MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor as well as preparation method and application thereof

Abstract

The invention provides an MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor as well as a preparation method and application thereof, the MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor provided by the invention has the advantages of simple preparation method, high membrane stability, low detection limit and the like, and has important significance on liver health condition analysis.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical detection technology, and in particular to a MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor and a preparation method and application thereof. Background Art

[0002] Liver disease is one of the most serious diseases that endanger human health. It is reported that there are approximately 400 million chronic hepatitis B patients and approximately 200 million chronic hepatitis C virus patients. In recent years, with the application of histology and proteomics, determining the prognosis of liver cancer patients from the aspects of molecular level, liver cancer etiology, pathogenesis, biological characteristics and new treatment methods has become a hot topic in current oncology research. Studies have confirmed that upregulation of cholyglycine (CG) is associated with hepatocellular carcinoma (HCC). Therefore, there is an urgent need for accurate and quantitative CG detection in patients suspected of liver disease.

[0003] Glycocholic acid is a combination of bile acid and glycine and is one of the main components of bile acid. Studies have found that glycocholic acid is a new specific diagnostic biomarker for hepatocellular carcinoma. The level of glycocholic acid in serum is a good indicator of liver function in liver disease. It is very important to accurately detect the level of glycocholic acid in the body. Commonly used detection methods include enzyme-linked immunosorbent assay (ELISA) and latex enhanced immunoturbidimetry. Although these methods have high sensitivity and accuracy for the detection of glycocholic acid, they require a lot of time to prepare samples, high capital costs and experimental personnel with technical foundations to operate. Therefore, for a large number of clinical test samples, it is necessary to develop some efficient, economical and easy-to-operate detection methods to meet the growing pressure of clinical testing. Summary of the invention

[0004] The first object of the present invention is to provide a method for preparing a MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor.

[0005] Specifically, the preparation method of the MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor comprises the following steps: (1) Prepare MWCNTs-MoS2 with ethanol solution, add 2-8% (preferably 5%) nafion solution thereto, and disperse by ultrasonication to obtain an electrode modification solution; (2) The glassy carbon electrode (GCE) was polished with Al2O3 of different particle sizes, and then ultrasonically cleaned to obtain an activated GCE electrode; (3) applying the electrode modification liquid droplets to the surface of the activated GCE electrode and allowing it to dry to obtain a GCE / MWCNTs-MoS2 electrode; (4) Using HAuCl4 aqueous solution as the electrolyte, the GCE / MWCNTs-MoS2 / AuNPs electrode was prepared by electrodeposition; (5) drop-coating the antibody solution onto the surface of the GCE / MWCNTs-MoS2 / AuNPs electrode and allowing it to react to obtain a GCE / MWCNTs-MoS2 / AuNPs / Anti-CG electrode; (6) The BSA solution was drop-coated on the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG electrode interface and allowed to react to obtain the MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor.

[0006] Preferably, in step (1), in the electrode modification solution, the ratio of MWCNTs to MoS2 is 25 wt %-400 wt %, more preferably 100 wt %~200 wt %. At this ratio, it is beneficial to increase the electron transfer efficiency of the sensor.

[0007] Preferably, the ethanol solution is prepared with deionized water, and more preferably the concentration of the ethanol solution is 30-60%.

[0008] Preferably, the amount of the 2%-8% nafion solution is 50 μL per mL.

[0009] Preferably, the material is ultrasonically dispersed, with a suitable power of 9-12 (preferably 11) W / L and an ultrasonic time of 20-40 min.

[0010] In step (1), after the electrode modification solution is prepared, it is stored at a low temperature of 2-6°C (preferably 4°C).

[0011] Preferably, in step (2), 0.3-0.35 μm Al2O3 and 0.05-0.1 μm Al2O3 are used in sequence to grind and polish the glassy carbon electrode GCE on the suede surface. Under the above conditions, a more ideal polishing effect can be achieved.

[0012] Preferably, the ultrasonic cleaning is performed with an ethanol solution and deionized water for 2 to 3 min each, wherein the concentration of the ethanol solution is preferably 95%.

[0013] Preferably, in step (3), the concentration of the electrode modification solution is 1.2-1.8 mg / mL, preferably 1.5 mg / mL; the volume used is 4-8 μL, preferably 5 μL.

[0014] Preferably, in step (4), the concentration of the HAuCl4 aqueous solution is 1-2 mM, preferably 1.5 mM; and / or the deposition time of the electrodeposition method is 30-120 s, preferably 60-80 s.

[0015] Under the above electrodeposition conditions, a dense layer of nano-gold particles can be deposited on the electrode surface.

[0016] Preferably, in step (4), the initial voltage of the electrodeposition is -0.1 V.

[0017] Preferably, in step (5), the concentration of the antibody solution is 80-120 μg / mL, preferably 100 μg / mL.

[0018] Preferably, the amount of the antibody solution is 8-12 μL, preferably 10 μL.

[0019] Preferably, the reaction time of the static reaction (ie, the modification time of the antibody solution) is 15 to 90 min, preferably 1 h.

[0020] Preferably, in step (6), the concentration of the BSA solution is 0.5-1.5 w / v%, preferably 1 w / v%, and the amount used is 8-12 μL, preferably 10 μL.

[0021] Preferably, in step (6), the reaction time of the static reaction is 15 to 90 min, preferably 1 h.

[0022] Under the above conditions, step (6) can be used to seal the electrode to prevent the generation of nonspecific signals.

[0023] The second object of the present invention is to provide a MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor prepared by the above preparation method.

[0024] The present invention makes full use of the specificity of antigen-antibody binding and the conductivity and dispersibility of multi-walled carbon nanotubes to prepare a MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor with significantly improved specificity, accuracy and sensitivity for glycocholic acid detection. Based on the sensor provided by the present invention, the accurate determination of the content of glycocholic acid in serum can be indirectly achieved, providing an efficient and feasible solution for evaluating the content of glycocholic acid in serum.

[0025] The third object of the present invention is to provide the application of the above-mentioned MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor in the detection of glycocholic acid.

[0026] The fourth purpose of the present invention is to provide a specific electrochemical detection method for glycocholic acid, which is beneficial to improve the accuracy of glycocholic acid detection, enhance the reproducibility between detection batches, and reduce the difficulty of detection.

[0027] The electrochemical detection method of glycocholic acid provided by the present invention utilizes the above-mentioned MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor to perform electrochemical determination of glycocholic acid.

[0028] The present invention establishes an electrochemical detection method and a standard curve of glycocholic acid by using the prepared MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor.

[0029] The method of the present invention performs electrochemical determination on mixed standard solutions of different concentrations, and draws a standard curve according to the relationship between the intensity of the oxidation current signal and the concentration of each mixed standard solution.

[0030] The specific reference of the "mixed standard solutions of different concentrations" herein is known to those skilled in the art, and the present invention does not impose any particular limitation thereto. As many concentration gradients as possible may be set to improve the accuracy; for example, 1 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL.

[0031] Preferably, the modified electrode for electrochemical determination is MWCNTs-MoS2 / AuNPs / Anti-CG.

[0032] Preferably, the method for detecting glycocholic acid provided by the present invention adopts a three-electrode detection system: the MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor is used as the working electrode, the calomel electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode.

[0033] Preferably, the method for detecting glycocholic acid provided by the present invention selects 0.4~0.8Mm (preferably 0.5Mm) K4Fe(CN)6 / K3Fe(CN)6 solution as the electrolyte.

[0034] The present invention selects the above three-electrode detection system, which can help increase the sensitivity and accuracy of detection.

[0035] Preferably, the scanning voltage is -0.4V~+0.6V, and the scanning rate is 50 mV / s.

[0036] The method for detecting glycocholic acid provided by the present invention breaks through the limitations of traditional glycocholic acid detection methods.

[0037] The beneficial effects of the present invention are: (1) The MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor provided by the present invention has the advantages of simple preparation method, high membrane stability and low detection limit.

[0038] (2) The MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor provided by the present invention has good reproducibility, selectivity and stability. The immunosensor has a wide linear detection range for CG (1 ~ 80 ng / mL) and a LOD of 1.25 pg / mL.

[0039] (3) The MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor provided by the present invention has high sensitivity and strong anti-interference ability. It shows good selectivity and accuracy in detecting CG in real serum samples, which is completely consistent with the results of latex enhanced immunoturbidimetry, indicating that the immunosensor has practical application value in liver disease detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 Magnified TEM images of MoS2 and MWCNTs-MoS2 composite materials; Figure A is the magnified TEM image of MoS2, and Figure B is the magnified TEM image of MWCNTs-MoS2 composite material.

[0042] Figure 2 SEM images of MWCNTs-MoS2, MWCNTs-MoS2 / AuNPs, MWCNTs-MoS2 / AuNPs / Anti-CG and MWCNTs-MoS2 / AuNPs / Anti-CG / BSA, among which Figure A is the SEM image of MWCNTs-MoS2, Figure B is the SEM image of MWCNTs-MoS2 / AuNPs, Figure C is the SEM image of MWCNTs-MoS2 / AuNPs / Anti-CG, and Figure D is the SEM image of MWCNTs-MoS2 / AuNPs / Anti-CG / BSA.

[0043] Figure 3 EDS image of MWCNTs-MoS2.

[0044] Figure 4 EDS image of MWCNTs-MoS2 / AuNPs.

[0045] Figure 5 EDS image of MWCNTs-MoS2 / AuNPs / Anti-CG.

[0046] Figure 6 EDS image of MWCNTs-MoS2 / AuNPs / Anti-CG / BSA.

[0047] Figure 7 Electrochemical characteristic spectrum of MWCNTs-MoS2 / AuNPs / Anti-CG sensor for detecting glycocholic acid. The electrolyte is 0.5 mM K4Fe(CN)6 / K3Fe(CN)6 solution. Three-electrode detection system: GCE / MWCNTs-MoS2 / AuNPs / Anti-CG modified electrode as working electrode, calomel electrode as reference electrode, platinum wire electrode as counter electrode, scanning voltage of -0.4v~+0.6v, scanning rate of 50 mV / s.

[0048] Figure 8 It is a schematic diagram of electrochemical detection signals of different electrodes.

[0049] Fig. 9 This is a schematic diagram of the electrochemical detection signal of the modification liquid MWCNTs-MoS2 ratio.

[0050] Fig.10 This is a schematic diagram of the effect of the time of glycocholic acid antibody modification on the electrode peak current.

[0051] Fig.11 This is a schematic diagram of the effect of 1% BSA blocking time on electrode peak current.

[0052] Fig.12 It is a schematic diagram of the relationship between electrodeposition time and electrochemical detection signal.

[0053] Fig.13 Schematic diagram of DPV and EIS response of electrodes obtained in different modification steps; Figure A is a schematic diagram of DPV response of different modification steps; Figure B is a schematic diagram of EIS response of different modification steps.

[0054] Fig.14 Schematic diagram of DPV response current of MWCNTs-MoS2 / AuNPs / Anti-CG sensor for CG detection of different concentrations.

[0055] Fig.15 It is a linear relationship diagram.

[0056] Fig.16 This is the result diagram of sensor selectivity.

[0057] Fig.17This is the result diagram of the sensor's anti-interference performance. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0060] Example 1 This embodiment provides a preparation method, comprising the following steps: 1. Accurately weigh 20 mg MoS2 and 10 mg MWCNTs and dissolve them in 20 mL 50% ethanol solution. Add 1 mL of nafion solution (concentration is 5%) and ultrasonicate for 30 min until the solution is evenly dispersed. The prepared composite material is stored at 4 °C.

[0061] Transmission electron microscopy (TEM) reveals the morphology of MoS2 and MWCNTs-MoS2. TEM images (see Figure 1 Figure A and Figure 1 Figure B in the figure shows that MoS2 is 0.5-1.5 μm long, 300-500 nm wide, has a sheet-like stacking structure, and has a relatively smooth surface; in the MWCNTs-MoS2 composite material, the MWCNTs are 300-400 nm long, 5-15 nm in diameter, and have a rod-like structure. Both are in the form of flakes and overlap each other, providing a large number of deposition sites for gold nanoparticles.

[0062] 2. Carefully polish the glassy carbon electrode (GCE) with 0.05 μm and 0.35 μm alumina powder respectively, and wash it continuously with water. After the polished electrode is blown dry, it is ultrasonically cleaned in pure water for 1 min, and the electrode after ultrasonic cleaning is blown dry to obtain an activated GCE electrode.

[0063] 3. Apply 5 μL of electrode modification solution (MWCNTs-MoS2 composite material solution) to the surface of the activated GCE electrode and dry it under a fluorescent lamp to obtain a GCE / MWCNTs-MoS2 electrode.

[0064] 4. Immerse the GCE / MWCNTs-MoS2 electrode in 1.5 mM HAuCl4 solution and perform electrodeposition using the it method with an initial voltage of -0.1 V and a deposition time of 60 s.

[0065] The electrode after electrodeposition was immersed in H2SO4 with pH=1, and the modified electrode was electrochemically characterized by the CV method. The scanning potential range was -0.4 V-1.4 V, and the scanning rate was 0.1 V / s. The reduction curve was observed to see if there was an obvious gold characteristic peak at 0.8 V.

[0066] 5. After successful modification, 10 μL of antibody solution (10 μL, 100 μg / mL) was added to the electrode surface and incubated at 4°C for 1 h to obtain the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG electrode.

[0067] 6. Drop the BSA solution (10 μL, 1 w / v%) onto the surface of the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG electrode and incubate at room temperature for 1 h (to avoid nonspecific interactions) to obtain the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG / BSA immunosensor.

[0068] In the preparation method provided in this embodiment, the modified electrode is cleaned with distilled water at the end of each modification step.

[0069] Example 2 This example is a TEM image of the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor prepared in Example 1.

[0070] Transmission electron microscopy (TEM) reveals the morphology of MoS2 and MWCNTs-MoS2. TEM images (see Figure 1 Figure A and Figure 1 Figure B in the figure shows that MoS2 is 0.5-1.5 μm long, 300-500 nm wide, has a sheet-like stacking structure, and has a relatively smooth surface; in the MWCNTs-MoS2 composite material, MWCNTs are 300-400 nm long, 5-15 nm in diameter, and have a rod-like structure. Both are in the form of flakes and overlap with each other, providing a large number of deposition sites for AuNPs.

[0071] Example 3 This example shows the SEM and EDS images of the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor prepared in Example 1.

[0072] Figure 2Representative SEM images of GCE / MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor. Figure 2 Figure A in the middle is the SEM image of MWCNTs-MoS2. Figure 2 Figure B is the SEM image of MWCNTs-MoS2 / AuNPs. Figure 2 Figure C in the middle is the SEM image of MWCNTs-MoS2 / AuNPs / Anti-CG. Figure 2 Figure D in the middle is the SEM image of MWCNTs-MoS2 / AuNPs / Anti-CG / BSA.

[0073] Figure 3 This is the EDS image of MWCNTs-MoS2. Figure 4 This is the EDS image of MWCNTs-MoS2 / AuNPs. Figure 5 This is the EDS image of MWCNTs-MoS2 / AuNPs / Anti-CG. Figure 6 EDS image of MWCNTs-MoS2 / AuNPs / Anti-CG / BSA.

[0074] The surface of the electrode prepared in this example was observed by SEM. Figure 2 As shown in Figure A, MoS2 is a lamellar structure, and MWCNTs is a tubular structure with a diameter of about 15nm. The uniform compound between MoS2 and MWCNTs can reduce the accumulation of MoS2 and obtain a nanostructure with an ultra-high specific area, which can provide many sites for subsequent material attachment. Its EDS image also shows the distribution of Mo and S elements. Figure 2 As shown in Figure B, gold nanoparticles are deposited on the surface of the modified material by electrodeposition, further increasing the specific surface area of ​​the modified material and providing a large number of binding sites for antibodies to bind to the electrode surface. The EDS image shows that there are Au elements on the electrode surface, indicating that gold nanoparticles have been successfully deposited on the electrode surface. Figure 2 As shown in Figure C, the volume of the gold nanoparticles on the electrode surface after antibody modification increases significantly, and the surface morphology becomes smoother. This is because the antibody is an immunoglobulin, and after combining with the gold nanoparticles, a larger spherical material is deposited on the electrode surface. In addition, the protein molecule contains a large amount of O and N elements. The peaks of O and N elements appear at the corresponding positions in the EDS image, which corresponds to the SEM results. BSA is a smaller protein molecule and can bind to multiple active sites on the electrode surface. Figure 2 As shown in Figure D, more small molecule proteins are deposited on the electrode surface to block potential active sites, causing the electrode to exhibit specific detection performance for glycocholic acid.

[0075] Example 4 This example is a clinical application example of the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor prepared in Example 1.

[0076] Human serum samples were collected from volunteers of the Beijing Agricultural College Hospital, and consent was obtained from all volunteers. This study adopted a random sampling method, and a total of 10 blood samples from healthy volunteers were collected, of which 7 were added with standard CG solution to establish CG disease samples for experimental research, as follows: 3 mL of fasting venous blood from volunteers was collected using a blood collection tube containing heparin anticoagulant and numbered, centrifuged for 15 min at a speed of 3000 rpm, and the supernatant was collected with a pipette and injected into an EP tube. The sample was sealed with a sealing film and stored in a refrigerator at -20°C for standby use. When used, the serum sample was diluted 1000 times with ultrapure water to eliminate the influence of the matrix effect on the experimental results to the greatest extent. The GCE / MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor prepared in Example 1 was immersed in the diluted serum sample, incubated at 37°C for 15 min, and then DPV detection was performed to record its electrochemical signal.

[0077] Test Example 1 This test example verifies the effect of different modified electrodes on the electrochemical detection performance of glycocholic acid. The specific operation is as follows: A 1 mg / mL MWCNTs solution, a 1 mg / mL MWCNTs-2 mg / mL MoS2 solution, and a 1 mg / mL MWCNTs-2 mg / mL C3N4 solution were prepared with a mixture of deionized water and ethanol in a ratio of 1:1, and 5% nafion solution was added to each solution. The solution was ultrasonicated for 30 min to prepare the electrode modification solution.

[0078] GCE activated electrodes were prepared according to the method provided in Example 1, and three modified electrodes, GCE / MWCNTs / Anti-CG, GCE / MWCNTs-MoS2 / Anti-CG, and GCE / MWCNTs-C3N4 / Anti-CG, were prepared respectively.

[0079] A three-electrode detection system was selected, i.e., GCE, GCE / MWCNTs / Anti-CG, GCE / MWCNTs-MoS2 / Anti-CG, and GCE / MWCNTs-C3N4 / Anti-CG electrodes were used as working electrodes, calomel electrode was used as reference electrode, platinum wire electrode was used as counter electrode, and 0.5 mM K4Fe(CN)6 / K3Fe(CN)6 solution was used as electrolyte for electrochemical detection. The results are shown in the figure. Figure 7 .

[0080] Figure 8This is the electrochemical characteristic peak current result when the concentration of glycocholic acid is 40 ng / mL. Figure 8 It can be seen that in 0.5mM K4Fe(CN)6 / K3Fe(CN)6 solution, no characteristic peak current was observed when glycocholic acid was detected using the GCE electrode (a); when glycocholic acid was detected using the GCE / MWCNTs / Anti-CG electrode (b), a characteristic peak current of glycocholic acid was generated, indicating that the MWCNTs-modified GCE electrode can effectively improve the electrocatalytic activity of glycocholic acid; when lactic acid was detected using the GCE / MWCNTs-MoS2 / Anti-CG electrode (c), the current signal increased significantly and the peak current was the strongest, indicating that the combined effect of MWCNTs-MoS2 further improved the electrocatalytic activity; when glycocholic acid was detected using the GCE / MWCNTs-C3N4 / Anti-CG (d) modified electrode, the electrochemical characteristic peak current decreased, indicating that the effect of MWCNTs-C3N4 reduced the electrocatalytic activity and electron transfer performance of the electrode surface. Therefore, the GCE / MWCNTs-MoS2 / Anti-CG modified electrode had the best effect.

[0081] Test Example 2 This test example verifies the effect of different ratios of MWCNTs-MoS2 in the electrode modification solution on the electrochemical detection performance of glycocholic acid. The specific operation is as follows: According to the electrode preparation method provided in Example 1, different MWCNTs-MoS2 ratios were used to verify the effect on the electrochemical detection signal. The results are shown in Fig. 9 .

[0082] from Fig. 9 It can be seen that the modification of MWCNTs-MoS2 at the electrode interface can significantly promote the interaction between glycocholic acid and antibodies. As the proportion of MoS2 increases, the peak current signal of glycocholic acid at the electrode interface also gradually increases, and the signal is strongest when the ratio (wt%) is 200%.

[0083] Test Example 3 This test example verifies the effect of different incubation times of glycocholic acid antibody on the electrochemical detection performance of glycocholic acid. The specific operation is as follows: According to the electrode preparation method provided in Example 1, different incubation times of glycocholic acid antibodies were used to verify the effect on the electrochemical detection performance of glycocholic acid. The results are shown in Fig.10 .

[0084] from Fig.10It can be seen that with the increase of the incubation time of glycocholic acid antibody on the electrode surface, the peak current signal on the electrode surface tends to be stable. When the modification time is 60 min, the peak current change of the prepared electrode surface is basically stable. Therefore, when preparing GCE / MWCNTs-MoS2 / AuNPs / Anti-CG modified electrode, the antibody incubation time of 60 min is the best.

[0085] Test Example 4 This test example verifies the effect of different blocking time of 1% BSA solution on the electrochemical detection performance of glycocholic acid. The specific operation is as follows: According to the electrode preparation method provided in Example 1, different 1% BSA solution blocking times were used to verify the effect on the electrochemical detection performance of glycocholic acid. The results are shown in Fig.11 .

[0086] from Fig.11 It can be seen that with the increase of BSA blocking time on the electrode surface, the peak current signal on the electrode surface tends to be stable. When the modification time is 60 min, the peak current change on the prepared electrode surface is basically stable. Therefore, when preparing GCE / MWCNTs-MoS2 / AuNPs / Anti-CG / BSA immunosensor, BSA incubation for 60 min is the best.

[0087] Test Example 5 This test example verifies the effect of different electrodeposition times on the electrochemical detection performance of glycocholic acid. The specific operation is as follows: According to the electrode preparation method provided in Example 1, different electrodeposition times were used to verify the effect on the electrochemical detection performance of glycocholic acid. The results are shown in Fig.12 .

[0088] from Fig.12 It can be seen that the electrodeposition time has a significant effect on the performance of the electrode interface. With the extension of the electrodeposition time, the detection performance of the prepared GCE / MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor for glycocholic acid improves, and the signal is the highest at 60s. Therefore, the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor is prepared with an optimal electrodeposition time of 60s.

[0089] Experimental Example 6 Electrochemical Characterization In this experimental example, the state of the working electrode before and after each modification step was characterized by DPV and EIS. Five states of the electrode were characterized, including the polished glassy carbon electrode GCE; the glassy carbon electrode modified with GCE / MWCNTs-MoS2; the glassy carbon electrode modified with GCE / MWCNTs-MoS2 / AuNPs; the glassy carbon electrode modified with GCE / MWCNTs-MoS2 / AuNPs / Anti-CG and the glassy carbon electrode modified with GCE / MWCNTs-MoS2 / AuNPs / Anti-CG / BSA.

[0090] DPV characterization results are as follows Fig.13 As shown. Fig.13 As shown in Figure A, the polished glassy carbon electrode GCE has a significant potassium ferrocyanide absorption peak near 0.2V. After drop-coating the MWCNTs-MoS2 material on the electrode surface, the electrode conductivity decreased slightly. This may be due to the lamellar structure of MoS2, which increases the electron transfer distance and leads to a decrease in conductivity. Compared with MWCNTs-MoS2, the DPV current signal intensity obtained by electrodeposition of nanogold is significantly enhanced. This is because AuNPs are deposited in the gaps between MoS2 layers, increasing the conductivity of the electrode surface. After the surface is fixed with antibodies and blocked with BSA, the electrode conductivity decreases. This is because the specific immune reaction between antigens and antibodies will form a larger non-conductive immune complex on the electrode surface, reducing the electron transfer efficiency on its surface; after BSA is blocked on the electrode surface, a non-conductive protein molecule layer is also formed, which ultimately manifests as a decrease in the DPV electrical signal.

[0091] Fig.13 Figure B shows the different modified electrodes at 5 mM [Fe(CN)6] 3- / 4-EIS response in a solution containing 0.1 M KCl. The electron transfer resistance (Ret) of the electrode surface is represented by the diameter of a semicircle in the impedance spectrum. The bare gold electrode shows a large semicircular area. When the electrode surface is modified with MWCNTs-MoS2 nanocomposite, the semicircle diameter is significantly reduced compared with the bare electrode, which proves that the electrode resistance of the modified nanocomposite is reduced, showing that the prepared MWCNTs-MoS2 has excellent conductivity and can accelerate the transfer of electrons on the electrode surface. The electrode resistance is further reduced after the electrode is electrodeposited with gold nanoparticles, indicating that the gold nanoparticles are successfully modified and the conductivity is further increased. After the CG antibody is fixed on the electrode surface, the semicircle diameter is significantly increased compared with the electrode with the modified material, and the electrode impedance increases. Because the antibody has biological activity, it inhibits the electron transfer of the redox probe on the modified electrode surface. After that, BSA is modified on the electrode surface to block the excess active binding sites on the electrode surface. Since BSA is a non-conductive biomolecule, the impedance value of the electrode surface is further increased, so the semicircle diameter is larger than that in the previous step, indicating that BSA is successfully modified on the electrode surface. The above results confirmed the successful preparation of the immunosensor.

[0092] Test Example 7 CG Antigen DPV Detection and Selectivity Test This experimental example verifies the anti-interference ability of the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG modified electrode (i.e., immunosensor, the same below).

[0093] Based on the electrochemical immunosensor constructed in this study, under the optimal experimental conditions, the 3- / 4- The aqueous solution of 0.1 mol / L KCl was used as the electrolyte solution. The concentration gradient of CG antigen standard solution was 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, and 80 ng / mL. The concentrations were measured three times in parallel and the average value of the measurement results was taken. The DPV detection spectrum was shown in the figure. Figure 14~Figure 17 As shown. Fig.14 It can be seen that the target detection object has a peak at a potential of about 0.15 V, and as the concentration of the CG antigen solution increases, the DPV response signal curve gradually rises.

[0094] Furthermore, the DPV response current signal difference ∆I corresponding to each concentration of CG antigen solution was calculated, and then the standard curve was fitted with the concentration of CG antigen solution as the horizontal axis (X ng / mL) and ∆I as the vertical axis, as shown in Fig.15As shown, the linear equation in the range of 1-6 ng / mL was calculated to be y = 2.47103x + 11.14443, with a linear correlation coefficient of r = 0.995, and the linear equation in the range of 10-80 ng / mL was calculated to be y = 0.13522x + 32.79891, with a linear correlation coefficient of r = 0.985.

[0095] 2 mL of 1 μM CG, glucose, uric acid, and ascorbic acid solutions, as well as 2 mL of 2 μM CG-glucose, CG-uric acid, CG-ascorbic acid, and CG-glucose-uric acid-ascorbic acid solutions were prepared, respectively, and then the prepared immunosensors were immersed in them and incubated in a heating device at 37 °C for 15 min.

[0096] like Fig.16 , Fig.17 As shown in the figure, through DPV detection, it was found that the current of the immunosensor increased significantly after the interaction with CG, while the current of other single interfering substances increased slightly, and the detection signal of the mixed interfering substance solution was basically consistent with the CG detection signal. This shows that the immunosensor has good anti-interference ability.

[0097] Test Example 8 Spike recovery experiment In order to measure the practical application value of the electrochemical immunosensor constructed in this study, the accuracy of the immunosensor was verified by a spike recovery experiment.

[0098] Serum samples from healthy humans were selected, and low, medium and high concentrations of CG antigen standard solutions (1 ng / mL, 10 ng / mL, and 20 ng / mL) were added to the serum samples, respectively. After being diluted 1000 times with pure water and mixed evenly, the constructed electrochemical immunosensor was used to perform DPV detection on the samples in turn. Each spiked sample was measured in parallel 3 times. The measured current signals and analysis results are shown in Table 1. The spiked recovery rates of actual samples were between 98.3% and 105.2%, and the corresponding relative standard deviations were between 0.72% and 3.09%, which met the requirements of the relevant standards for the spiked recovery results. This shows that the constructed immunosensor has good accuracy and can be applied to the detection of CG in actual serum samples.

[0099] Table 1

[0100] Experimental Example 9 Comparison of the detection performance of electrochemical immunosensor and immunoenhanced latex turbidimetry According to the electrode preparation method disclosed in Example 1, the detection performance of the immunosensor detection method was compared with that of the latex enhanced immunoturbidimetry.

[0101] A total of 7 positive samples (the concentration of glycocholic acid was 40 ng / mL after serum was diluted 1000 times, which can be diagnosed as an abnormal value) and 3 negative samples (normal serum) were set up and tested using two methods under label-free conditions. The results are shown in Table 2.

[0102] It can be seen from Table 2 that the detection rates of the immunosensor and latex enhanced immunoturbidimetry are consistent. Therefore, the immunosensor of the present invention has the performance of detecting actual samples.

[0103] Table 2

[0104] +: positive result; -: negative result; NC: negative control.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor, comprising the following steps: (1) MWCNTs-MoS2 is prepared with an ethanol solution, a 2-8% nafion solution is added thereto, and ultrasonication is performed to disperse the MWCNTs-MoS2 to obtain an electrode modification solution; (2) The glassy carbon electrode (GCE) was polished with Al2O3 of different particle sizes, and then ultrasonically cleaned to obtain an activated GCE electrode; (3) applying the electrode modification liquid droplets onto the surface of the activated GCE electrode and allowing it to dry to obtain a GCE / MWCNTs-MoS2 electrode; (4) Using HAuCl4 aqueous solution as the electrolyte, the GCE / MWCNTs-MoS2 / AuNPs electrode was prepared by electrodeposition; (5) drop-coating the antibody solution onto the surface of the GCE / MWCNTs-MoS2 / AuNPs electrode and allowing it to react to obtain a GCE / MWCNTs-MoS2 / AuNPs / Anti-CG electrode; (6) The BSA solution was drop-coated on the GCE / MWCNTs-MoS2 / AuNPs / Anti-CG electrode interface and allowed to react to obtain the MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor.

2. The preparation method according to claim 1, characterized in that: In the electrode modification solution, the ratio of MWCNTs to MoS2 is 25%-400wt%; and / or, the concentration of the electrode modification solution in step (3) is 1.2-1.8mg / mL, and the volume used is 4~8 μL.

3. The preparation method according to claim 1 or 2, characterized in that: In step (2), the glassy carbon electrode GCE is polished on the suede surface using 0.3-0.35 μm Al2O3 and 0.05-0.1 μm Al2O3 in turn.

4. The preparation method according to claim 1 or 2, characterized in that: In step (4), the concentration of the HAuCl4 aqueous solution is 1-2 mM; and / or the deposition time of the electrodeposition method is 30-120 s.

5. The preparation method according to claim 1 or 2, characterized in that: In step (5), the concentration of the antibody solution is 80-120 μg / mL; the amount of the antibody solution is 8-12 mL; and / or the reaction time of the static reaction is 15-90 min.

6. The preparation method according to claim 1 or 2, characterized in that: In step (6), the concentration of the BSA solution is 0.5-1.5 w / v%, the dosage is 8-12 mL, and / or the reaction time of the static reaction is 15-90 min.

7. A MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor, characterized in that: The method is prepared by any one of claims 1 to 6.

8. Use of the MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor according to claim 7 in the detection of glycocholic acid.

9. An electrochemical detection method for glycocholic acid, characterized in that: The MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor described in claim 7 is used to electrochemically determine glycocholic acid.

10. The electrochemical detection method according to claim 9, characterized in that: A three-electrode detection system was adopted: MWCNTs-MoS2 / AuNPs / Anti-CG immunosensor was used as the working electrode, calomel electrode was used as the reference electrode, and platinum wire electrode was used as the counter electrode; the scanning voltage was -0.4v~+0.6v, and the scanning rate was 50 mV / s.

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