Electrochemical immunosensor based on AuNPs / c-MWCNTs / PTH composite material and detection platform

Abstract

The invention discloses an electrochemical immunosensor based on an AuNPs / c-MWCNTs / PTH composite material and a detection platform, firstly, a gold nano solution and a carboxylated multi-walled carbon nanotube particle dispersion liquid are subjected to ultrasonic uniform mixing to prepare the AuNPs / c-MWCNTs composite material, then, an electron mediator thionine is electrically polymerized on the surface of a glassy carbon electrode by using a cyclic voltammetry to obtain a polythionine modified electrode, and finally, the electrochemical immunosensor based on the AuNPs / c-MWCNTs / PTH composite material is obtained. The preparation method comprises the following steps: firstly preparing an AuNPs / c-MWCNTs composite material, then dispensing the AuNPs / c-MWCNTs composite material on a polythionine modified electrode, and finally combining a recognition antigen with the composite material through a covalent bond, the detection range of the sensor on a toxoplasma gondii antibody is 1-1000pg / mL, the detection limit is 0.504 pg / mL, the sensor has good sensitivity, stability and specificity, a new detection technology is provided for diagnosis of toxoplasmosis, and the sensor has good application prospects. The important clinical application value is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of sensing materials, and particularly relates to an electrochemical immunosensor based on an AuNPs / c-MWCNTs / PTH composite material, and also relates to a detection platform containing the electrochemical immunosensor. Background Art

[0002] Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii (TG) that seriously endangers human health. According to epidemiological studies, Toxoplasma has infected more than 2 billion people. In China, there are significant regional differences in the seropositivity rate of the population, ranging from 5% to 20%. Although in immunocompetent hosts, most cases show subclinical infections, however, in immunosuppressed individuals and cases of primary infection during pregnancy, it may cause serious consequences such as fatal encephalitis, congenital malformations, and adverse pregnancy outcomes. At present, the main clinical treatment plan is based on pyrimethamine combined with sulfonamides. However, due to its bone marrow suppression toxicity, this limitation restricts its long-term application, and there is currently a lack of effective preventive vaccines. Under such circumstances, the development of efficient early diagnosis techniques has become the key to blocking the spread of the disease. However, traditional detection methods, such as indirect fluorescent antibody assay (IFA) and enzyme-linked immunosorbent assay (ELISA), have many defects, such as poor sensitivity (detection limit > 1 IU / mL), complex operation procedures (requiring multiple-step incubation), and long detection cycles (> 6 h), and are difficult to meet the actual needs of on-site rapid screening. Therefore, the development of a new detection system that simultaneously has high sensitivity, simplicity of operation, and rapid response has extremely important clinical application value. Summary of the Invention

[0003] In view of this, one of the purposes of the present invention is to provide an electrochemical immunosensor based on an AuNPs / c-MWCNTs / PTH composite material; the second purpose of the present invention is to provide a detection platform containing the electrochemical immunosensor.

[0004] To achieve the above purposes, the present invention provides the following technical solutions:

[0005] 1. An electrochemical immunosensor based on an AuNPs / c-MWCNTs / PTH composite material, and the preparation method is as follows:

[0006] First, the gold nanoparticle solution and the carboxylated multi-walled carbon nanotube particle dispersion were ultrasonically mixed evenly to prepare the AuNPs / c-MWCNTs composite material. Then, the electron mediator thionine was electro-polymerized on the surface of the glassy carbon electrode by cyclic voltammetry to form a uniform negatively charged polythionine film on its surface, obtaining the polythionine modified electrode. Then, the AuNPs / c-MWCNTs composite material was drop-coated onto the polythionine modified electrode. Finally, the recognition antigen was combined with the composite material through covalent bonds to obtain an electrochemical immunosensor for detecting the target antibody based on the AuNPs / c-MWCNTs / PTH composite material.

[0007] In some embodiments of the present invention, the weight percentage of carbon element in the AuNPs / c-MWCNTs composite material is 80%-90%.

[0008] In some embodiments of the present invention, the weight percentage of oxygen element in the AuNPs / c-MWCNTs composite material is 4%-5%.

[0009] In some embodiments of the present invention, the weight percentage of gold element in the AuNPs / c-MWCNTs composite material is 2%-3%.

[0010] In some embodiments of the present invention, the volume ratio of the gold nanoparticle solution to the carboxylated multi-walled carbon nanotube particle dispersion is 1:1.

[0011] In some embodiments of the present invention, the preparation method of the gold nanoparticle solution is the sodium citrate reduction of chloroauric acid method.

[0012] In some embodiments of the present invention, the concentration of the carboxylated multi-walled carbon nanotube particle dispersion is 1 mg / mL.

[0013] In some embodiments of the present invention, the recognition antigen is the Toxoplasma gondii surface antigen SAG1.

[0014] 2. A detection platform containing the electrochemical immunosensor, using a platinum plate electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, the electrochemical immunosensor as the working electrode, and a potassium ferricyanide solution as the electrolyte.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention constructs a Toxoplasma gondii antibody electrochemical sensor based on an AuNPs / c-MWCNTs / PTH composite material modified GCE, and studies its detection performance. The electropolymerization of thionine monomer solution (THI) was characterized, and a polythionine film could be polymerized within a certain voltage range. The AuNPs / c-MWCNTs composite solution was scanned by SEM, and its morphology showed a large specific surface area that could provide more electrochemically active sites, and the material was mainly distributed with C, O, and Au as the main elements. The mixed solution of gold nanoparticles and carbon nanotubes was physically adsorbed on the polythionine electrode by the drop-coating method, and the prepared electrode was activated with EDC / NHS crosslinker to carboxylate. 10 μL of 100 μg / mL TG SAG1 recombinant antigen was added dropwise to the modified electrode. After the antigen was fixed to the electrode, non-specific binding sites were blocked with skim milk powder. The constructed electrochemical sensor could be used for subsequent tests.

[0017] The electrochemical characterization of the sensor was carried out by cyclic voltammetry, indicating that the sensor was successfully constructed. To obtain a better current response, the number of polymerization cycles of thionine, the coating amount of the modifier, and the incubation time of antigen-antibody were optimized respectively. The optimal conditions were 30 cycles of polymerization, a coating amount of 10 μL, and an incubation time of 60 min. Under these conditions, the DPV method was used to detect the Toxoplasma gondii antibody solution. The sensitivity test results showed that the peak current had a good linear correlation with the antibody solution concentration. The linear equation was y = -15.53x + 138.096, R 2 = 0.9837, and the detection limit was 0.504 pg / mL (S / N = 3); specific detection was carried out with different antibody solutions, and the results showed that the sensor had a large current difference only with the solution containing TG SAG1 antibody, indicating that the sensor had good specificity; the repeatability test was carried out by testing the sensor at the same concentration 4 times, and the relative standard deviation RSD = 3.08%, indicating that the sensor had good repeatability; clinical tests showed that the sensor had an obvious current decrease only in the positive serum samples of Toxoplasma gondii, indicating that the sensor had an obvious specific binding with the Toxoplasma gondii antibody. Compared with the detection value of the negative serum, the P value < 0.001, showing a significant difference. It had good clinical application value for the rapid detection of Toxoplasma gondii antibodies in women during pregnancy preparation or pregnancy and people with low immunity. Brief Description of the Drawings

[0018] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

[0019] Figure 1 For the expression identification of TG SAG1 recombinant protein; M: protein molecular weight standard; 1: supernatant of empty bacteria; 2: precipitate of empty bacteria; 3: supernatant after ultrasonic treatment of the strain; 4: precipitate after ultrasonic treatment of the strain.

[0020] Figure 2 SDS-PAGE electrophoresis results of purified TG SAG1 protein; M: Protein molecular mass; 1: Supernatant after dissolution in 8M urea; 2: Precipitate after dissolution in 8M urea; 3: Flow-through; 4 - 6: Eluate with 200mM imidazole; 7: Eluate with 100mM imidazole.

[0021] Figure 3 Identification of recombinant TG SAG1 protein by Western-blot; M: Protein molecular mass standard; 1: Purified recombinant TG SAG1 protein; 2: BL21(DE3) control group.

[0022] Figure 4 Electrophoresis identification diagram of monoclonal antibody; M: Protein molecular mass standard; 1: Monoclonal antibody against TG SAG1.

[0023] Figure 5 Component diagram of AuNPs / c-MWCNTs / PTH / GCE electrochemical immunosensor.

[0024] Figure 6 Cyclic voltammograms of thionine monomer solution at different scan rates (a) and cyclic voltammogram of thionine monomer solution (b).

[0025] Figure 7 Cyclic voltammogram of electropolymerization of polythionine.

[0026] Figure 8 SEM characterization and EDS elemental mapping of AuNPs / c-MWCNTs composite solution.

[0027] Figure 9 Cyclic voltammograms and slope diagrams of bare glassy carbon electrode (a - b) and modified electrode (c - d) at different scan rates.

[0028] Figure 10 CV diagrams during the electrode surface modification process; a: After modification with AuNPs / c-MWCNTs / PTH; b: After antigen immobilization; c: After blocking with skim milk powder; d: After antigen-antibody reaction; e: Bare electrode.

[0029] Figure 11 Diagram of condition optimization; a: Influence of the number of thionine polymerization cycles on current; b: Influence of modifier coating amount on current; c: Influence of antigen-antibody incubation time on current.

[0030] Figure 12 DPV curves of different concentrations of TG SAG1 antibody.

[0031] Figure 13 Linear relationship of different concentrations of TG SAG1 antibody.

[0032] Figure 14 This is a specific experiment for electrochemical sensors.

[0033] Figure 15 This is a repeatability experiment for electrochemical sensors.

[0034] Figure 16 This is a bar graph of the peak current changes in clinical experiments. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0036] Experimental reagents: N-hydroxysulfosuccinimide (NHS) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., carbodiimide (EDC) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., thionine was purchased from Shanghai Yien Chemical Technology Co., Ltd., ethylene glycol was purchased from Shanghai Lutian Biotechnology Co., Ltd., carboxylated multi-walled carbon nanotubes were purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and MES buffer was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0037] The present invention uses genetic engineering technology to prepare the Toxoplasma gondii surface membrane protein SAG1 antigen with strong specificity and high activity, purifies and identifies the SAG1 monoclonal antibody ascites prepared in the early stage, and uses it as a reagent raw material to construct an electrochemical biosensor based on AuNPs / c-MWCNTs / PTH modified glassy carbon electrode. Au has good conductivity and combines with Toxoplasma gondii antigen through Au-S bond to enhance the electron transfer ability. A large number of carboxylation chemical sites on the surface of carboxylated multi-walled carbon nanotubes (c-MWCNTs) can improve the dispersibility of carbon nanotubes, form a uniform conductive network, increase the effective surface area of ​​the electrode, and the carboxyl group can be covalently combined with the antigen amino group through the activation of EDC / NHS to achieve directional fixation of the antigen, improve stability, thereby enhancing the electrochemical signal and then improving the sensitivity and selectivity of the detection. The polythiocyanate (PTH) produced by electropolymerization has a strong charge transfer ability to amplify the detection signal, and has a strong adhesion on the electrode surface, good stability, and can be used as an electron transfer medium of the electrochemical sensor. The combination of the three materials forms a three-dimensional conductive network with an efficient electron transmission path, which reduces interface resistance, enhances electrochemical response, and lowers detection limits, aiming to achieve rapid detection of Toxoplasma antibodies.

[0038] Example 1. Preparation of Toxoplasma gondii TG SAG1 recombinant protein and SAG1 monoclonal antibody

[0039] TG SAG1 is highly expressed on the surface of Toxoplasma gondii tachyzoites (the main proliferative form in the acute infection stage), and it is one of the major surface antigens of Toxoplasma gondii. Due to its high expression level, it is likely to trigger the host immune response. Therefore, SAG1 is an ideal diagnostic target for antibody detection. Referring to the SAG1 gene sequence (accession number: S76248.1) published in Genbank, its codons were optimized, and the TG SAG1 gene was chemically synthesized. Using the BamHI / XhoI restriction enzyme sites, the target gene was cloned into the pET32a(+) vector to construct the pET32a(+)-TGSAG1 recombinant plasmid. In this invention, the pET32a(+)-TG SAG1 plasmid was transformed into competent Escherichia coli BL21(DE3) cells. After inoculation into LB medium, single colonies were selected for expanded culture, and IPTG was used for induction expression. Electrophoresis showed ( Figure 1 ), the TG SAG1 recombinant protein was mainly expressed in the form of inclusion bodies. Therefore, urea was needed to dissolve the inclusion bodies. The dissolved sample was purified by a protein purifier. Since the recombinant protein has a HIS tag and can bind to the nickel column, finally, when the imidazole concentration was 200 mM, a large amount of the target protein could be eluted. The purified protein band was clear without impurity bands, indicating that a relatively pure recombinant protein (concentration 0.3824 - 0.3961 mg / mL) was obtained. After specific identification by Western-blot, the recombinant protein specifically bound to the mouse anti-His tag monoclonal antibody, indicating that the recombinant protein had good specificity and could provide an effective reagent for subsequent detection. Using the Western blot method, hybridization was carried out with a mouse anti-His tag monoclonal antibody (1:5000), and a clear band could be observed at 55 kDa, indicating that the recombinant protein had good specificity ( Figure 3 ).

[0040] After the SAG1 monoclonal antibody ascites prepared in our laboratory previously was purified by Protein G affinity chromatography, SDS-PAGE showed characteristic bands of 25 kDa (light chain) and 50 kDa (heavy chain) ( Figure 4 ). Using the BCA method, the antibody concentration was measured to reach 0.839 - 0.8738 mg / mL. The purified monoclonal antibody had a titer of 1:512000 measured by indirect ELISA. The purified monoclonal antibody could be used as an effective reagent for subsequent sensitivity testing and specificity detection of methods.

[0041] Example 2. Preparation of AuNPs / c-MWCNTs composite

[0042] The preparation of the gold nanoparticle solution was carried out by the sodium citrate reduction method of chloroauric acid. First, 1 ml of the original chloroauric acid solution (mass concentration 1%) placed in a round-bottom flask was mixed with 99 ml of deionized water, and the mixture was heated to boiling. Subsequently, 4 ml of freshly prepared sodium citrate (mass fraction 1%) solution was quickly added to the flask, and the mixture was stirred until the solution color changed from purple to wine red. Heating was stopped after about 15 minutes. After the solution cooled to room temperature, it was placed in the refrigerator for later use.

[0043] 5 mg of carboxylated multi-walled carbon nanotubes (c-MWCNTs) was weighed and added to 5 mL of ethylene glycol. After ultrasonic dispersion for 30 minutes, a 1 mg / mL c-MWCNTs dispersion was prepared. This solution was mixed with the gold nanoparticle solution at a ratio of 1:1 (v / v), and ultrasonic treatment was carried out for 1 h to obtain the composite material (AuNPs / c-MWCNTs) dispersion.

[0044] Example 3. Preparation of the sensor

[0045] (1) Pretreatment of the glassy carbon electrode: The glassy carbon electrode was placed on a suede pad, a small amount of ultrapure water was added, and it was polished in the shape of an "8" with alumina powder to remove surface impurities. Then, it was ultrasonically cleaned successively with an ethanol-nitric acid solution (volume ratio 1:1) and ultrapure water for 10 minutes, and the washed electrode was allowed to dry naturally.

[0046] (2) Polythionine-modified electrode (PTH / GCE): The polished bare glassy carbon electrode was placed in a 5 mM thionine (THI) solution, and cyclic voltammetry was used to scan 30 cycles in the voltage range of -0.4 to 1.2 V at a scanning rate of 80 mV / s. After scanning, it was rinsed clean with ultrapure water and dried at room temperature. A blue film (PTH) appeared on the electrode surface, which was the polythionine film.

[0047] (3) Nanogold / carboxylated multi-walled carbon nanotube / polythionine-modified electrode (AuNPs / c-MWCNTs / PTH): 10 μL of the composite material dispersion prepared in Example 1 was aspirated with a pipette and dropped onto the polythionine-modified electrode, and it was allowed to dry naturally.

[0048] (4) Activation of carboxyl groups with EDC / NHS cross-linking agent: The modified glassy carbon electrode was placed in an NHS / EDC mixed amino activation solution for 1 h and dried at room temperature.

[0049] (5) Antigen binding: At room temperature, 10 μL of 100 μg / mL TG SAG1 antigen was evenly dropped onto the glassy carbon electrode modified with AuNPs / c-MWCNTs / PTH and left overnight at 4 °C. The next day, the electrode was rinsed with 0.1 M PBS buffer, and then 10 μL of skim milk blocking solution was added and incubated at room temperature for 2 h. The non-specific binding sites between the TG SAG1 antigen and the electrode surface were blocked. After blocking, it was washed with PBS and stored at 4 °C for later use.

[0050] The preparation process of the sensor is as Figure 5 shown below.

[0051] Example 4. Characterization of the electrode material

[0052] (1) Electrochemical polymerization characterization of thionine

[0053] Place the bare glassy carbon electrode in a 5 mM thionine monomer solution (pH = 6.5), and use cyclic voltammetry to scan within a certain potential range for 30 cycles to determine the potential value at which thionine monomers can undergo electrochemical polymerization, and record the detection data. The preparation method of the 5 mM thionine solution (THI) is as follows: Take 0.144 thionine in a beaker, add PBS (pH = 6.5) to dissolve it, and then make up the volume to 100 mL.

[0054] Control the potential scanning range of the thionine monomer electrolyte between -0.38 V and 0.45 V, and perform CV scanning at a scanning rate between 25 mV / s and 600 mV / s. From Figure 6 , a, it can be seen that there is a pair of redox peaks on the i-E curve, and the peak current is proportional to the square root of the scanning rate, indicating that the electrode reaction rate is diffusion-controlled. Figure 6 , b shows that at a potential of -0.38 V to -0.45 V and scanning for 20 cycles, the oxidation peak appears at -0.108 V, the reduction peak appears at -0.146 V, the peak current and peak potential are almost independent of the number of scanning times. After the scanning is completed, there is no blue polymer film on the working electrode. The reversible redox peak is formed by the oxidation and reduction properties of thionine itself. Within this potential range, thionine does not undergo electrochemical polymerization.

[0055] However, when the potential scanning range is widened to (-0.4 V to 1.2 V) and the scanning rate is 80 mV / s, within this potential range, the GCE electrode in the thionine solution Figure 7 shows that the peak current on the i-E curve gradually increases with the increase in the number of scanning times. After the scanning is completed, it is found that there is a blue polymer film on the working electrode, and this blue film is polythionine (PTH).

[0056] (2) Electron microscopy characterization of the composite material

[0057] Use field emission scanning electron microscopy (SEM: Zeiss, model: G300) and energy-dispersive X-ray (EDS: Oxford xplore30) to perform morphological and Mapping scans on the gold nanoparticles / carboxylated multi-walled carbon nanotube composite material solution for structural characterization and EDS element distribution.

[0058] The SEM results are as Figure 8 shown. Due to the large specific surface area of carbon nanotubes, the active sites of the electrode are greatly increased. Through energy chromatography (EDS) analysis, from Figure 8, from c to g and Table 1, it can be seen that the surface of the composite material mainly contains C, O, and Au elements.

[0059] Table 1. Elemental composition analysis table of the surface of AuNPs / c-MWCNTs composite material solution

[0060]

[0061] Example 5. Electrochemical characterization of the electrochemical immunosensor

[0062] The electrochemical system connected to the electrochemical workstation adopts a three-electrode system. The platinum sheet electrode is the counter electrode, the Ag / AgCl electrode is the reference electrode, and the modified electrode is the working electrode. Put them into the electrolyte to form a three-electrode system for electrochemistry.

[0063] (1) Perform cyclic voltammetry on the bare glassy carbon electrode and the glassy carbon electrode modified with AuNPs / c-MWCNTs / PTH in a 5 mM potassium ferricyanide solution, and scan at different scan rates (10 mV, 30 mV, 50 mV, 80 mV, 100 mV, 150 mV, 200 mV) respectively. The scanning voltage is -0.5 to 0.8 V. The electrochemical active area of the modified electrode and the bare glassy carbon electrode can be compared according to the Randles-Sevcik equation. Preparation method of 5 mM potassium ferricyanide solution: Weigh 0.8237 g of K3[Fe(CN)6], 1.056 g of K4[Fe(CN)6], and 3.727 g of KCl, add ultrapure water and dissolve it on a magnetic stirrer. After complete dissolution, make up the volume to 500 mL.

[0064] Compare the modified electrode and the bare electrode at different scan rates. As Figure 9 shown, the peak current Ip of the modified electrode has a linear relationship with the square root of the scan rate, indicating a diffusion-controlled process. According to the slope between the modified electrode and the bare electrode in the Randles-Sevcik equation, the effective active area of the modified electrode is approximately twice that of the bare electrode active area.

[0065] (2) Place the bare electrode, the electrode modified with AuNPs / c-MWCNTs / PTH, the electrode after antigen immobilization, the electrode after blocking with skim milk powder, and the electrode after antigen-antibody reaction into a 5 mM potassium ferricyanide solution containing 0.1 mol / L KCl electrolyte respectively, and scan by CV method. The scanning potential is set to -0.5 to 0.8 V, and the CV diagrams are compared and analyzed to verify whether the electrode modification is successful.

[0066] The chemical behavior of the modified electrode is characterized by CV method. As Figure 10As shown, curves a - e are respectively the AuNPs / c - MWCNTs / PTH modified glassy carbon electrode, the modified electrode with antigen immobilized on it, the modified electrode after being blocked with skim milk powder, the modified electrode after antigen - antibody reaction, and the bare electrode. The redox peaks of the electrode modified with AuNPs / c - MWCNT / PTH increase significantly, indicating that the material has been successfully modified onto the electrode. With the step - by - step modification of antigen, skim milk powder, and antibody, the redox peak current gradually decreases because a biofilm is formed on the electrode surface, which hinders electron transfer, indicating that the antigen has also been successfully immobilized on the modified electrode.

[0067] Example 6. Detection of Electrochemical Immunosensor and Optimization of Experimental Conditions

[0068] By means of physical adsorption and electropolymerization, the AuNPs / c - MWCNTs / PTH composite material is used to modify the GCE. Then, the Toxoplasma gondii antigen is firmly immobilized on the surface of the AuNPs / c - MWCNTs / PTH / GCE electrode. The unbound sites are blocked with a blocking solution. Different concentrations of anti - TG SAG1 antibody are dropped onto the working electrode and tested in a 5 mM potassium ferricyanide solution. The magnitude of the induced current on the working electrode is detected by differential pulse voltammetry (DPV) to achieve the detection of Toxoplasma gondii antibody. The test conditions are a scanning voltage range of - 0.3 - 0.5 V, a potential increment of 4 mV, an amplitude of 50 mV, and a pulse period of 0.5 s. Through the CHI760E electrochemical workstation, the current response signal of the sensing interface can be recorded in real - time.

[0069] During the experiment, the effects of the number of thionine polymerization cycles, the coating amount of the modifier, the antigen - antibody binding time, etc. on the determination of anti - TG SAG1 antibody are explored. Through comparative experiments, the optimal test conditions are finally determined. Using differential pulse voltammetry (DPV), through the current response values under different conditions, the difference in current ΔI = I0 - I d , where I0 is the current response value after being blocked with skim milk powder, and I d is the current response value of the detection. The results are as Figure 11 shown. When the number of thionine electropolymerization cycles is 30, the coating amount of the modified material is 10 μL, and the antigen - antibody incubation time is 60 min, a larger response signal can be obtained, thereby improving the sensitivity of the sensor.

[0070] Example 7. Sensitivity, Specificity, Repeatability, and Clinical Detection of Electrochemical Immunosensor

[0071] (1) Sensitivity

[0072] After immobilizing the TG SAG1 recombinant protein on the working electrode, 10 μL of Toxoplasma gondii antibody solutions with concentrations of 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, and 1000 pg / mL were respectively added dropwise to the blocked working electrode. The mixture was incubated in a constant temperature environment at 37 °C for 60 minutes to allow sufficient antigen-antibody reaction, and then differential pulse voltammetry (DPV) was used for relevant detection.

[0073] The sensitivity of the sensor was detected by DPV method, and the responses of the sensor to different concentrations of TG SAG1 antibody were recorded. As Figure 12 shown, as the concentration increased, the current response value gradually decreased, indicating that the specific reaction occurring on the electrode surface increased, thus preventing the transfer of electrons. Figure 13 It was shown that there was a good linear relationship between the concentration of Toxoplasma gondii antibody in the range of 1 pg / mL - 1000 pg / mL and the peak current. The linear equation was y = -15.53x + 138.096, and R 2 = 0.9837. The detection limit was measured to be 0.504 pg / mL under the condition of signal-to-noise ratio (S / N = 3).

[0074] (2) Specificity

[0075] After the electrode surface was modified with electrode materials and the TG SAG1 antigen was immobilized and blocked, rabies virus antibody (RV), pseudorabies virus antibody (PRV), brucellosis antibody, and TG SAG1 antibody and a mixture of the four antibodies were respectively modified for detection. Differential pulse voltammetry was used to record the detection data to judge the specificity of the electrochemical sensor.

[0076] DPV measurements were respectively carried out with three non-specific antibodies, TG SAG1 antibody, and a mixed solution of the four antibodies to detect the current difference ΔI between the current response value and the current response value after blocking with skim milk powder. As Figure 14 shown, the interfering substances in the non-specific antibody solution were relatively complex. The redox-active substances contained in it were oxidized / reduced at a specific potential, resulting in an increase in the background current and a negative current difference. Only in the solution containing Toxoplasma gondii antibody would a specific reaction occur. After the antigen-antibody reaction, a composite film was formed, resulting in a decrease in the peak current, indicating that the sensor had good specificity.

[0077] (3) Repeatability

[0078] The electrode materials were modified on the working electrode. After immobilizing and blocking the TG SAG1 antigen, repetitive DPV tests were carried out with the same concentration of Toxoplasma gondii antibody solution, scanning continuously four times, with an interval of 15 minutes each time. The repeatability of the electrochemical sensor was judged according to the standard deviation of the four detection results.

[0079] The results are as Figure 15 shown. The differences in the four test results are small, with an RSD of 3.08%, indicating that the sensor has good repeatability.

[0080] (4) Clinical detection

[0081] Three collected clinical positive sera and three negative sera were divided into three groups according to their concentrations, with three parallel controls set for each group. Differential pulse voltammetry was used to record the detection data to judge the accuracy of the sensor for clinical detection.

[0082] The detection results are shown in Table 2. The change amount of the first group of positive samples reached 61.03 μA, and the current change value of the negative serum was 15.47 μA; the current change value of the second group of positive sera was 39.81 μA, and the current change of the negative serum was 5.93 μA; the current change value of the third group of positive sera was 31.7 μA, and the negative serum value was 4.67 μA. Calculate the P values between the positive and negative sera in each group. P < 0.001, as Figure 16 shown, indicating a significant difference between the positive and negative samples.

[0083] Table 2. Clinical detection result diagram

[0084]

[0085] To further evaluate the clinical application value of this sensor, this study was compared with the biosensors for Toxoplasma gondii antibody detection reported in the current literature, as shown in Table 3. The sensitivity levels of the existing Toxoplasma gondii antibody biosensors are all at the pg / mL level. Compared with the traditional ELISA method (sensitivity at the ng / mL level), while the sensitivity is improved, the detection time is shortened to several minutes, and no enzyme labeling is required. Clinically, the detection of Toxoplasma gondii antibody is usually qualitative, and the negative range value is usually 0 - 1 (the unit varies depending on the method). The biosensor in this study has an approximate detection range as that of the research in foreign journals, and there is a significant difference in differentiating negative and positive sera in clinical detection. However, the operation process of this study is simpler, and the relative standard deviation is 3.08%, with good repeatability, which has good clinical application value for the rapid detection of Toxoplasma gondii antibodies in women during pregnancy preparation or pregnancy and people with low immunity.

[0086] Table 3 Comparison of this study with the published Toxoplasma gondii antibody biosensors

[0087]

[0088] The above-described embodiments are merely preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. An electrochemical immunosensor based on AuNPs / c-MWCNTs / PTH composite material, characterized in that: The preparation method is as follows: First, the gold nanoparticle solution and the carboxylated multi-walled carbon nanotube particle dispersion are ultrasonically mixed evenly to obtain the AuNPs / c-MWCNTs composite material. Then, the electron mediator thionine is electropolymerized on the surface of the glassy carbon electrode by cyclic voltammetry to form a uniform negatively charged polythionine film on its surface, obtaining the polythionine modified electrode. Then, the AuNPs / c-MWCNTs composite material is drop-coated onto the polythionine modified electrode. Finally, the recognition antigen is covalently bonded to the composite material to obtain an electrochemical immunosensor for detecting the target antibody based on the AuNPs / c-MWCNTs / PTH composite material.

2. The electrochemical immunosensor based on the AuNPs / c-MWCNTs / PTH composite material according to claim 1, wherein: The weight percentage of carbon element in the AuNPs / c-MWCNTs composite material is 80%-90%.

3. The electrochemical immunosensor based on the AuNPs / c-MWCNTs / PTH composite material according to claim 1, characterized in that: The weight percentage of oxygen element in the AuNPs / c-MWCNTs composite material is 4%-5%.

4. The electrochemical immunosensor based on the AuNPs / c-MWCNTs / PTH composite material according to claim 1, wherein: The weight percentage of gold element in the AuNPs / c-MWCNTs composite material is 2%-3%.

5. The electrochemical immunosensor based on the AuNPs / c-MWCNTs / PTH composite material according to claim 1, characterized in that: The volume ratio of the gold nanoparticle solution to the carboxylated multi-walled carbon nanotube particle dispersion is 1:

1.

6. The electrochemical immunosensor based on the AuNPs / c-MWCNTs / PTH composite material according to claim 1, wherein: The preparation method of the gold nanoparticle solution is the sodium citrate reduction of chloroauric acid method.

7. The electrochemical immunosensor based on the AuNPs / c-MWCNTs / PTH composite material according to claim 1, characterized in that: The concentration of the carboxylated multi-walled carbon nanotube particle dispersion is 1 mg / mL.

8. The electrochemical immunosensor based on the AuNPs / c-MWCNTs / PTH composite material according to claim 1, characterized in that: The recognition antigen is the Toxoplasma gondii surface antigen SAG1.

9. A detection platform comprising the electrochemical immunosensor according to any one of claims 1 to 8, characterized in that: Using a platinum sheet electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the electrochemical immunosensor according to any one of claims 1 to 8 as the working electrode, and using a potassium ferricyanide solution as the electrolyte.

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