Electrochemical immunosensor for amplifying detection signal by utilizing deposition reaction of nano-enzymatic electroactive molecule ferrocene-tyramine as well as preparation method and application of electrochemical immunosensor

Abstract

The invention belongs to the technical field of biosensors, and relates to an electrochemical immunosensor for amplifying a detection signal by utilizing a nano-enzymatic electroactive molecule ferrocene-tyramine deposition reaction as well as a preparation method and application of the electrochemical immunosensor. According to the invention, an ultra-sensitive split type biosensor based on 96-well plate and nano-enzyme catalytic electroactive molecule ferrocene-tyramine deposition signal amplification is used, the electroactive molecule ferrocene-tyramine is used as a signal amplification element and is labeled on a signal antibody, and a target antigen is detected through a sandwich immune reaction among a capture antibody probe, a target antigen and the signal antibody. The significant amplification of a current detection signal and the sensitive detection of a target antigen are realized. The sensor and ELISA (enzyme-linked immunosorbent assay) are simultaneously used for detecting the SMP30 antibody level in the serum of a clinical liver cancer patient, the result shows consistency, the recovery rate is 93-104%, and the sensor has great popularization and application value for portable and rapid detection of tumor markers.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors and relates to an electrochemical immunosensor which utilizes nano-enzyme-catalyzed electroactive molecule ferrocene-tyramine deposition reaction to amplify detection signals, as well as a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the greatest threats to human health, with a mortality rate nearly equal to its morbidity. The incidence of HCC is increasing annually due to numerous risk factors, insidious early symptoms, and poor prognosis. Therefore, the development of sensitive, rapid, and specific new HCC biomarkers and detection tools is crucial for the early detection and diagnosis of HCC. Senescence marker protein-30 (SMP30), also known as calcineurin, is primarily expressed in normal human hepatocytes and renal tubular epithelial cells. However, SMP30 expression is lower in HCC tissue than in adjacent HCC tissue. Although traditional tumor markers are directly produced by cancer cells, tumors can induce an immune response, producing autoantibodies against tumor-associated antigens. The expression level of these antibodies is closely correlated with tumor progression, making them useful as diagnostic markers even when the concentration of tumor-associated antigens is low. Studies have shown that SMP30 antibodies are significantly expressed in early-stage HCCs that are negative for AFP. Therefore, SMP30 antibodies are considered an attractive new target for HCC and a reliable tumor marker for tracking and diagnosing early-stage HCC.

[0003] Nanozymes are artificial enzymes with nanostructured dimensions and are prepared from single-component nanomaterials. They exhibit catalytic performance similar to natural enzymes and unique stability, while also being able to modify their surface structure according to the conditions of use. Over the past few decades, various types of nanozymes have been designed. Based on their composition and catalytic activity mechanisms, they are mainly divided into metal particle nanozymes, metal compound nanozymes, non-metal nanozymes, and other types: (i) Nanozymes composed of metal nanoparticles such as gold, silver, platinum, and palladium exhibit excellent catalytic performance in heterogeneous reactions and, without any modification, exhibit ultra-high peroxidase-like activity. Their catalytic process is related to pH, the amount of substrate in the reaction system, and the degree of activation. (ii) Metal compound nanozymes, including bimetallic particle alloys and complexes of metals and other nanomaterials, exhibit ultra-high catalytic performance and multi-enzyme activity through the spatial synergistic effects of multiple nanomaterials during catalysis. (iii) Non-metal nanozymes. Some carbon-based nanomaterials, such as graphene, fullerenes, and quantum dots, are considered a new direction in nanozyme research due to their unique enzyme-mimicking catalytic capabilities. Its catalytic ability is mainly due to the internal electron density and electron transfer efficiency or band gap effect of the nanomaterial. (iv) In addition, there is a study that has constructed a nanozyme with nanochannels through etching methods, and its catalytic redox ability has been improved many times. At present, more and more nanozymes with excellent catalytic performance are used to develop biosensors for detecting tumor markers. In order to meet the requirements of clinical tumor marker diagnosis, the specificity and sensitivity of nanozymes in the design of tumor marker sensing analysis are also new areas that need to be explored in the future. In addition, the potential utilization value of nanomaterials in the construction of functional biosensors still needs to be explored.

[0004] Biosensors for detecting tumor markers are widely used in molecular diagnostics due to their reliable results and rapid analysis speed. However, most tumor markers are inert protein molecules, making them difficult to detect directly during the assay process. Enzymes are often labeled on recognition probes as exogenous signal molecules for enzyme-catalyzed signal amplification, playing a key role in sandwich biosensor systems. Ferrocene (Fc), an organic transition metal compound composed of two cyclopentadienyl rings connected to a central iron atom, exhibits reversible redox states and high electrochemical activity, making it an ideal electrochemical signal molecule. Therefore, to further improve sensor performance for analyzing target molecules, electroactive molecules based on Fc and its derivatives are widely used in the construction of electron transfer mediator biosensors. In recent years, numerous biosensors for ultrasensitive trace analysis based on enzyme-catalyzed signal amplification have been reported. Tyramide signal amplification (TSA) is an early technology for enhancing fluorescence immunohistochemical analysis through in situ amplification. At present, its in situ amplification characteristics are widely used in sensor strategies. In its reaction principle, the enzyme catalyzes tyramine to form a covalent binding site in the presence of H2O2. The newly added tyramine and its complex can stably bind to it in a short period of time, promoting the geometric amplification of the original signal.

[0005] There is currently no report on the synthesis of a new ferrocene-based electroactive substance, ferrocenetyroxide, as an electroactive probe to develop a biosensor analysis platform for the specific detection of the liver cancer marker GPC3. Summary of the Invention

[0006] The present invention addresses the above-mentioned issues by providing an electrochemical immunosensor, a method for preparing and applying the nanoenzyme-catalyzed electroactive ferrocene-tyramide deposition reaction to amplify detection signals. Using a mouse monoclonal antibody as the analyte, a split-type biosensor model for detecting the liver cancer tumor marker SMP30 was proposed based on independent immunoreactions in a 96-well plate and the nanoenzyme GO-Hemin-catalyzed ferrocene-tyramide deposition reaction. The feasibility of the split-type biosensor was explored, and the sensor model was subsequently used for quantitative analysis of SMP30 antibodies. In the presence of the target substance, the nanoenzyme's efficient, stable, and rapid enzymatic activity synergistically interacted with the excellent redox properties of ferrocene-tyramide, resulting in the deposition of a large amount of the electroactive substance ferrocene-tyramide on the electrode surface, significantly altering the current signal output. The split-type biosensor's specific and sensitive analytical performance enables rapid detection of SMP30 antibodies and also provides new insights into the sensing and analysis of other tumor markers.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is: An electrochemical immunosensor that utilizes nanoenzyme-catalyzed electroactive molecule ferrocene-tyramine deposition reaction to amplify detection signals. The electrochemical immunosensor is an ultra-sensitive split biosensor based on a 96-well plate and nanoenzyme-catalyzed electroactive molecule ferrocene-tyramine deposition signal amplification. The electroactive molecule ferrocene-tyramine is used as a signal amplification element and labeled with a signal antibody. Through a sandwich immune reaction between a capture antibody probe, a target antigen, and the signal antibody, significant amplification of the current detection signal and sensitive detection of the target antigen are achieved.

[0008] The present invention also provides a method for preparing the electrochemical immunosensor described above, which specifically comprises the following steps: (1) Preparation of immune complex GO / Hemin-Ab2: S1. Dissolve carboxylated graphene oxide in ultrapure water, disperse by ultrasonication, centrifuge, and take the supernatant. S2, then dissolving hemin in dimethylformamide, mixing with the supernatant obtained in step S1, ultrasonically treating, separating the precipitate and the supernatant by centrifugation, washing twice with PBS buffer, and drying in an oven to obtain GO / Hemin, then dispersing the obtained hemin-functionalized GO / Hemin in ultrapure water, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) after ultrasonication, stirring at room temperature, and then adding polyclonal antibody, reacting, and then centrifuging to separate the supernatant and precipitate, washing with PBS, and finally drying in a vacuum dryer and resuspending in PBS solution to prepare the final immune complex GO / Hemin-Ab2 for later use; (2) Preparation of GE / Au NPs / MPA / Tyr modified gold electrode: 1) Polish the gold electrode with alumina slurry, ultrasonically clean the cleaned gold electrode in anhydrous ethanol, then ultrasonically clean it in ultrapure water, and repeat this alternating ultrasonication three times; 2) Electrochemically clean the three-electrode system simultaneously in 0.5 M H2SO4 solution, perform electrochemical cyclic voltammetry (CV) slow scan, then charge and discharge the cell. Connect the cell, perform CV fast scan, and finally perform CV slow scan again. Repeat the above steps twice, and wash with ddH2O three times. 3) GE was immersed in HAuCl4 solution and Au NPs were electrodeposited to form Au NPs-modified GE / Au NPs. GE / AuNPs were incubated in 3-mercaptopropionic acid overnight to obtain GE / Au NPs / MPA. EDC-NHS solution was dropped onto the surface of GE / Au NPs / MPA and incubated at room temperature. Tyr solution was then added dropwise to the surface of GE / Au NPs / MPA and incubated at room temperature. Tyramine was fixed on the electrode surface by the condensation reaction between carboxyl and amino groups. After rinsing with water, an Au NPs / MPA / Tyr-modified gold electrode (GE / AuNPs / MPA / Tyr) was obtained. (3) Incubate the target Ag at room temperature to allow Ab1 to react with Ag in a specific immune manner; then add the GO-Hemin-Ab2 prepared in step (1) and incubate for 1 hour; then add 14 μL of 1 mg.mL -1 Fc-Tyr, 14 μL 1 mg / mL -1 A mixed solution of Tyr and 14 μL 50 mM H2O2 was continuously added dropwise, and the GE / Au NPs / MPA / Tyr obtained in step (2) was also added. The reaction was continued at 37°C for 30 minutes to obtain the electrochemical immunosensor.

[0009] It is further explained that in step S1, the ratio of the carboxylated graphene oxide to ultrapure water is 1 mg:1 mL, ultrasonic dispersion is performed for more than 3 hours, and centrifugation is performed at 3000 rpm for 30 minutes.

[0010] Further, in step S2, the amount of hemin used is 26.076 mg, the amount of dimethylformamide used is 4 mL, the ultrasonic treatment is performed at 130 kHz for 3 hours at room temperature, and then the precipitate and the supernatant are separated by centrifugation at 13000 rpm for 30 minutes; the ratio of GO / Hemin to ultrapure water is 1 mg:mL; the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 500 μL, stirred at room temperature for 4 hours, and then 30 μL of a polyclonal antibody with a concentration of 1 mg / mL is added. The entire mixture system is shaken at 4°C for 24 hours, and then centrifuged at 4°C and 3000 rpm for 30 minutes to separate the supernatant and precipitate, washed twice with PBS, and finally dried in a vacuum dryer for 12 hours and resuspended in 2 mL of 0.01 M PBS solution to prepare the final immune complex GO / Hemin-Ab2, which is stored at 4°C for later use.

[0011] It is further explained that in step 1), the fineness of the aluminum oxide slurry is 0.3 μm and / or 0.05 μm; and the ultrasonic cleaning is performed for 5 minutes.

[0012] Further explanation: in step 2), the three-electrode system includes a gold electrode, a platinum wire counter electrode, and an Ag / AgCl reference electrode.

[0013] Further explanation, in step 2), the scanning range of the electrochemical cyclic voltammetry slow scan is -0.25~1.5V, and the scanning rate is 100mV / s; the voltage of the electrolytic cell is 2V, 5s and -0.25V, 10s; the scanning range of the CV fast scan is -0.25~1.5V, and the scanning rate is 400mV / s.

[0014] Further explanation, in step 3), the specific operation is to immerse the pretreated graphene GE in HAuCl4 solution, electrodeposit Au NPs at a potential of -0.5 V for 120s to form Au NPs-modified GE / Au NPs, and rinse the electrode with water three times. The GE / Au NPs are incubated in 300 μL of 10 mM 3-mercaptopropionic acid at 4 ° C overnight to obtain GE / Au NPs / MPA. 40 μL of 10 mM EDC-NHS solution is dropped on the surface of GE / Au NPs / MPA and incubated at room temperature for 40 min, and then washed with water. Next, 40 μL of 1 mg.mL -1 Tyr solution was added dropwise to the GE / Au NPs / MPA surface and incubated at room temperature for 60 min. Tyramine was fixed on the electrode surface by the condensation reaction between carboxyl and amino groups. After rinsing with water, a GE / Au NPs / MPA / Tyr-modified gold electrode was obtained.

[0015] Further explanation: in step (3), the target Ag is 2.5 μg / mL, 100 μL of soluble HIS-SMP30 protein is added to the bottom of a 96-well microplate and incubated at 4°C overnight. Then, 0.5%, 100 μL of BSA blocking solution is added and blocked at 37°C for 1 h. Subsequently, 100 μL of Ab1 of different concentrations is added to the bottom of the microplate and bound at 37°C for 1 h.

[0016] The present invention also provides a use of the electrochemical immunosensor as described above or the electrochemical immunosensor prepared by any of the methods described above in an in vitro diagnostic product.

[0017] Due to the adoption of the above technical solution, the present invention has the following beneficial effects: The present invention constructs an ultra-sensitive split biosensor based on a 96-well plate and nanozyme-catalyzed electroactive molecule ferrocene-tyramide (Fc-Tyr) deposition signal amplification for the detection of a new liver cancer marker, the senescence marker protein-30 antibody (SMP30 antibody). It is reported that the SMP30 antibody has a high positive expression in hepatocellular carcinoma serum. The functional electroactive substance Fc-Tyr has both the enzyme cascade catalytic activity of tyramide signal amplification (TSA) and the excellent redox performance of ferrocene. In terms of design, the 96-well plate independent immune response system is used to coordinate the nanozyme to efficiently catalyze Fc-Tyr deposition, reducing the steric hindrance caused by direct incubation of the sample on the electrode surface. The introduced nanozyme triggers TSA, thereby inducing a large amount of Fc-Tyr to be deposited layer by layer on the electrode surface. The constructed sensing and analysis platform verifies the concept of split biosensor with nanoenzyme-catalyzed signal amplification. The SMP30 antibody split biosensor shows high selectivity and sensitivity to SMP30 antibodies in the range of 0.1 ng / mL to 1 μg / mL. Finally, the sensor was used together with ELISA to detect the SMP30 antibody levels in the serum of clinical liver cancer patients. The results showed consistency, with a recovery rate of 93-104%, which has great promotion and application value for portable and rapid detection of tumor markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 (A) Schematic diagram of the modification principle of the gold electrode of the present invention; (B) Schematic diagram of the reaction principle of GO-Hemin coupled antibody; (C) Schematic diagram of the construction principle of the SMP30 antibody electrochemical biosensor.

[0019] Figure 2 These are transmission electron microscopy analysis images of graphene oxide (I), GO-Hemin composite materials (II, III), and GO-Hemin composite nanomaterials of the present invention.

[0020] Figure 3 Characterization diagrams of GO-Hemin and its antibody-coupled complex of the present invention. (A) FT-IR (B) CV (C) UV–vis.

[0021] Figure 4 The catalytic performance analysis diagram of GO-Hemin of the present invention. (A) Absorbance spectrum and image of TMB color reaction catalyzed by different nanomaterials (B) DPV response of H2O2 decomposition catalyzed by different nanozymes.

[0022] Figure 5Graphs showing the electrochemical behavior of the biosensor prepared in this invention. (A) CV and (B) EIS. (C) CV and (D) DPV: Current monitoring of the self-assembly process on the electrode surface in the presence and absence of target molecules. (E) CV curves of GE / Au NPs / MPA / Tyr / Fc-Tyr at different scan rates. (F) Statistical analysis of CV curves at different scan rates.

[0023] Figure 6 These are scanning electron micrographs of the electrochemical sensor of the present invention at various modification stages: (I) bare gold electrode, (II) GE / AuNPs, (III) GE / Au NPs / MPA, (IV) GE / Au NPs / MPA / Tyr, and (V) GE / Au NPs / MPA / Tyr / Fc-Tyr.

[0024] Figure 7 Atomic force microscopy images (A) and X-ray energy spectra (B) of the electrochemical sensor of the present invention at different modification stages: (I) bare gold electrode, (II) GE / Au NPs, (III) GE / Au NPs / MPA, (IV) GE / Au NPs / MPA / Tyr, and (V) GE / Au NPs / MPA / Tyr / Fc-Tyr.

[0025] Figure 8 (A) X-ray photoelectron spectra of Au elements in the sensor at different modification stages of the present invention; (B) X-ray photoelectron spectra of Fe elements in the sensor at different modification stages. (I) Bare gold electrode, (II) GE / Au NPs, (III) GE / Au NPs / MPA, (IV) GE / Au NPs / MPA / Ty (V) GE / Au NPs / MPA / Tyr / Fc-Tyr.

[0026] Figure 9 Comparison of optimized conditions of the present invention. (A) Potential for Au NP deposition. (B) Time for Au NP deposition. (C) CV curves for depositing Au NPs at different concentrations. (D) DPV curves for depositing Au NPs at different concentrations. (E) Effect of different Au NP concentrations on electrochemical deposition. Error bars represent ±SD of three measurements.

[0027] Figure 10 Comparison of optimized conditions for the present invention. (A) Tyramide concentration. (B) Effect of temperature on electrochemical deposition. (C) Effect of nanozyme-labeled secondary antibody conjugate concentration on Fc-Tyr deposition. (D) Effect of hydrogen peroxide and (E) Fc-Tyr:Tyr concentration ratio on sensor electrochemical response. Error bars represent ±SD of triplicate measurements.

[0028] Figure 11(A) DPV curves of mouse monoclonal antibodies at different concentrations. (B) Relationship between peak current and mouse monoclonal antibody concentration. (C) Calibration curve of peak current intensity versus the logarithm of mouse monoclonal antibody concentration. (D) DPV curves of SMP30 antibodies at different concentrations. (E) Relationship between peak current and SMP30 antibody concentration. (F) Calibration curve of peak current intensity versus the logarithm of SMP30 antibody concentration. (G) Detection of the split biosensor for target molecules SMP30 antibody and other 1 μg mL -1 Protein selectivity, p < 0.0001. (H) SMP30 antibody content was detected in three parallel experiments with different concentrations of the target molecule SMP30 antibody (0.1, 10, and 100 ng / mL, respectively). (I) Stability analysis of the SMP30 antibody split biosensor, p > 0.05. Error bars represent triplicate measurements ± SD.

[0029] Figure 12 The present invention (A) uses the SMP30 antibody electrochemical biosensor to analyze the SMP30 antibody expression level in clinical serum samples and (B) the standardized heat map drawn by the relative expression level of SMP30 antibodies in serum samples. N=5, based on healthy people (1), AFP-positive liver cancer patients (2-6), AFP-negative liver cancer patients (7-11) and AFP-positive non-liver cancer patients (12-16). (C) Standard curve of the SMP30 antibody quantitative kit; (D) Results of SMP30 antibodies in samples from healthy people, AFP-positive liver cancer patients, AFP-negative liver cancer patients and AFP-positive non-liver cancer patients, N=5. OD represents optical density; Health, healthy people; liver cancer (AFP + ), AFP-positive liver cancer patients; liver cancer(AFP-), AFP-negative liver cancer patients; non-liver cancer(AFP + ), AFP-positive non-hepatocellular carcinoma patients. DETAILED DESCRIPTION

[0030] The specific implementation of the invention is further described below with reference to the accompanying drawings.

[0031] All reagents and instruments of the present invention can be purchased on the market.

[0032] Reagents: Tyramine (Tyr) and 3-mercaptopropionic acid (MPA) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Sodium perchlorate, p-hydroxybenzoic acid (PHBA), bovine serum albumin (BSA), and hydrogen peroxide solution were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Hemin, sodium carbonate, and sodium bicarbonate were purchased from Aladdin Reagent (Shanghai) Co., Ltd. Chloroauric acid (HAuCl4) was a product of Sinopharm Chemical Reagent Company. Bovine serum albumin (BSA), human IgG (hIgG, D110501), alpha-fetoprotein (AFP), N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) were purchased from Shanghai Sangon Biotechnology Service Co., Ltd. (Shanghai, China). Soluble SMP30 protein was purchased from Aviva. Anti-His tag mouse monoclonal antibody was purchased from Kangwei Century. RGN Monoclonal Antibody (4B9) was purchased from Thermo Fisher Scientific. Goat anti-human IgG (H+L) secondary antibody was purchased from Beijing Novi Biotechnology. All other reagents were of reagent or analytical grade and used without further purification. All solutions were prepared with 18 MΩ ultrapure water and purified using a Milli-Q purification system (Millipore, USA).

[0033] Equipment: Ultraviolet-visible spectrophotometry (UV-vis) detection was performed using a UH5300 spectrophotometer (Hitachi, Japan). Fourier transform infrared spectroscopy (FT-IR) was performed using a Nicolet iS10 (Thermo Fisher Scientific, USA). The surface morphology of the electrodes at different modification stages was examined using a scanning electron microscope (SEM) with an SU8020 (Hitachi, Japan). Atomic force microscopy (AFM) images were acquired using an Agilent 5500 microscope (Agilent, USA). X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250Xi spectrometer (Thermo Fisher Scientific, USA). All electrochemical measurements were performed at room temperature using a CHI660E electrochemical analyzer (Shanghai Chenhua Instrument, China). All electrochemical measurements used a three-electrode system consisting of a gold electrode, a saturated calomel electrode, and a platinum wire electrode, with the gold electrode as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire electrode as the counter electrode. Cyclic voltammetry (CV) was performed in phosphate buffer solution (PBS) (containing 0.01 M Na2HPO4 and Na2HPO4, pH 7.4, respectively) containing 5 mM K3Fe(CN)6 / K4Fe(CN)6 and 0.1 M KCl at a scan rate of 100 mV.s -1, with a potential window of -0.8 to 0.8 V. Differential pulse voltammetry (DPV) was performed in PBS (pH 7.4) containing 0.2 M NaClO4 with a scan rate of 100 mV.s -1 , the potential window is 0 to 0.8V.

[0034] Example 1 An electrochemical immunosensor that utilizes nanoenzymatic electroactive molecule ferrocene-tyramine deposition reaction to amplify detection signals comprises the following steps: (1) Preparation of GO-Hemin and its conjugated antibody complex First, 4 mg of carboxylated graphene oxide (GO / COOH) was dissolved in 4 mL of ultrapure water (1 mg / mL) and ultrasonically dispersed for 3 hours. The mixture was then centrifuged at 3000 rpm for 30 minutes, and the supernatant was collected. 26.076 mg of hemin (10 mM) was then dissolved in 4 mL of dimethylformamide (DMF). The mixture was thoroughly mixed with the supernatant and ultrasonicated for 3 hours (room temperature, 130 kHz). The precipitate and supernatant were separated by centrifugation (13000 rpm for 30 minutes), washed twice with PBS buffer, and dried in an oven to obtain the GO / hemin solution for future use. Then, 2 mg of the obtained hemin-functionalized GO / Hemin was dispersed in 2 mL of ultrapure water (1 mg / mL), and 500 μL of EDC 1-ethyl-(3-dimethylaminopropyl)carbodiimide (100 mM) and NHS (N-hydroxysuccinimide) (100 mM) were added after sonication. The mixture was stirred at room temperature for 4 h, and then 30 μL of a 1 mg / mL polyclonal antibody was added. The entire mixture was shaken at 4 ° C for 24 h, and then the supernatant and precipitate were separated by centrifugation (4 ° C, 3000 rpm, 30 min), washed twice with PBS, and finally dried in a vacuum dryer for 12 h and resuspended in 2 mL of 0.01 M PBS solution to prepare the final immune complex GO / Hemin-Ab2, which was stored at 4 ° C for use ( Figure 1 B).

[0035] (2) Pretreatment of gold electrodes First, a gold electrode was polished using 0.3 μm and 0.05 μm alumina slurries. The cleaned gold electrode was ultrasonically cleaned in anhydrous ethanol for 5 minutes, followed by ultrasonic cleaning in ultrapure water for 5 minutes, alternating ultrasonic cleaning three times. The three-electrode system (gold electrode, platinum counter electrode, Ag / AgCl reference electrode) was simultaneously placed in an electrolytic cell filled with 0.5 M H2SO4 solution for electrochemical cleaning. Electrochemical cyclic voltammetry (scan range -0.25 to 1.5 V, scan rate 100 mV / s) was performed. The cell was then charged and discharged, and the cell was connected (2 V, 5 s and -0.25 V, 10 s). A CV fast scan was performed (scan range -0.25 to 1.5 V, scan rate 400 mV / s), followed by another CV slow scan. The above steps were repeated twice, and the electrode was washed three times with ddH2O.

[0036] Next, the pretreated graphene GE was immersed in 5mL of 0.01% HAuCl4 solution. Au NPs were electrodeposited at a potential of -0.5V for 120s to form Au NPs-modified GE (GE / Au NPs), and the electrode was rinsed with water three times. GE / Au NPs were incubated in 300μL of 10mM 3-mercaptopropionic acid at 4°C overnight to form an MPA molecular film on the surface of GE / Au NPs. GE / Au NPs / MPA was rinsed with water, and 40μL of 10mM EDC-NHS solution was dropped on the surface of GE / Au NPs / MPA and incubated at room temperature for 40min, and then washed with water. Next, 40μL of 1mg.mL -1 Tyr solution was added dropwise to the GE / Au NPs / MPA surface and incubated at room temperature for 60 min. Tyramine was fixed on the electrode surface by the condensation reaction between carboxyl and amino groups. After rinsing with water, a GE / Au NPs / MPA / Tyr modified gold electrode was obtained ( Figure 1 A).

[0037] (3) Preparation of electrochemical sensors The developed electrochemical immunosensor integrated detection system includes an immune reaction part that captures the target molecule and a signal amplification part that nanoenzymatically catalyzes the deposition reaction of the electroactive molecule ferrocene-tyramine. The working mechanism of this split electrochemical immunosensor is as follows: Figure 1As shown in C. First, the soluble HIS-SMP30 protein (Ag) (2.5μg / mL, 100μL) was added to the bottom of a 96-well microplate and incubated at 4°C overnight. Secondly, BSA blocking solution (0.5%, 100μL) was added and blocked at 37°C for 1h. Subsequently, 100μL of monoclonal antibody (Ab1) of different concentrations was added to the bottom of the microplate and bound at 37°C for 1h. Then 100μL of the prepared GO-Hemin-Ab2 was added to the bottom of the microplate and incubated under the same conditions for 1h. Before each incubation step, the microplate was gently rinsed with PBST solution and patted dry. Then the mixed solution (14μL 1mg.mL -1 Fc-Tyr, 14 μL 1 mg / mL -1 Tyr and 14 μL of 50 mM H₂O₂ were dripped onto the bottom of a microplate. Simultaneously, the resulting GE / Au NPs / MPA / Tyr solution was inserted into the microplate and allowed to react at 37°C for 30 minutes. After the reaction, the electrochemical immunosensor was gently rinsed with water, and the electrochemical reaction was recorded using DPV. All data were calculated in triplicate.

[0038] Example 2: Electrochemical verification test All blood samples were provided by the First Affiliated Hospital of Guangxi Medical University (Nanning, China) and approved by the hospital's ethics committee. Before the experiment, samples were placed in centrifuge tubes and centrifuged at 3000 rpm for 15 minutes to obtain serum. Anti-SMP30 antibodies were detected using the electrochemical immunosensor constructed in Example 1, following the experimental procedure described above.

[0039] 1.1 Characterization of GO-Hemin and its conjugated antibody composite nanomaterials The morphology of the prepared GO / Hemin samples was described using TEM, FT-IR and CV techniques. Figure 2 As shown in the figure, the carboxylated graphene oxide is in the form of flakes after ultrasound. Compared with GO, dark granular substances can be seen on the surface of GO functionalized with hemin, indicating that Hemin is successfully attached to the GO surface. The two are stably combined through π-π bonds. The nanocomposite material is evenly dispersed under the microstructure, which improves its stability and catalytic performance. Subsequently, FT-IR spectroscopy was used to characterize the preparation of GO / Hemin composite. The carboxylated GO has a 3441cm -1 There is an obvious broad absorption peak around 1633cm -1 A strong stretching vibration peak of the C=O bond in COOH appears nearby. Similarly, the FT-IR spectrum of Hemin has a peak at 3441 cm -1 and 1633cm -1There are similar functional group characteristic absorption peaks at 1500-500cm. Further analysis of the spectrum of the synthesized composite nanomaterial GO / Hemin shows that the characteristic peak intensity of the GO / Hemin spectrum is significantly reduced compared with GO and Hemin. This is attributed to the fact that some functional groups reacted and their number decreased during the synthesis process. -1 There are a large number of similar peaks in the region, which fully demonstrates the successful binding of GO and Hemin ( Figure 3 A). In addition, the synthesized GO / Hemin was characterized by electrochemical methods. GO and GO / Hemin samples were added to the cleaned screen-printed electrodes, respectively, and incubated at room temperature for 1 hour before CV detection in 0.1M PBS solution. Comparing the CV graphs of the bare electrode and the electrode incubated with GO, the electrode incubated with GO / Hemin showed a clear reduction peak at a potential of approximately -0.37V. This is due to the electron transfer process of iron in the hemin structure ( Figure 3 B).

[0040] Finally, to prove the successful synthesis of GO / Hemin and Ab2 conjugates, UV–vis was used to characterize the main components of GO / Hemin-Ab2. Figure 3 As shown in Figure C, after the nanomaterial GO / Hemin was coupled with the antibody, a new absorption peak appeared at 280 nm, confirming that Ab2 was successfully bound to GO / Hemin.

[0041] 1.2 Characterization of nanozyme catalytic activity The catalytic activity of nanozymes is extremely important for the sensitivity of biosensors. Therefore, the peroxidase-like activity of GO-Hemin is crucial for the analytical performance of the developed electrochemical immunosensor. First, we used the classic 3,3',5,5'-tetramethylbenzidine (TMB) oxidation experiment to analyze the peroxidase-like activity of GO-Hemin. Under the same conditions of H2O2 and TMB, Figure 4 As shown in the UV-vis spectrum of A, GO-Hemin and GO-Hemin-Ab2 groups showed strong absorption bands at 370 and 652 nm. This is mainly because the nanozyme GO-Hemin can decompose H2O2 to produce hydroxyl radicals, which can combine with the substrate TMB in the solution to oxidize it to generate a blue product oxTMB, and a specific absorption band appears under UV-vis. This result confirms that the prepared GO-Hemin has peroxidase-like activity. In order to study the catalytic activity of GO-Hemin nanomaterials in electrochemical reactions, we incubated several nanomaterials on the treated screen-printed electrodes under the same conditions for 1 hour, and then performed DPV detection in H2O2 solution. The results are shown in Figure 2. Figure 4As shown in Figure B, the electrocatalytic H2O2 abilities of several nanomaterials under the same conditions are different. Among them, GO has almost no current response, while GO-Hemin has the strongest DPV response and the highest electrochemical signal. This fully demonstrates that GO-Hemin has good electrocatalytic performance and can realize sensitive analysis of target molecule detection of the developed electrochemical immunosensor.

[0042] 1.3 Study on electrochemical behavior of biosensors CV and EIS of electrochemical immunosensors are used to characterize the assembly process of electrochemical immunosensors, such as Figure 5 As shown in A and B, in 5.0mmol / L[Fe(CN)6] 3- / 4- In PBS, electron transfer between the bare gold electrode and the redox probe was excellent. After gold nanoparticle deposition, the gold electrode's specific surface area increased, and the noble metal's excellent conductivity resulted in minimal changes in the current signal. Subsequently, with continued incubation with MPA and Tyr, the current gradually decreased and the resistance increased. In particular, after the electrochemical deposition of Fc-Tyr, a large amount of Fc-Tyr was deposited on the electrode surface, resulting in significant changes in the CV and EIS patterns. This is because the large amount of Fc-Tyr deposited on the electrode surface further hindered charge transfer between the redox probe and the electrode. These EIS and CV results confirm the successful layer-by-layer assembly of the constructed biosensor electrode. To further validate the feasibility of the developed biosensing strategy, CV and DPV responses were measured in the presence and absence of an anti-His-Tag mouse monoclonal antibody. Figure 5 As shown in C and D, in the absence of anti-His tag mouse monoclonal antibody, almost no current response was observed in DPV, and since no Fc-Tyr deposition occurred on the electrode surface, the ferrocyanide electron transfer process was not restricted. On the contrary, when the target analyte anti-His tag mouse monoclonal antibody was added, a strong DPV response was generated. This change was mainly attributed to the complete immune reaction after the addition of anti-His tag mouse monoclonal antibody. The introduced nanozyme GO / Hemin triggered the electroactive molecule Fc-Tyr to be deposited on the GE / Au NPs / MPA / Tyr electrode surface in the detection solution system. The CV detection results were highly consistent with the DPV results, which fully demonstrated the successful assembly of the sensor electrode and confirmed the feasibility of the proposed immunosensing strategy. In addition, in order to study the GE / Au NPs / MPA / Tyr / Fc-Tyr assembled electrode / electrolyte interface reaction kinetics, different scan rates (150-450mV / s) were used to detect the CV curve. Figure 5As shown in Figures E and F, the peak current increases linearly with scan rate, demonstrating that the redox reaction on the electrode surface during the nanozyme-induced Fc-Tyr deposition reaction is a typical surface-controlled process. These results demonstrate the successful construction of the split-type biosensor and the feasibility of the signal amplification strategy based on the nanozyme-induced ferrocene-tyramide deposition reaction introduced by the immune reaction in a 96-well plate.

[0043] 1.4 Characterization of biosensor electrode surface assembly In order to understand the changes in morphology and elements during the electrode modification stage during the sensor construction process, the sensor was characterized by analytical techniques such as scanning electron microscopy (SEM), atomic force microscopy (AFM), energy dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS). First, the electrode morphology at each modification stage was analyzed using a scanning electron microscope. Figure 6 As shown, the bare gold surface is essentially smooth, with no visible material present. With each nanoparticle modification step, the electrode surface gradually becomes rougher, with evenly distributed granular material deposited on the electrode surface visible under a microscope. The differences in the electrode surface at each modification stage clearly demonstrate the successful sensor assembly. In particular, the GE / Au NPs / MPA / Tyr / Fc-Tyr electrode exhibits distinct granular material compared to the bare gold electrode, demonstrating the successful Fc-Tyr deposition reaction and the feasibility of this sensor fabrication method.

[0044] Atomic force microscopy is one of the methods to characterize electrodes and observe their surface morphology and structural changes. Through image observation, we can understand parameters such as electrode surface height, three-dimensional structure, and surface morphology at each modification stage. Figure 7 A is an atomic force microscope image of the sensor at each modification stage during the assembly process, and the images are arranged from top to bottom as (I) to (V). It can be observed from the figure that as the particles on the electrode surface are continuously modified, the electrode surface becomes rougher from the initial simple flatness, and the electrode height continues to increase. After the deposition of Fc-Tyr in the final stage, the electrode height increases significantly. This result shows that the Fc-Tyr deposition reaction is successful. At the same time, we can also observe from the three-dimensional image that the modification of each stage of the sensor causes different changes on the electrode surface. These AFM parameter change images intuitively prove that the SMP30 antibody sensor was successfully constructed. EDS is a method to characterize the elements on the surface of the sensor electrode, such as Figure 7As shown in Figure B, EDS was used to analyze the elemental content of the materials modified on the electrode surface, including C, N, O, Fe and Au. We mainly focused on the changes in the content of the characteristic elements Fe and Au at each modification stage. After the deposition of Au NPs, the EDS image showed a significant increase in the content of the Au element. With the continuous modification of the subsequent steps, the Au element was covered and gradually decreased. When the Fe element was not added, the EDS image showed that no Fe element was detected. After the deposition of Fc-Tyr in the final stage, the EDS spectrum showed an energy peak of the Fe element. Compared with other modification stages, the percentage content of the Fe element changed significantly ( Figure 7 BI~V comparison), this change in element content fully demonstrates that Fc-Tyr is successfully deposited on the electrode surface.

[0045] The metal elements Fe and Au at different modification stages of the electrode surface were analyzed by X-ray photoelectron spectroscopy. Figure 8 In Figure A, the bare gold electrode clearly exhibits significant Au characteristic peaks, located at approximately 83 and 87 eV. After the deposition of Au NPs, these peaks intensify, increasing the electrode surface area and conductivity. With each subsequent modification step, the Au characteristic peaks decrease, reaching a significant decrease after the Fc-Tyr deposition reaction. This is likely due to the large amount of material modified on the gold electrode surface, which covers the elemental Au and weakens the Au XPS signal. Figure 8 B is the XPS characterization spectrum of elemental Fe. The image shows that the characteristic peak of Fe appears only after the Fc-Tyr deposition reaction occurs in the final stage, with the peak value at approximately 708eV. No characteristic peak of Fe appears in other stages. This result indicates that Fc-Tyr is successfully modified on the electrode surface and the sensor is successfully assembled.

[0046] 1.5 Test Condition Optimization In order to improve the analytical performance of the constructed biosensor and enhance its detection sensitivity, based on the key factors affecting the current response, our research team optimized the preparation conditions of the sensor, including the concentration of deposited gold nanoparticles, the potential of deposited gold nanoparticles and their deposition time, tyramine concentration, the concentration ratio of ferrocene tyramine to tyramine (Fc-Tyr:Tyr), GO-Hemin-Ab2 concentration, H2O2 concentration, and Fc-Tyr deposition reaction temperature. In the electroplating method, the potential and time of depositing nanoparticles have a great influence on the electrode performance. Changing the potential and time of depositing gold nanoparticles, such as Figure 9A and B, with the increase of deposition potential and deposition time, the DPV response value first increases and then decreases. This change may be due to the fact that under the action of potential, gold nanoparticles form a diffusion layer on the electrode surface. When the potential is too high or the deposition time is too long, the reversible reaction equilibrium condition of the metal substance in the diffusion layer and the electrolyte solution is broken, resulting in a decrease in the DPV electrical signal response. Therefore, -0.5V potential and 120s deposition time are selected as the optimal reaction conditions for electrodeposition of gold nanoparticles. Gold nanoparticles were deposited using HAuCl4 solutions of different concentrations and CV ( Figure 9 C) and DPV( Figure 9 D and Figure 9 E) Method testing compared the effect of concentration on electrodeposition results. The above results show that the current response value varies with the concentration of HAuCl4 solution. As the concentration increases, the DPV current response value continues to increase. The sensor obtains the strongest DPV current response signal at a concentration of 0.1%. This result may be due to the deposition of gold nanoparticles increasing the electrode specific surface area and enhancing the electrode conductivity. Subsequently, increasing the concentration of the deposited HAuCl4 solution slightly decreases the current response value. This may be due to the formation of steric hindrance caused by the dense arrangement of the deposited HAuCl4 particles at too high a concentration. Therefore, the appropriate gold nanoparticle deposition concentration can effectively improve the sensitivity of the constructed sensor for detecting SMP30 antibodies. Therefore, 0.1% HAuCl4 solution was selected as the optimal experimental condition for gold nanoparticle deposition.

[0047] Tyramide on the electrode surface serves as the initial binding site for Fc-Tyr deposition reaction. Too low a concentration will result in incomplete Fc-Tyr deposition reaction. On the contrary, too high a concentration will lead to greater steric hindrance, reducing the current response value. Therefore, different concentrations of tyramide were tested and the corresponding current values ​​were recorded. The results showed that at a tyramide concentration of 5 mg / mL, the DPV current response value was the highest ( Figure 10 A). Enzyme catalytic reaction temperature and enzyme amount are key factors affecting sensor performance. This study conducted DPV tests in the temperature range of 4-70℃. The results are as follows Figure 10 As shown in B, the DPV current response value of the nanozyme-catalyzed Fc-Tyr deposition reaction is the highest at 37°C. This is because too low or too high a temperature will affect the enzyme activity and limit the enzyme catalytic reaction. Similarly, after the immune reaction and electrochemical deposition reaction of different concentrations of enzyme-labeled antibodies, the DPV test results show that the current response is best when the concentration is 1μg / mL ( Figure 10C) In the Fc-Tyr deposition reaction, hydrogen peroxide concentration is also a key factor in the sensor's analytical performance. GO-Hemin catalyzes the substrate hydrogen peroxide to deposit the electroactive substance Fc-Tyr on the electrode surface. Therefore, the level of hydrogen peroxide concentration affects the deposition efficiency of the electroactive substance Fc-Tyr. When the concentration is too low, it affects the enzyme catalytic efficiency, while when it is too high, it inhibits the Fc-Tyr deposition reaction. Figure 10 As shown in Figure D, when the hydrogen peroxide concentration is 50mM, the nanozyme catalytic deposition effect is the best and the current value is the largest, so 50mM hydrogen peroxide is selected as the optimal experimental condition. In order to obtain a higher current signal, this study optimized the concentration ratio of ferrocene tyramine to tyramine (Fc-Tyr:Tyr), as shown in Figure 4. Figure 10 As shown in Figure E, it can be observed that excessively high concentrations of tyramine or ferrocenyltyramine increase steric hindrance on the electrode surface, hindering Fc-Tyr deposition. Excessively low concentrations lead to incomplete Fc-Tyr deposition and a reduced current response. The current response is highest when the Fc-Tyr:Tyr ratio is 1:1, so 1:1 is selected as the optimal Fc-Tyr:Tyr concentration ratio.

[0048] According to the above optimization results, the relationship between the DPV current response signal of the electrochemical sensor and the experimental conditions under different experimental conditions was obtained, and the optimal reaction conditions were obtained. The results are shown in Table 1.

[0049] Table 1 Summary of experimental condition optimization results Experimental variables Optimization range Optimal conditions <![CDATA[Concentration of HAnO4 (%)]]> 0.0001～1 0.1 An NPs deposition potential (V) -0.7～-0.3 -0.5 An NPs deposition time (s) 30～480 120 Tyr concentration (mg / mL) 0.01～10 5 Fc-Tyr:Tyr 1：3～3：1 1：1 <![CDATA[GO-Hemin-Ab2 concentration (μg / mL)]]> 0.01～5 1 <![CDATA[H2O2 concentration (mM)]]> 1～100 50 Fc-Tyr deposition temperature (℃) 4～70 37 1.6. Sensor performance analysis After optimizing the experimental conditions of the constructed split biosensor and obtaining the optimal reaction conditions, the analytical performance of the split biosensor was tested. Figure 11 As shown in A and B, as the concentration of anti-His tag mouse monoclonal antibody increases, the DPV current response value also increases. Figure 11 C shows the relationship between the DPV current response value and the logarithm of the concentration of anti-His tag mouse monoclonal antibody (log C) in the concentration range of 0.1 ng / mL to 10 μg / mL. Ab-鼠 ) have a good linear relationship. The linear regression equation obtained by linear fitting is Y=0.7442X+0.9623(R 2=0.9973), the detection limit of the anti-His tag mouse monoclonal antibody is the detection limit (LOD) of 0.3453 ng / mL (S / N=3), and the calculation formula of LOD is: LOD=3sb / q, sb is the standard deviation of 3 blank samples, q is the slope of the standard curve, and the mouse model test results show that the sensing system has high sensitivity. The electrochemical sensing analysis equipment constructed in this article has good detection capabilities for mouse monoclonal antibodies. As far as we know, there are currently no electrochemical sensors for detecting human SMP30 antibodies in the clinic. Therefore, in order to test the applicability of the constructed sensor, we used the DPV detection method under the above-mentioned optimal experimental conditions to test the analytical performance of the sensor for the target human SMP30 antibody. Figure 11 As shown in D and E, as the concentration of SMP30 antibody increases, the DPV current response value continues to increase accordingly. Figure 11 F shows the relationship between the DPV current response value and the logarithm of the SMP30 antibody concentration (log C Ab-SMP30 ) have a good linear relationship. The linear regression equation obtained by linear fitting is Y=1.2411X+0.9235(R 2 =0.9906), the detection limit of SMP30 antibody was 0.1681 ng / mL (S / N=3), and the LOD was calculated as: LOD=3sb / q, where sb is the standard deviation of three blank samples and q is the slope of the standard curve. The SMP30 antibody model detection results showed that this sensing strategy is also applicable to the detection of human SMP30 antibodies.

[0050] 1.7 Stability, specificity and reproducibility test of SMP30 antibody split biosensor In order to investigate the specificity and anti-interference ability of the SMP30 antibody split biosensor based on 96-well plate immunoreaction and GO-Hemin nanoenzyme-induced Fc-Tyr deposition, the same concentrations of BSA, hIgG, AFP, CNN2 antibody (AbCNN2), SMP30 antibody (AbSMP30) and a mixture of all substances (Mix) were selected for detection evaluation. Figure 11 G shows that the experimental group whose target substance is SMP30 antibody produces a strong DPV current response. On the contrary, the DPV current response values ​​of other substances are much lower than the detection results of the SMP30 antibody group. At the same time, the DPV current response value of the mixed group is almost close to the DPV current response result of the SMP30 antibody group. This result fully demonstrates that the constructed split biosensor has good selectivity for SMP30 antibody and is not affected by other substances. In addition, three parallel tests were performed on three concentrations of SMP30 antibody (0.1, 10, 100 ng / mL) to verify the repeatability of the sensor ( Figure 11H), the relative standard deviation (RSD) of the DPV response current of each concentration group from low to high was 4.22%, 0.3% and 1.5%, respectively. This result shows that the SMP30 antibody split biosensor has good repeatability. In order to investigate the stability of the constructed split biosensor, the same batch of sensor probes were stored at 4°C for 5 days, 10 days, and 15 days, and then the sensors were tested by DPV at the corresponding time points. The results showed that the sensor probes still maintained a high current signal response value within two weeks ( Figure 11 I).

[0051] 1.8 Serological testing In order to demonstrate the applicability of the constructed SMP30 antibody split electrochemical sensor based on 96-well plate immunoreaction and Fc-Tyr deposition reaction in actual sample detection, our research team used the constructed sensor to detect 4 groups of clinical serum samples (healthy people; AFP-positive liver cancer patients; AFP-negative liver cancer patients; AFP-positive non-liver cancer patients). Figure 12 As shown in A, we observed that the SMP30 antibody levels in the serum of AFP-positive liver cancer patients (No. 2-6) were slightly higher than those in the serum of healthy individuals (No. 1) and AFP-negative non-liver cancer patients (No. 12-16). It is worth noting that relatively significant SMP30 antibody levels were detected in the serum of AFP-negative liver cancer patients (No. 7-11), and the generated normalized signal heat map ( Figure 12 B) The results also showed that only the serum samples from patients with liver cancer showed a significant current response, while the other samples did not show a substantial current response. The biosensor detection of SMP30 antibodies in the actual serum samples was compared with the ELISA immunoassay results, and the results were consistent ( Figure 12 C and D), and the recovery rate of the split electrochemical biosensor was calculated to be 93-104% (Table 2). Therefore, these results demonstrate that the constructed split electrochemical biosensor has good discrimination and detection capabilities for serum SMP30 antibody levels in HCC patients, further demonstrating that SMP30 antibodies can serve as a potential candidate liver cancer tumor marker.

[0052] Table 2. Recovery rate of SMP30 antibody detection in serum samples The split biosensor constructed by the present invention has good detection capabilities for monoclonal antibodies, and has good repeatability, specificity, anti-interference and stability. Based on the above significant analytical performance, this study tested different concentrations of mouse and human monoclonal antibodies, and the results confirmed the universal applicability of the constructed biosensor strategy for the detection of different types of biological molecules. The SMP30 antibody is a new candidate liver cancer tumor marker. Its liver cancer diagnosis research is in the basic stage. Existing research results have shown its limitations in detection in serum samples, and current laboratory detection methods are mainly based on colorimetry. Therefore, in order to improve its detection sensitivity, the inventors have attempted to combine the specificity of traditional immunoassays with the sensitivity of electrochemistry to develop a new sensing platform for detecting antibodies. The experimental results also confirmed that the sensor constructed by the crystallization of the present invention is capable of sensitive and specific detection of low-abundance target molecules.

[0053] Furthermore, the inventors used the biosensor developed by the present invention to test clinical serum samples. By analyzing related research conducted by other research groups, the inventors designed a protocol for detecting SMP30 antibodies in four different clinical serum samples, focusing on the key words "whether the patient is a liver cancer patient" and "AFP results." The inventors also used a commercially available test kit as a control for the biosensor's detection results. The results showed consistency between the two experimental methods, fully demonstrating the potential application value of the biosensor developed by the present invention in clinical serum sample testing.

[0054] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. An electrochemical immunosensor that utilizes nano-enzyme-catalyzed electroactive molecule ferrocene-tyramine deposition reaction to amplify detection signals, characterized in that: The electrochemical immunosensor is an ultra-sensitive split biosensor based on a 96-well plate and nanozyme-catalyzed electroactive molecule ferrocene-tyramine deposition signal amplification. The electroactive molecule ferrocene-tyramine is used as a signal amplification element and labeled with a signal antibody. Through the sandwich immune reaction between the capture antibody probe, the target antigen and the signal antibody, significant amplification of the current detection signal and sensitive detection of the target antigen are achieved.

2. A method for preparing an electrochemical immunosensor as claimed in claim 1, characterized in that: The specific steps include: (1) Preparation of immune complex GO / Hemin-Ab2: S1. Dissolve carboxylated graphene oxide in ultrapure water, disperse by ultrasonication, centrifuge, and take the supernatant. S2. Then dissolve hemin in dimethylformamide, mix with the supernatant obtained in step S1, treat by ultrasonication, separate the precipitate and the supernatant by centrifugation, wash twice with PBS buffer, and dry in an oven to obtain GO / Hemin. Then, disperse the obtained hemin functionalized GO / Hemin in ultrapure water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide after ultrasonication, stir at room temperature, and then add polyclonal antibody. The reaction was then centrifuged to separate the supernatant and precipitate, washed with PBS, and finally dried in a vacuum dryer and resuspended in PBS solution to prepare the final immune complex GO / Hemin-Ab2 for use; (2) Preparation of GE / AuNPs / MPA / Tyr modified gold electrode: 1) Polishing the gold electrode (GE) with alumina slurry, ultrasonically cleaning the polished GE in anhydrous ethanol, and then ultrasonically cleaning it in ultrapure water, and repeating this alternating ultrasonication for 3 times; 2) Placing the three-electrode system in an electrolytic cell of 0.5MH2SO4 solution for electrochemical cleaning, using electrochemical cyclic voltammetry to slowly scan, and then charging and discharging the electrolytic cell, and connecting the electrolytic cell to , followed by CV fast scan, and finally CV slow scan again, the above operation was repeated twice, and ddH2O was washed three times; 3) the GE treated in the first step was immersed in HAuCl4 solution, AuNPs were electrodeposited to form AuNPs-modified GE / AuNPs, GE / AuNPs were incubated in 3-mercaptopropionic acid overnight to obtain GE / AuNPs / MPA, EDC-NHS solution was dropped on the surface of GE / AuNPs / MPA and incubated at room temperature, then Tyr solution was dropped on the surface of GE / AuNPs / MPA and incubated at room temperature, tyramine was fixed on the electrode surface by the condensation reaction of carboxyl and amino groups, and then rinsed with water, Obtaining an AuNPs / MPA / Tyr modified gold electrode (GE / AuNPs / MPA / Tyr); (3) incubating the target Ag at room temperature to allow Ab1 to undergo a specific immune reaction with Ag; then dripping the GO-Hemin-Ab2 prepared in step (1) and incubating for 1 hour, and then dripping a mixed solution of 14 μL 1 mg.mL-1 Fc-Tyr, 14 μL 1 mg.mL-1 Tyr, and 14 μL 50 mM H2O2, and at the same time adding the GE / AuNPs / MPA / Tyr obtained in step (2), and continuing the reaction at 37°C for 30 minutes to obtain the electrochemical immunosensor.

3. The preparation method according to claim 2, characterized in that: In step S1, the ratio of carboxylated graphene oxide to ultrapure water is 1 mg: 1 mL, ultrasonic dispersion is performed for more than 3 hours, and centrifugation is performed at 3000 rpm for 30 minutes.

4. The preparation method according to claim 2, characterized in that: In step S2, the amount of hemin used is 26.076 mg, the amount of dimethylformamide used is 4 mL, the ultrasonic treatment is carried out at 130 KHz for 3 hours at room temperature, and then the precipitate and the supernatant are separated by centrifugation at 13000 rpm for 30 minutes; the ratio of GO / Hemin to ultrapure water is 1 mg:mL; the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is 500 μL, stirred at room temperature for 4 hours, and then 30 μL of polyclonal antibody with a concentration of 1 mg / mL is added, the entire mixture system is shaken at 4°C for 24 hours, and then centrifuged at 4°C and 3000 rpm for 30 minutes to separate the supernatant and precipitate, washed twice with PBS, and finally dried in a vacuum dryer for 12 hours and resuspended in 2 mL of 0.01 M PBS solution to prepare the final immune complex GO / Hemin-Ab2, which is stored at 4°C for use.

5. The preparation method according to claim 2, characterized in that: In step 1), the fineness of the alumina slurry is 0.3 μm and / or 0.05 μm; and the ultrasonic cleaning is performed for 5 minutes.

6. The preparation method according to claim 2, characterized in that: In step 2), the three-electrode system includes a gold electrode, a platinum wire counter electrode, and an Ag / AgCl reference electrode.

7. The preparation method according to claim 2, characterized in that: In step 2), the scanning range of the electrochemical cyclic voltammetry slow scan is -0.25 to 1.5 V, and the scanning rate is 100 mV / s; the voltage of the electrolytic cell is 2 V, 5 s and -0.25 V, 10 s; the scanning range of the CV fast scan is -0.25 to 1.5 V, and the scanning rate is 400 mV / s.

8. The preparation method according to claim 2, characterized in that: In step 3), the specific operation is to immerse the gold electrode (GE) pretreated in the first step in a HAuCl4 solution, electro-deposit AuNPs at a potential of -0.5V for 120s to form AuNPs-modified GE / AuNPs, and rinse the electrode with water 3 times, incubate GE / AuNPs in 300μL10mM3-mercaptopropionic acid at 4°C overnight to obtain GE / AuNPs / MPA, drop 40μL10mM EDC-NHS solution on the surface of GE / AuNPs / MPA and incubate at room temperature for 40min, and rinse with water, then drop 40μL1mg.mL-1Tyr solution on the surface of GE / AuNPs / MPA and incubate at room temperature for 60min, use the condensation reaction of carboxyl and amino groups to fix tyramine on the electrode surface, and after rinsing with water, obtain a GE / AuNPs / MPA / Tyr-modified gold electrode.

9. The preparation method according to claim 2, characterized in that: In step (3), the target Ag is 2.5 μg / mL, 100 μL of soluble HIS-SMP30 protein is added to the bottom of a 96-well microplate and incubated at 4°C overnight, then 0.5%, 100 μL of BSA blocking solution is added and blocked at 37°C for 1 hour, and then 100 μL of Ab1 at different concentrations is added to the bottom of the microplate and combined at 37°C for 1 hour.

10. Use of the electrochemical immunosensor according to claim 1 or the electrochemical immunosensor prepared by the method according to any one of claims 2 to 9 in an in vitro diagnostic product.

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