak anode current I. pa代 The concentration of the CEA solution to be tested is obtained by fitting the equation.

[0012] Beneficial Effects: This invention provides an electrochemical sensor based on aptamer-assisted surface-imprinted self-assembled monolayer membrane of antifouling peptide, its preparation method, and detection applications. It can be used for the identification and detection of protein biomarkers such as CEA. In preparation, an aptamer-protein complex is first pre-immobilized on a gold surface via gold-sulfur bonds. Then, an antifouling peptide containing polyproline is immobilized around the complex. After removing the template protein using a weakly acidic solution, a highly specific imprinted cavity is efficiently formed on the gold surface. This imprinted cavity can specifically capture target proteins such as CEA, allowing direct detection of carcinoembryonic antigen concentrations as low as 0.1 ng / mL using electrochemical impedance spectroscopy. This method utilizes a surface-imprinted self-assembled monolayer membrane, a novel material formed by the co-assembly of protein template molecules and organic monomers on a substrate surface. This material holds promise as a replacement for traditional imprinted polymers used in protein detection. To enhance sensor performance, this invention employs a self-assembly strategy, assembling disordered GOx / ConA structures in situ on the sensor surface to form an ordered superstructure polymer. Specifically, it uses concanavalin A (ConA) as a recognition element and cross-linking agent to induce glucose oxidase (GOx) to assemble in situ on the electrode surface that captures the glycoprotein CEA. This in-situ formation of ConA-GOx assemblies increases sensitivity by 100 times, achieving high-precision, high-efficiency, high-sensitivity, and high-selectivity signal amplification and detection of the target glycoprotein CEA. The proposed surface-imprinted self-assembled monolayer membrane strategy opens new avenues for designing aptamer / molecular imprinted sensors, enabling dual recognition and precise detection of biomarkers. This provides crucial information for the preparation of imprinted materials and the development of innovative biosensor platforms. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of an impedance sensor based on a surface-imprinted self-assembled monolayer film.

[0014] Figure 2 (A) EIS curves of the aptamer-CEA modified gold electrode before (curve 1) and after (curve 2) modification with antifouling peptide, followed by washing (curve 3) and CEA rebinding (curve 4) EIS curves. (B) Impedance changes after eight washing / binding cycles.

[0015] Figure 3 (A) EIS curves of CEA at different concentrations detected by impedance method. (B) Relationship between Ret and CEA concentration.

[0016] Figure 4(A) Schematic diagram of target analyte detection via GOx signal amplification. (B) Cyclic voltammetry: CEA-binding electrode before ConA treatment (curve 1) and after treatment (curve 2); CEA-binding electrode after GOx treatment (curve 3); CEA-binding electrode after sequential ConA and GOx treatment (curve 4); CEA-binding electrode after ConA / GOx mixed treatment (curve 5). (C) Electrochemical impedance spectroscopy: Conditions for curves 1-5 are the same as in Figure B.

[0017] Figure 5 (A) LSV curves for different concentrations of CEA. (B) I pa Relationship with CEA concentration. The inset shows the linear portion of the fitted curve.

[0018] Figure 6 Selectivity comparison chart of CEA detection by signal amplification sensor: The concentration of CEA is 250 pg / mL, and the concentration of other proteins is 10 ng / mL. The protein samples are dissolved and diluted with 50 mM phosphate buffer (pH 7.4). Detailed Implementation

[0019] The present invention will now be described in a clear and complete manner with reference to the accompanying drawings and embodiments.

[0020] Example 1 A schematic diagram illustrating the fabrication principle of imprinted electrodes is shown below. Figure 1 As shown, aptamers are used as imprinting carriers for template proteins to enhance the recognition ability of the imprinted cavity. The imprinting of the template protein is achieved in a one-step process: first, a thiolized aptamer-protein complex is adsorbed onto a gold surface; then, an antifouling peptide is assembled through gold-sulfur interactions, forming a hybrid self-assembled monolayer with an ordered structure and high surface density. The template protein can then be easily removed with an acidic solution. This process promotes the formation of the imprinted cavity without removing the fixed aptamer and antifouling peptide; the biocompatible imprinted cavity and the antifouling peptide monolayer provide multiple binding sites, exhibiting excellent specific capture capability.

[0021] Fabrication of imprinted gold electrodes An aptamer-CEA conjugate was prepared by mixing 5 μM aptamer with 1 mg / mL CEA. To form an imprinted self-assembled monolayer, a clean 2 mm disc gold electrode was pre-incubated in 20 μL of the aptamer-CEA conjugate for 12 hours. Then, 10 μL of 50 μM antifouling peptide (amino acid sequence EKEKEKEKPPPPC) was added to the solution to block the unoccupied gold surface around the aptamer-CEA conjugate. After 12 hours of incubation, 10 μL of 500 μM cysteine ​​solution was added to the mixture to further block the unreacted gold surface and eliminate the non-specific adsorption of small thiol molecules in the complex biological matrix. After another 2 hours of incubation, the electrode was washed with 10 mM hydrochloric acid under gentle sonication to remove the CEA template, thus forming an aptamer-mediated antifouling peptide imprinted self-assembled monolayer electrode, which is the electrochemical sensor electrode for glycoprotein detection.

[0022] Example 2: Detection of CEA To facilitate impedance-based detection of CEA, the prepared aptamer / antifouling peptide-imprinted electrode was incubated with a specific concentration of CEA for 30 minutes. After thorough rinsing with phosphate buffer, the imprinted electrode was placed in a 5 mM solution of 0.1 M potassium chloride [Fe(CN)6]. 3− / [Fe(CN)6] 4− EIS measurements were performed in a (1:1) solution using a CHI 660E electrochemical workstation. In the electrochemical apparatus, the imprinted electrode served as the working electrode, a platinum wire as the auxiliary electrode, and a silver / silver chloride electrode as the reference electrode. After the test, the electrode was washed with 10 mM hydrochloric acid under gentle ultrasonication to release the captured CEA on the imprinted surface, thereby promoting the regeneration of the sensing imprinted electrode.

[0023] Feasibility analysis of impedance method detection results Monitoring [Fe(CN)6] using electrochemical impedance spectroscopy (EIS) 3− / 4− The electron transfer at the electrode interface was investigated to study the template removal and target analyte rebinding capabilities. For example... Figure 2 As shown in Figure A, the impedance changes of the gold electrode modified with the aptamer-CEA complex before and after assembly of the antifouling peptide were analyzed (curves 1 and 2). The Ret increased after assembly of the antifouling peptide, indicating that the antifouling peptide can restrict [Fe(CN)6]. 3− / 4− Electron transfer occurred. Ret decreased after the template CEA was removed from the self-assembled membrane by acid washing (curve 3). When the imprinted electrode was co-incubated with the CEA solution (curve 4), Ret increased, indicating that the rebinding of the target on the imprinted cavity hindered [Fe(CN)6]. 3− / 4−Electron transfer. The electrostatic and hydrogen bonding interactions between the aptamer and the protein are pH-dependent. Studies have shown that proteins captured by the aptamer on the sensor surface can be eluted with acidic or alkaline solutions. This study found that washing the electrode with 10 mM hydrochloric acid effectively removed the captured CEA, leading to regeneration of the blot surface. No significant change in Ret was observed after 8 regeneration / detection cycles. Figure 2 B). Therefore, a single sensing electrode can detect multiple samples, significantly improving detection throughput and reducing analysis time and cost.

[0024] Sensitivity of impedance method for detecting CEA like Figure 3 As shown, to investigate the sensitivity of the biosensor prepared in Example 1, different concentrations of CEA were measured using the biosensor. The Ret value gradually increased with increasing CEA concentration, indicating that the biosensor can be used for the quantitative detection of CEA. Figure 3 A). Within the concentration range of 0.1–2.5 ng / mL, the linear equation is Ret = 20884 + 5414[CEA] (ng / mL) Figure 3 (B) The lowest detectable concentration of this method is within the normal range of CEA concentration in the blood of healthy adults (0–2.5 ng / mL). Therefore, this imprinted biosensor shows great potential for CEA detection in real-world samples.

[0025] Example 3: CEA signal amplification and detection technology Although label-free detection of targets can be achieved through impedance changes induced by CEA, its sensitivity is still lower than other electrochemical sensing methods. To meet the needs of quantitative detection of low-abundance analytes, the detection sensitivity can be improved through signal amplification by enzymes, nanomaterials, and supramolecular technologies. CEA is a glycoprotein that can bind to various recognition elements, including antibodies, aptamers, lectins, and borate materials. Concanavalin A (ConA) is a tetrameric lectin protein that can bind to mannose / glucose units and can trigger the assembly of dimer and tetrameric glycoproteins. Glucose oxidase (GOx) is a homodimeric protein that can be used as a signal marker for various biosensors. This study found that CEA captured by the electrode can be recognized by ConA through lectin-glucose interactions, thereby achieving the capture of homodimeric GOx. Figure 4 A) The exposed glycosyl chains of GOx can capture free ConA through lectin-glycan interactions. The surface-attached ConA can then recruit more GOx molecules through the same interaction, thereby promoting the in-situ formation of ConA / GOx assemblies on the electrode surface and achieving high-precision, high-efficiency, high-sensitivity, and high-selectivity signal amplification and detection of the target protein.

[0026] Enzyme-catalyzed signal amplification detection method: First, the imprinted electrode prepared in Example 1 was incubated in a CEA solution of a specific concentration for 30 min to allow CEA to bind to the imprinted electrode; then, the electrode was incubated in 10 μL of 0.1 mg / mL ConA solution for 10 min, followed by the addition of 20 μL of 0.1 mg / mL GOx to the solution for another 30 min; after thorough rinsing with ultrapure water, the imprinted electrode was used as the working electrode, a platinum wire as the auxiliary electrode, and a silver / silver chloride electrode as the reference electrode. The three electrodes were placed in a phosphate buffer solution containing 50 μM ferrocene methanol and 5 mM glucose. The other ends of the three electrodes were connected to an electrochemical workstation for linear sweep voltammetry (LSV) measurement.

[0027] Using ferrocene methanol (Fc) as a redox medium, the bound GOx can be determined by the electrocatalytic oxidation of glucose. For example... Figure 4 As shown in Figure B, the CEA-bound electrode (curve 1) and the CEA-bound electrode treated with ConA or GOx (curves 2 and 3) exhibit reversible redox peaks, attributed to the redox reaction of ferrocene methanol. When the electrode corresponding to curve 2 is co-incubated with GOx, its anodic current increases significantly while its cathodic current decreases (curve 4), which is a typical enzyme-co-electrocatalytic redox peak in the ferrocene / glucose system. When the CEA-bound electrode is incubated with a ConA / GOx mixture (curve 5), the anodic current is higher than that of a single ConA-GOx conjugate (curve 4), indicating that the electrochemical signal is amplified by multiple GOx molecules. Further verification of the above amperometric results was performed using EIS. Figure 4 As shown in C, the adhesion of ConA to the CEA binding electrode leads to R et The impedance value increased (curves 1 and 2), while direct incubation of the CEA-bound electrode with GOx did not cause a significant change in impedance (curve 3). This indicates that GOx capture depends on the crosslinking agent ConA. Subsequent capture of GOx on the ConA / CEA-bound electrode (curve 4) led to an increase in R... et The value increases. Notably, when the CEA-bound electrode is incubated with the ConA / GOx mixture (curve 5), R... et The value change was significantly higher than that of incubation with ConA and GOx sequentially (curve 4), indicating that the ConA / GOx assembly is effective for [Fe(CN)6]. 3− / 4− The electron transfer inhibition effect is better than that of a single ConA-GOx conjugate.

[0028] Sensitivity of signal amplification method The sensitivity of the signal amplification method was evaluated by measuring different concentrations of CEA. Figure 5As shown in Figure A, increasing CEA concentration leads to a gradual increase in anolyte current, indicating that higher CEA concentrations can promote the capture of more GOx molecules. Based on the relationship between anolyte current (Ipa) and CEA concentration, a good linear fit curve was obtained in the range of 1–250 pg / mL. Figure 5 B). The linear equation is I pa = 0.25 + 0.01 [CEA] (pg / mL). The lowest detectable concentration is lower than that of the previous impedance method, and this value is comparable to or even lower than that of other electrochemical biosensors that employ enzymes and nanomaterials for signal amplification. The high sensitivity can be attributed to the in-situ formed ConA / GOx complex having multiple catalytic sites. In addition, the signal amplification method in this study is simple to operate and does not require the use of modified enzymes and nanomaterials.

[0029] Selectivity of signal amplification To evaluate the selectivity of this signal amplification method, we analyzed other protein biomarkers, including prostate-specific antigen (PSA), alpha-fetoprotein (AFP), antigen 125 (CA125), and human serum albumin (HSA). Figure 6 As shown, none of the tested proteins caused significant changes in the anolyte current (columns 1-5), indicating that the imprinted electrode has excellent specificity for CEA detection. The coexistence of other proteins did not affect CEA determination (column 6). Therefore, the imprinted self-assembled monolayer membrane with dual recognition elements exhibits excellent selectivity and anti-interference properties in target detection. Furthermore, considering the high sensitivity of this method, multiple dilutions of real samples are expected to effectively reduce potential interference from other biological matrices.

[0030] In summary, this patent develops an electrochemical sensor based on a dual-recognition strategy using an aptamer / imprinted self-assembled monolayer membrane for the electrochemical detection of the biomarker glycoprotein. This patent's dual-recognition element, based on aptamers and molecularly imprinted polymers, enhances target binding efficiency, avoids non-specific adsorption, and improves analytical accuracy. The surface-imprinted self-assembled monolayer membrane, formed by the co-assembly of template molecules and organic monomers on the substrate surface, holds promise as a replacement for traditional imprinted polymers. Antifouling peptides play a crucial role in forming biocompatible cavities, enhancing target binding and eliminating non-specific adsorption. Compared to previous aptamer / molecularly imprinted polymer dual-recognition systems, the antifouling peptide-imprinted self-assembled monolayer membrane offers several significant advantages, such as avoiding the use of organic solvents for polymerization, providing mild template removal conditions, and exhibiting excellent mass transfer performance of proteins within and outside the imprinted cavity. Furthermore, concanavalin A can simultaneously serve as a target recognition element and a cross-linking agent, triggering the in-situ assembly of glucose oxidase (GOx) on the electrode surface, with the ConA / GOx complex acting as a signal marker to amplify the signal. This simple signal amplification strategy improves detection sensitivity by 100-fold. This study provides important support for the preparation of imprinted materials and the development of novel biosensing platforms.

Claims

1. An electrochemical sensor for glycoprotein detection, characterized in that, include Gold electrode; An aptamer, attached to a gold electrode, can form a conjugate with a glycoprotein; Antifouling peptides attach to the gold electrode and surround the aptamer to form an imprinted cavity for capturing glycoproteins.

2. The electrochemical sensor for glycoprotein detection according to claim 1, characterized in that, The sequence of the aptamer is: 5'-HS-C3-ATACCAGCTTATTCAATT-3'.

3. The electrochemical sensor for glycoprotein detection according to claim 1, characterized in that, The sequence of the antifouling peptide is: EKEKEKEPPPPC.

4. The electrochemical sensor for glycoprotein detection according to claim 1, characterized in that, The glycoprotein is carcinoembryonic antigen (CEA).

5. A method for amplifying and detecting the enzyme catalytic signal of carcinoembryonic antigen (CEA), characterized in that, Includes the following steps: (1) Prepare the electrochemical sensor electrode as described in claim 4; (2) The sensor electrode was placed in a CEA standard solution of known concentration and incubated for a certain time t4; then the electrode was placed in a ConA solution and incubated for a certain time t5, and then GOx solution was added to the solution and incubated for a certain time t6; after being taken out and thoroughly rinsed with ultrapure water, the sensor electrode was used as the working electrode, the platinum wire as the auxiliary electrode, and the silver / silver chloride electrode as the reference electrode. The three electrodes were placed in a phosphate buffer containing ferrocene methanol and glucose and linear sweep voltammetry (LSV) was performed; after the measurement was completed, the sensor electrode was regenerated. (3) Change the concentration of the CEA standard solution and repeat step (2) at least three times to establish I. pa - [CEA] standard curve and obtain the fitting equation; (4) Using the CEA solution to be tested, repeat the LSV measurement in step (2), and calculate the peak anode current I. pa Substitute the values ​​into the fitting equation to obtain the concentration of the CEA solution to be tested.

6. The method for amplifying and detecting the enzyme catalytic signal of carcinoembryonic antigen (CEA) according to claim 5, characterized in that, The concentration of the ConA solution in step (2) is 0.1 mg / mL, and the concentration of the GOx solution is 0.1 mg / mL.

7. The method for amplifying and detecting the enzyme catalytic signal of carcinoembryonic antigen (CEA) according to claim 5, characterized in that, In step (2), the concentration of ferrocene methanol in the phosphate buffer is 50 μM and the concentration of glucose is 5 mM.

8. The method for amplifying and detecting the enzyme catalytic signal of carcinoembryonic antigen (CEA) according to claim 5, characterized in that, In step (2), t4 is 20-40 min.

9. The method for amplifying and detecting the enzyme catalytic signal of carcinoembryonic antigen (CEA) according to claim 5, characterized in that, In step (2), t5 is 5-15 min and t6 is 20-40 min.

10. The method for amplifying and detecting the enzyme catalytic signal of carcinoembryonic antigen (CEA) according to claim 5, characterized in that, The regeneration of the sensor electrode in step (2) is achieved by washing the sensor electrode with 10 mM hydrochloric acid to release the CEA it has captured.

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