A high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance and its preparation method and application

Abstract

The present invention discloses a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance, and a preparation method and application thereof, belonging to the field of biosensor technology. The present invention uses a high-photocurrent signal-responsive FTO / ZnIn2S4 photoanode and a FTO / CuInS2 / Pt photocathode as signal conversion elements, modifies the capture antibody corresponding to the target antigen on the photocathode, and uses a zwitterionic peptide with a sequence of SKSESKSEPPPPC as an anti-biomacromolecule pollution interface brush to prevent non-specific adsorption of biomacromolecules such as proteins on the electrode surface to generate false positive signals and reduce the specificity and sensitivity of the detection. Through the specific immune reaction between the capture antibody and the target antigen, the specific detection of the target antigen is achieved. The high-photocurrent signal-responsive FTO / ZnIn2S4 photoanode can generate a large photocurrent signal, and the dual-photoelectrode immunosensor can detect the target antigen with high sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and relates to a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance, and a preparation method and application thereof. Background Art

[0002] Accurate early diagnosis of common major diseases can timely detect and effectively prevent and control diseases, thereby effectively protecting people's health. Photoelectrochemical immunosensor is an emerging sensing and analysis technology developed by organically combining electrochemical analysis and photoelectrochemical technology. This technology has the advantages of simple equipment, low cost, simple operation process, and easy miniaturization; at the same time, it also has the characteristics of low background interference, higher sensitivity, and self-powered system. Based on these characteristics, photoelectrochemical immunosensor technology is easier to perform real-time and rapid on-site analysis and detection, which has aroused more and more research interest.

[0003] Based on the properties of the photoelectrode, photoelectrochemical immunosensors can be divided into two sensing categories: photoanode sensing and photocathode sensing. Since photoanode immunosensors rely on electrons as the main charge carriers in the system, a large photocurrent signal is generated when an electron donor is present, and thus photoanode immunosensors tend to have higher sensitivity. However, since the hole oxidation reaction occurs at the photoanode interface, the photoanode immunosensor has weak anti-interference ability to reductants such as glutathione, dopamine, and ascorbic acid that coexist in biological samples. However, although the photocathode immunosensor can overcome the disadvantage of poor anti-interference of the photoanode immunosensor, because the electron reduction reaction occurs at the photocathode interface, the potential coexisting reductant will not be oxidized to produce an interference signal. Although photocathode immunosensors are more capable of accurate detection in complex biological matrices, in actual complex samples such as serum solutions and cell tissue fluids, in addition to the possible dissolution of small molecule reducing agents such as glutathione, dopamine, and ascorbic acid, there are also multi-component biomacromolecules such as proteins. Photocathode immunosensors cannot resist the nonspecific adsorption of biomacromolecules such as proteins on the electrode surface. In addition, the photocathode uses holes as the main charge carrier and cannot use electron donors to provide electrons, resulting in significantly weaker photocurrent signals and lower sensitivity. In order to prevent the nonspecific adsorption of biomacromolecules such as proteins on the electrode surface to produce false positive signals and reduce the specificity and sensitivity of detection, the anti-pollution performance of peptides has begun to attract people's attention.

[0004] It has been demonstrated that the zwitterionic peptide sequence EKEKEKEPPPPC, which contains alternating negatively charged glutamic acid (E) and positively charged lysine (K), can effectively prevent nonspecific biomacromolecules from being adsorbed on the electrode surface through hydrophobic interactions and electrostatic effects to a certain extent due to its strong hydrophilicity and electrical neutrality, thus showing excellent anti-fouling ability.

[0005] The latest data show that the incidence of breast cancer is 24.2% of female cancers worldwide, ranking first among female cancers. In China, the incidence of breast cancer is particularly obvious. Cancer antigen 15-3 (CA15-3) is one of the most important biomarkers for breast cancer, and its sensitive and accurate detection has important clinical significance for early diagnosis and prognosis evaluation. Therefore, the present invention uses CA15-3 as the target detection object, which has certain representativeness. Summary of the invention

[0006] In view of this, one of the purposes of the present invention is to provide a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance in response to the problems in the prior art. The sensitivity of the dual-photoelectrode immunosensor can be improved by utilizing a high photocurrent signal response photoanode, and the zwitterionic peptide with a sequence of SKSESKSEPPPPC can be used to reduce the nonspecific adsorption of biomacromolecules on the surface of the photocathode, thereby improving the specificity and sensitivity of the photocathode for target antigen detection. This can not only realize the anti-interference ability of the dual-photoelectrode immunosensor against reducing agents coexisting in biological samples, but also prevent the nonspecific adsorption of biomacromolecules such as proteins on the electrode surface to generate false positive signals and reduce the specificity and sensitivity of the detection.

[0007] The second object of the present invention is to provide a method for preparing a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance, wherein the preparation process has mild conditions, simple steps, easy operation and low cost.

[0008] The third purpose of the present invention is to provide an anti-pollution dual-photoelectrode photoelectrochemical immunosensor for use in the preparation of in vitro diagnostic products, which can detect CA15-3, the most important biomarker for breast cancer, in vitro, with low consumption of biological samples and significant non-specific adsorption effect against biological macromolecules such as proteins.

[0009] In order to achieve one of the above purposes, the technical solution of the present invention is as follows:

[0010] A highly sensitive dual-photoelectrode immunosensor with anti-biomacromolecule fouling performance, including high photocurrent signal response FTO / ZnIn 2 S 4 Photoanode and FTO / CuInS 2 / Pt photocathode, the FTO / CuInS 2 The surface of the FTO / CuInS photocathode was first modified with the capture antibody corresponding to the target antigen, and then the modified FTO / CuInS 2 The / Pt photocathode was further modified with a zwitterionic peptide with the sequence SKSESKSEPPPPC.

[0011] Furthermore, the concentration of the zwitterionic peptide with the sequence SKSESKSEPPPPC is 0.1-0.5 mg / mL.

[0012] Furthermore, the concentration of the capture antibody is 100-250 μg / mL.

[0013] Furthermore, the target antigen is preferably CA15-3.

[0014] In order to achieve the second of the above objectives, the present invention adopts the following technical solution:

[0015] A method for preparing a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance comprises the following steps:

[0016] S1. Grow ZnIn on FTO conductive glass by hydrothermal method 2 S 4 Nanosheets, preparation of high photocurrent signal response FTO / ZnIn 2 S 4 The FTO conductive glass is SnO doped with fluorine. 2 Conductive glass;

[0017] S2. Electrodeposition of CuInS on FTO conductive glass 2 , and obtain FTO / CuInS 2 Electrode, Pt nanoparticles are modified to the FTO / CuInS by electrostatic adsorption 2 On the electrode, FTO / CuInS was prepared 2 / Pt photocathode;

[0018] S3, modifying the capture antibody corresponding to the target antigen to the FTO / CuInS 2 / Pt photocathode, the zwitterionic peptide with the sequence SKSESKSEPPPPC was applied to the modified FTO / CuInS 2 The / Pt photocathode was further modified to obtain a high-sensitivity dual-photoelectrode immunosensor with resistance to biomacromolecule contamination.

[0019] Furthermore, the specific steps of S1 are: using ZnSO 4 、InCl 3 and C 2 H 5 NS were used as Zn source, In source and S source, respectively, wherein the ZnSO 4The concentration is 9 to 18 mM, and the molar ratio of the Zn source, the In source and the S source is fixed to be 1:2:4. The mixed solution is stirred for reaction for 30 to 60 minutes to obtain a precursor solution, and then the precursor solution is transferred to a high-pressure reactor, and the FTO conductive glass is vertically placed in the high-pressure reactor. Under sealed conditions, it is maintained at 160° C. for 8 to 14 hours, and naturally cooled to room temperature to form a uniform film on the surface of the FTO conductive glass. The uniform film is washed with water and ethanol in turn, and dried at 60° C. for 1 hour to obtain the high photocurrent signal response FTO / ZnIn 2 S 4 Photoanode.

[0020] Furthermore, the specific steps of S2 are: using the FTO conductive glass as a working electrode, a platinum wire as a counter electrode, and a saturated calomel electrode as a reference electrode, inserting the FTO conductive glass into a 2 , 4mM InCl 3 and 12 mM sulfur powder in a dimethyl sulfoxide deposition solution, the deposition potential is -1.0 V to -1.50 V, and then a one-step deposition method is used for 20 to 40 minutes to obtain the FTO / CuInS 2 The FTO / CuInS electrode was cleaned with deionized water. 2 After the electrode, 20 μL of Pt nanoparticle solution was added to the FTO / CuInS 2 The electrode was dried at room temperature to obtain the FTO / CuInS 2 / Pt photocathode.

[0021] Furthermore, the specific steps of S3 are: in the FTO / CuInS 2 20-25 μL of the capture antibody corresponding to the target antigen was added to the Pt photocathode for modification, and incubated at 4°C for 10-15 h; the modified FTO / CuInS was quenched with 10 mM, pH 7.4 phosphate buffer. 2 After the / Pt photocathode is cleaned, 20 to 25 μL of the zwitterionic peptide solution with the sequence SKSESKSEPPPPC is added dropwise to the modified FTO / CuInS 2 / Pt photocathode and incubated at 4°C for 4 to 8 hours to obtain a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance.

[0022] In order to achieve the third of the above objectives, the present invention adopts the following technical solutions:

[0023] The present invention provides the use of the anti-pollution dual-photoelectrode photoelectrochemical immunosensor in the preparation of in vitro diagnostic products.

[0024] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following excellent effects:

[0025] 1) The FTO / CuInS dual-photoelectrode immunosensor with high sensitivity and anti-biomacromolecule pollution performance provided by the present invention 2 / Pt photocathode modified with a zwitterionic peptide with the sequence SKSESKSEPPPPC, the zwitterionic peptide embedding neutral serine (S) between the charged E and K can effectively block the electrostatic attraction of the E and K side chains, and the hydrophilicity is further enhanced, so it has a better ability to resist biomacromolecule pollution. Therefore, it can be used as an anti-biomacromolecule pollution interface brush to prevent the nonspecific adsorption of biomacromolecules such as proteins on the electrode surface to produce false positive signals and reduce the specificity and sensitivity of the detection. By capturing the specific immune reaction between the antibody and the target antigen, the specific detection of the target antigen is achieved, and the high photocurrent signal response FTO / ZnIn 2 S 4 The photoanode can generate a larger photocurrent signal, overcoming the FTO / CuInS 2 / Pt photocathode cannot utilize electron donors to provide electrons, resulting in a significantly weak photocurrent signal and low sensitivity. The dual photoelectrode immunosensor has the unique advantages of obvious photocurrent signal for target antigen detection, high sensitivity, and good anti-biomacromolecule contamination performance.

[0026] 2) The highly sensitive dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance of the present invention does not require purification for the detection of target antigens, is convenient and rapid, and is a high-performance immunosensor that integrates the advantages of photocathode immunosensors and photoanode immunosensors. It not only provides an efficient sensing mode for photoelectrochemical biosensors, but also can effectively improve the accuracy of disease marker detection in actual biological samples, which has far-reaching significance for in vitro diagnosis of diseases.

[0027] 3) The method for preparing the high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance provided by the present invention has mild conditions, simple steps, easy operation and low cost during the preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the embodiment will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1The FTO / ZnIn corresponding to the different concentrations of Zn source in Example 1 2 S 4 Current signal response of the photoanode.

[0030] Figure 2 For different CuInS in Example 1 2 Deposition time corresponding to FTO / CuInS 2 / Pt photocathode current signal response diagram.

[0031] Figure 3 The Ab-modified FTO / CuInS corresponding to the different capture antibody incubation concentrations in Example 1 2 / Pt photocathode current signal response diagram.

[0032] Figure 4 For FTO / ZnIn in Example 2 2 S 4 Scanning electron micrograph of the photoanode.

[0033] Figure 5 For FTO / ZnIn in Example 2 2 S 4 X-ray diffraction pattern of the photoanode.

[0034] Figure 6 FTO / CuInS in Example 3 2 Scanning electron micrograph of Pt photocathode.

[0035] Figure 7 FTO / CuInS in Example 3 2 X-ray diffraction pattern of Pt / Pt photocathode.

[0036] Figure 8 This is a current signal response diagram of the photocathode preparation process with anti-biomacromolecule pollution performance in Example 4.

[0037] Fig. 9 This is a fluorescence microscope image of the photocathode with anti-biomacromolecule contamination performance in Example 4.

[0038] Fig.10 This is a photocurrent signal diagram of the high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance in Example 5 for detecting CA15-3.

[0039] Fig.11 This is a standard curve diagram of the high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance for CA15-3 detection in Example 5.

[0040] Fig.12This is a graph showing the anti-interference experimental data of the high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance in Example 6.

[0041] Fig.13 This is a graph showing the anti-biomacromolecule contamination ability of the dual photoelectrode immunosensor modified with two zwitterionic peptides, SKSESKSEPPPPC and EKEKEKEPPPPC, in Example 7. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0043] First of all, it should be noted that the current signal response in the experimental process of the following examples was tested on a photoelectrochemical system, as follows:

[0044] The emission wavelength is 430nm and the light intensity is 350W / m 2 The LED light was used as the excitation light source, which was turned on and off every 10 seconds. The photocurrent was recorded by an electrochemical workstation. When testing the current signal response of the photoanode, a two-electrode system was used: a photoanode with a modified area of ​​0.7mm×0.7mm was used as the working electrode, and a platinum wire electrode was used as the counter electrode; when testing the current response of the photocathode, a two-electrode system was used: a photocathode with a modified area of ​​0.7mm×0.7mm was used as the working electrode, and the optimized FTO / ZnIn 2 S 4 The photoanode serves as the counter electrode; and there is no external voltage applied to the system, and the system is self-powered.

[0045] Example 1

[0046] Optimization of detection conditions

[0047] 1. ZnSO 4 Solution concentration

[0048] FTO / ZnIn 2 S 4 The current signal response of the photoanode has an important influence on the detection sensitivity of the final anti-fouling dual photoelectrode immunosensor. 2 S 4 The preparation process parameters of the photoanode were optimized:

[0049] Due to ZnIn 2 S 4The deposition amount on the electrode can be adjusted by the precursor concentration. Under the condition that the molar ratio of Zn source, In source and S source in the precursor is fixed at 1:2:4, the concentration of the precursor Zn source is optimized. 4 、InCl 3 and C 2 H 5 NS were used as Zn source, In source and S source respectively, ZnSO 4 The concentrations of the solutions were 6mM, 9mM, 12mM, 15mM, 18mM, and 21mM, respectively. The mixed solution was stirred for 30 minutes to obtain a precursor solution, which was then transferred to a high-pressure reactor. The FTO conductive glass was placed vertically in a high-pressure reactor, kept at 160°C for 12 hours under sealed conditions, and naturally cooled to room temperature. The uniform film on the FTO conductive glass was washed with water and ethanol in turn, and dried at 60°C for 1 hour to obtain FTO / ZnIn 2 S 4 Photoanode.

[0050] By performing photocurrent characterization tests, it can be obtained, as shown in the attached Figure 1 As shown, ZnSO 4 When the concentration is 15mM, FTO / ZnIn 2 S 4 The current signal response of the photoanode is the best, which is due to the appropriate thickness of ZnIn 2 S 4 It can effectively improve the photoelectric conversion efficiency of the photoanode, while excess ZnIn 2 S 4 It will significantly increase the probability of charge recombination, leading to electron annihilation. Therefore, ZnSO 4 The concentration of 15mM is the optimal preparation process parameter.

[0051] 2. CuInS 2 Deposition time

[0052] FTO / CuInS 2 As one of the signal conversion elements, the Pt photocathode has an important influence on the detection performance of the final prepared dual photoelectrode immunosensor with high sensitivity and anti-biomacromolecule pollution performance. 2 The preparation process parameters of Pt photocathode were optimized:

[0053] CuInS 2 The modification amount can be adjusted by the deposition time, so for CuInS 2 The electrochemical deposition time was optimized. CuInS was deposited on FTO conductive glass using a constant potential method and an electrochemical workstation. 2The FTO conductive glass was used as the working electrode, the platinum wire as the counter electrode, and the saturated calomel electrode as the reference electrode. The FTO conductive glass was inserted into the 4mM CuCl 2 , 4mM InCl 3 The FTO / CuInS was prepared by a one-step method in a dimethyl sulfoxide deposition solution containing 12 mM sulfur powder at a deposition potential of -1.30 V for 20 min, 25 min, 30 min, 35 min, and 40 min, respectively. 2 After cleaning the electrode with deionized water, 20 μL of Pt nanoparticle solution was added to the FTO / CuInS 2 The electrode was dried at room temperature to obtain FTO / CuInS 2 / Pt photocathode.

[0054] By performing photocurrent characterization tests, it can be obtained, as shown in the attached Figure 2 As shown, when CuInS 2 When the deposition time of FTO / CuInS increases from 20min to 30min, 2 / Pt photocathode current signal response gradually increased. However, when the deposition time exceeded 30min, the current signal response gradually decreased due to the obvious increase in the electron movement diffusion resistance. Therefore, CuInS 2 The electrochemical deposition time of 30 min is the optimal preparation process parameter.

[0055] 3. Capture antibody incubation concentration

[0056] The amount of capture antibody modified on the sensing electrode has a significant impact on the quantitative detection range of the anti-fouling dual-photoelectrode immunosensor, so the preparation process parameters of the modified capture antibody were optimized:

[0057] Since the modification amount of capture antibody on the sensing electrode can be reflected by its incubation concentration on the electrode, the incubation concentration of capture antibody was optimized. 2 20 μL of capture antibodies with concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL were added to the Pt / FTO photocathode and incubated at 4 °C for 12 h. The electrode was washed with phosphate buffer (10 mM, pH 7.4) to obtain capture antibody-modified FTO / CuInS 2 / Pt photocathode.

[0058] By performing photocurrent characterization tests, it can be obtained, as shown in the attached Figure 3As shown, the incubation concentration of the capture antibody needs to be greater than or equal to 100 μg / mL, so as to ensure that the capture antibody is fully anchored on the sensing electrode to obtain the best quantitative detection range. Therefore, a capture antibody greater than or equal to 100 μg / mL is selected as the optimal incubation concentration.

[0059] Example 2

[0060] FTO / ZnIn 2 S 4 Preparation of photoanode

[0061] 15 mM ZnSO 4 、30mM InCl 3 and 60 mM CH 3 CSNH 2 As Zn source, In source and S source respectively, dissolved in deionized water, stirred for 30 minutes to prepare the precursor solution, and transferred to a 25mL polytetrafluoroethylene-lined stainless steel autoclave. Put the FTO conductive glass vertically into the autoclave, keep it at 160℃ for 12 hours under sealed conditions, and cool it naturally to room temperature. Wash the uniform film on the FTO conductive glass with water and ethanol in turn, and dry it at 60℃ for 1 hour to obtain FTO / ZnIn 2 S 4 Photoanode.

[0062] Scanning electron microscopy images are attached. Figure 4 As shown in the figure, it can be seen that ZnIn 2 S 4 The nanosheets are interconnected to form a mesh-like film. The nanosheets grow almost vertically to the substrate, and the thickness of the nanosheet layer is about 50nm. The elemental mapping analysis in the inset also shows that FTO / ZnIn 2 S 4 Successful preparation of the photoanode.

[0063] And the FTO / ZnIn 2 S 4 The X-ray diffraction of the photoanode is shown in the attached figure. Figure 5 As shown in the figure, the diffraction peaks at 28.9°, 32.4° and 47.2° correspond to hexagonal ZnIn 2 S 4 (PDF No.72-0773) (102), (104) and (112) crystal planes. In addition to the characteristic diffraction peaks of the FTO conductive glass substrate, no diffraction peaks of other impurity phases were found, which also proves that FTO / ZnIn 2 S 4 The photoanode was successfully prepared.

[0064] Example 3

[0065] FTO / CuInS 2 Preparation of Pt / Pt photocathode

[0066] CuInS was deposited on FTO conductive glass using a constant potential method using an electrochemical workstation. 2 The FTO conductive glass was used as the working electrode, the platinum wire as the counter electrode, and the saturated calomel electrode as the reference electrode. The FTO electrode was inserted into a 4mM CuCl 2 , 4mM InCl 3 and 12 mM sulfur powder in dimethyl sulfoxide deposition solution, the deposition potential was -1.30 V, and FTO / CuInS was obtained by one-step deposition for 30 min. 2 After cleaning the electrode with deionized water, 20 μL of Pt nanoparticle solution was added to the FTO / CuInS 2 The electrode was dried at room temperature to obtain FTO / CuInS 2 / Pt photocathode.

[0067] It should be noted that the Pt nanoparticle solution is prepared by adding 9 mg of sodium citrate to 45 mL of 0.223 mM H 2 PtCl 6 In the aqueous solution, 5 mL of 30 mM NaBH 4 The aqueous solution was stirred for 30 min in dark conditions.

[0068] Scanning electron microscopy images are attached. Figure 6 As shown in A, a large number of particles with a size of 5 to 10 nm are connected to form a porous nanofilm. The elemental mapping analysis in the inset also shows that FTO / CuInS 2 Successful preparation of the electrode.

[0069] Scanning electron microscope as attached Figure 6 As shown in B, after being modified with Pt nanoparticles, CuInS 2 The surface of the porous nanofilm is covered with a large number of small particles. The elemental mapping analysis in the illustration also further proves that FTO / CuInS 2 / Pt photocathode was successfully prepared.

[0070] And the FTO / CuInS 2 The X-ray diffraction spectrum of the Pt photocathode is shown in the attached figure. Figure 7 As shown, curve a is the X-ray diffraction spectrum of the FTO electrode substrate, and curve b is the modified CuInS 2 The diffraction peaks at 27.8°, 46.2°, 54.7° and 57.4° correspond to chalcopyrite CuInS 2Curve c is the spectrum after modification with Pt. The diffraction peaks at 39.7° and 46.2° correspond to the (111) and (220) crystal planes of pure Pt phase (PDFNo.04-080), respectively, and no diffraction peaks of other impurity phases are found, which proves that FTO / CuInS 2 / Pt photocathode was successfully prepared.

[0071] Example 4

[0072] FTO / CuInS with anti-biomacromolecule contamination performance 2 Preparation of Pt / Pt photocathode

[0073] FTO / CuInS prepared in Example 3 2 20 μL of 100 μg / mL capture antibody Ab was added to the / Pt photocathode and incubated at 4°C for 12 h. After cleaning with phosphate buffer (10 mM, pH 7.4), 20 μL of 0.2 mg / mL zwitterionic peptide solution with the sequence of SKSESKSEPPPPC was added to the Ab-modified electrode and incubated at 4°C for 6 h to block the active sites and form an anti-fouling interface brush, thus obtaining FTO / CuInS with anti-biomacromolecule pollution performance. 2 / Pt photocathode.

[0074] The FTO / CuInS with anti-biomacromolecule pollution performance was tested by current signal response. 2 The successful preparation of Pt photocathode was verified, as shown in the attached Figure 8 As shown. 2 S 4 In the photoelectric test with the photoanode as the counter electrode, the FTO electrode has a weaker current signal response (curve a); the electrodeposited CuInS 2 After that, the current signal response increases (curve b), because CuInS 2 The narrow band gap of about 1.54eV can effectively absorb visible light; after further modification with Pt nanoparticles, the current signal response is significantly enhanced (curve c), which is because the good conductivity and oxygen reduction reaction catalytic ability of Pt nanoparticles promote the charge transfer on the electrode surface; after incubation with capture antibodies and blocking the active sites with zwitterionic peptides, the current signal response gradually decreases (curves d and e), which is because the obvious steric hindrance of the two hinders the charge transfer. Therefore, the current signal response test proves that FTO / CuInS has the performance of resisting biomacromolecule contamination. 2 / Pt photocathode was successfully prepared.

[0075] Fluorescence microscopy images are attached. Fig. 9 As shown, FTO / CuInS 2 After the FTO / CuInS / Pt photocathode was incubated with bovine serum albumin labeled with fluorescent isothiocyanate (FITC-BSA), it showed obvious green fluorescence (Figure A), indicating that the photocathode had obvious nonspecific adsorption; while the FTO / CuInS / Pt photocathode modified with a zwitterionic peptide with the sequence SKSESKSEPPPPC, which has anti-biomacromolecule contamination performance, 2 After the FTO / CuInS photocathode was incubated in FITC-BSA for 2 h, no obvious green fluorescence was observed under the fluorescence microscope (Figure B), which proves that the FTO / CuInS photocathode has the ability to resist biomacromolecule contamination. 2 / Pt photocathode can effectively resist the adsorption of nonspecific proteins and has excellent anti-pollution performance.

[0076] Example 5

[0077] Highly sensitive dual-photoelectrode immunosensor with resistance to biomacromolecule contamination for detection of target antigen CA15-3

[0078] The FTO / CuInS prepared in Example 4 with anti-biomacromolecule contamination performance 2 / Pt photocathode was incubated with 20 μL of target antigens of different concentrations at room temperature for 1 h, so that CA15-3 and the capture antibody had a specific immune reaction, and after washing with phosphate buffer (10 mM, pH = 7.4), the FTO / ZnIn prepared in Example 2 2 S 4 The photoanode combination can obtain the high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance.

[0079] The final prepared high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule fouling performance was used for photocurrent signal detection in phosphate buffer (pH 7.4, 0.1 M) containing 0.1 M ascorbic acid (AA) as an electron donor.

[0080] The test results show that as the concentration of target antigen CA15-3 increases, the corresponding photocurrent signal gradually decreases, indicating that more target antigen CA15-3 and capture antibody specific recognition hinders charge transfer, such as Fig.10 As shown in the figure; and the concentration of the target antigen CA15-3 is in the range of 0.1pg / mL to 100ng / mL, the change value of the photocurrent signal is linearly related to the logarithm of the target antigen concentration, as shown in the attached figure. Fig.11 As shown, the linear fitting equation is △I=42.40+8.34logC CA15-3(ng / mL), the linear correlation coefficient is 0.9983, and the experimental minimum detection limit is 0.027pg / mL, which shows that the high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule pollution performance prepared by the present invention using a photocathode and a photoanode has high sensitivity to the target detection substance.

[0081] Example 6

[0082] Anti-interference performance test of high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance

[0083] In order to prove that the anti-pollution dual-photoelectrode immunosensor has excellent anti-interference performance, including interference with reducing small molecules and protein macromolecules, common reducing small molecules: dithiothreitol (DTT), glutathione (GSH), ascorbic acid (AA), and common protein macromolecules: tissue polypeptide-specific antigen (TPS), neuron-specific enolase (NSE), cardiac troponin I (cTnI) were selected as typical interferences for verification experiments. The specific operation is as follows:

[0084] 10 mM DTT, GSH, AA and 1 ng / mL TPS, NSE, cTnI were added separately or mixed into 10-fold diluted serum containing 0.1 ng / mL CA15-3 standard sample, and the dual photoelectrode immunosensor prepared by the present invention was used for detection according to the above method. The photocurrent signal changes were as shown in the attached figure. Fig.12 As shown. The results show that there is no significant difference between the test results of the presence of interfering substances such as DTT, GSH, AA, TPS, NSE, cTnI and their mixtures and the photocurrent signal change value of the target CA15-3 alone, and the error is kept within 5%. This proves that the anti-pollution dual-photoelectrode immunosensor prepared by the present invention has the ability to resist the interference of reducing small molecules and biological macromolecules while having high sensitivity. It has excellent application potential in the actual detection of complex biological samples and can achieve rapid, sensitive, accurate and efficient detection of target antigens.

[0085] Example 7

[0086] Comparison of the anti-biomacromolecule fouling ability of dual photoelectrode immunosensors modified with two zwitterionic peptides, SKSESKSEPPPPC and EKEKEKEPPPPC

[0087] In order to verify that the SKSESKSEPPPPC zwitterionic peptide used in the present invention has better anti-biomacromolecule contamination ability than the reported EKEKEKEPPPPC zwitterionic peptide. Under the same experimental conditions, dual photoelectrode immunosensors modified by two zwitterionic peptides, SKSESKSEPPPPC and EKEKEKEPPPPC, were prepared and tested in actual serum samples with different dilution concentrations. The photocurrent signal change rate is shown in the attached figure. Fig.13 As shown. The results show that in actual serum samples of different dilution concentrations, the dual photoelectrode immunosensor prepared by using the SKSESKSEPPPPC zwitterionic peptide of the present invention has a lower current signal change rate than the comparative zwitterionic peptide EKEKEKEPPPPC, indicating that the nonspecific adsorption capacity of biomacromolecules in actual serum samples is weaker. This directly proves that the SKSESKSEPPPPC zwitterionic peptide used in the present invention has a better ability to resist biomacromolecule contamination.

[0088] The above description of the disclosed embodiments and experimental examples enables professionals in the field to implement or use the present invention. Various modifications to these embodiments will be apparent to professionals in the field, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A highly sensitive dual-photoelectrode immunosensor with resistance to biomacromolecule contamination, characterized in that: The invention comprises a high photocurrent signal responsive FTO / ZnIn2S4 photoanode and a FTO / CuInS2 / Pt photocathode, wherein the surface of the FTO / CuInS2 / Pt photocathode is firstly modified by a capture antibody corresponding to the target antigen, and then the modified FTO / CuInS2 / Pt photocathode is further modified by a zwitterionic peptide with a sequence of SKSESKSEPPPPC; and the concentration of the capture antibody is 100-250 μg / mL.

2. The high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance according to claim 1, characterized in that: The concentration of the zwitterionic peptide with the sequence SKSESKSEPPPPC is 0.1-0.5 mg / mL.

3. The high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance according to claim 1, characterized in that: The target antigen is CA15-3.

4. A method for preparing a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. FTO conductive glass is grown with ZnIn2S4 nanosheets by a hydrothermal method to prepare a high photocurrent signal response FTO / ZnIn2S4 photoanode, wherein the FTO conductive glass is fluorine-doped SnO2 conductive glass; S2, electro-depositing CuInS2 on FTO conductive glass to obtain a FTO / CuInS2 electrode, and modifying Pt nanoparticles onto the FTO / CuInS2 electrode by electrostatic adsorption to prepare a FTO / CuInS2 / Pt photocathode; S3. The capture antibody corresponding to the target antigen is modified onto the FTO / CuInS2 / Pt photocathode, and the modified FTO / CuInS2 / Pt photocathode is further modified with the zwitterionic peptide with the sequence SKSESKSEPPPPC to obtain a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance.

5. The method for preparing a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance according to claim 4, characterized in that: The specific steps of S1 are: using ZnSO4, InCl3 and C2H5NS as Zn source, In source and S source respectively, wherein the concentration of ZnSO4 is 9-18 mM, and the molar ratio of Zn source, In source and S source is fixed at 1:2:4, the mixed solution is stirred to react for 30-60 min to obtain a precursor solution, and then the precursor solution is transferred to a high-pressure reactor, and the FTO conductive glass is vertically placed in the high-pressure reactor, maintained at 160°C for 8-14 h under sealing conditions, and naturally cooled to room temperature to form a uniform film on the surface of the FTO conductive glass, and the uniform film is washed with water and ethanol in turn, and dried at 60°C for 1 h to obtain the high photocurrent signal response FTO / ZnIn2S4 photoanode.

6. The method for preparing a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance according to claim 4, characterized in that: The specific steps of S2 are: using the FTO conductive glass as the working electrode, the platinum wire as the counter electrode, and the saturated calomel electrode as the reference electrode, inserting the FTO conductive glass into a dimethyl sulfoxide deposition solution containing 4 mM CuCl2, 4 mMInCl3 and 12 mM sulfur powder, with a deposition potential of -1.0 V to -1.50 V, and then using a one-step deposition method for 20 to 40 min to obtain the FTO / CuInS2 electrode, washing the FTO / CuInS2 electrode with deionized water, and then dripping 20 μL of Pt nanoparticle solution to the FTO / CuInS2 electrode, drying at room temperature to obtain the FTO / CuInS2 / Pt photocathode.

7. The method for preparing a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance according to claim 4, characterized in that: The specific steps of S3 are: adding 20 to 25 μL of the capture antibody corresponding to the target antigen to the FTO / CuInS2 / Pt photocathode for modification, and incubating at 4°C for 10 to 15 h; cleaning the modified FTO / CuInS2 / Pt photocathode with 10 mM, pH 7.4 phosphate buffer, adding 20 to 25 μL of the zwitterionic peptide solution with the sequence of SKSESKSEPPPPC to the modified FTO / CuInS2 / Pt photocathode, and incubating at 4°C for 4 to 8 h to obtain a high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance.

8. Use of the high-sensitivity dual-photoelectrode immunosensor with anti-biomacromolecule contamination performance according to any one of claims 1 to 3 in the preparation of in vitro diagnostic products.

Images