Photoelectrochemical immunosensor for detecting HE4 and preparation method and application thereof
阅读说明：本技术 一种检测he4的光电化学免疫传感器及其制备方法和应用 (Photoelectrochemical immunosensor for detecting HE4 and preparation method and application thereof ) 是由 沈艳飞 陈开洋 张越 于 2019-11-08 设计创作，主要内容包括：本发明公开了一种检测HE4的光电化学免疫传感器及其制备方法和应用,该光电化学免疫传感器由由Nb2共价结合到MWCNTs-PDA-AuNPs修饰后的基底电极上,HE4抗原与Nb2特异性结合,Nb3@nPCN-224与HE4抗原特异性结合制得。本发明利用光电化学免疫法提高了传感器的抗干扰性,同时使用的光活性物质nPCN-224具有很好的生物相容性,可以负载大量的生物分子,还具有很强的光电活性,以此来提高传感器的灵敏度。本发明制备的光电免疫传感器,操作简单,反应迅速,灵敏度高,抗干扰性强,稳定性佳,能够实现对于痕量抗原HE4的检测,为开发出用于临床诊断的优异平台提供了新思路。(The invention discloses a photoelectrochemical immunosensor for detecting HE4, and a preparation method and application thereof, wherein the photoelectrochemical immunosensor is prepared by covalently bonding Nb2 to a substrate electrode modified by MWCNTs-PDA-AuNPs, specifically bonding an HE4 antigen and Nb2, and specifically bonding Nb3@ nPCN-224 and HE4 antigen. The invention improves the anti-interference performance of the sensor by utilizing the photoelectrochemistry immunity method, and simultaneously, the used photoactive substance nPCN-224 has good biocompatibility, can load a large amount of biomolecules and also has strong photoelectric activity, thereby improving the sensitivity of the sensor. The photoelectric immunosensor prepared by the invention has the advantages of simple operation, rapid reaction, high sensitivity, strong anti-interference performance and good stability, can realize the detection of the trace antigen HE4, and provides a new idea for developing an excellent platform for clinical diagnosis.)
1. The photoelectrochemical immunosensor for detecting HE4 is characterized in that Nb2 is covalently bonded on a substrate electrode modified by MWCNTs-PDA-AuNPs, an HE4 antigen is specifically bonded with Nb2, and Nb3@ nPCN-224 and HE4 antigen are specifically bonded to prepare the photoelectrochemical immunosensor.
2. The photoelectrochemical immunosensor of claim 1, wherein the substrate electrode is an indium tin oxide semiconductor electrode.
3. The photoelectrochemical immunosensor according to claim 1, wherein the MWCNTs-PDA-AuNPs are added to the MWCNTs dispersion by DA, stirred to react to form MWCNTs-PDA dispersion, and then HAuCl is added4And Na3C6H5O7·2H2And adding O into the dispersion of the MWCNTs-PDA to react to generate the MWCNTs-PDA-AuNPs.
4. The photoelectrochemical immunosensor of claim 1, wherein the Nb3@ nPCN-224 is formed from H2TCPP、ZrOCl2·8H2Dissolving O and BA in DMF, heating to react to obtain nPCN-224, and labeling nPCN-224 with Nb3 to obtain Nb3@ nPCN-224.
5. A method for preparing the photoelectrochemical immunosensor for detection of HE4 according to claim 1, comprising the steps of:
(1) fixing a substrate material: dripping MWCNTs-PDA-AuNPs dispersion liquid on the surface of ITO, drying, cleaning with ultrapure water, and drying;
(2) anchoring recognition molecule: dripping mixed solution of EDC/NHS on the surface of the substrate electrode obtained in the step (1), washing by PBS solution after standing at room temperature, dripping the recognition molecule capture antibody Nb2 on the substrate electrode after washing, and washing by PBS solution after incubation;
(3) non-specific site blocking: dripping BSA solution on the surface of the electrode obtained in the step (2), sealing for 0.5-1h at the temperature of 25-37 ℃, and then washing with PBS solution;
(4) incubation of target molecule HE 4: dropwise adding HE4 antigen solution onto the electrode obtained in the step (3), reacting at 25-37 ℃ for 30-70min, and washing with PBS solution to remove the antigen which is not specifically bound;
(5) construction of photoelectrochemical immunosensor: and (3) dropwise adding the Nb3@ nPCN-224 solution to the surface of the electrode obtained in the step (4) for specific reaction, incubating at 25-37 ℃ for 30-70min, cleaning with a PBS solution, and airing to obtain the photoelectrochemical immunosensor for detecting HE 4.
6. The method for preparing the photoelectrochemical immunosensor according to claim 5, wherein the concentration of the MWCNTs-PDA-AuNPs in the step (1) is preferably 0.1-1 mg/mL.
7. The method for preparing the photoelectrochemical immunosensor according to claim 5, wherein the concentration of the NHS in the step (2) is 5 to 20mg/mL, the concentration of the EDC is 10 to 30mg/mL, and the concentration of the Nb2 is 1 to 40 μ g/mL.
8. The method for preparing the photoelectrochemical immunosensor according to claim 5, wherein the incubation in the step (2) is performed at 3.8-4.2 ℃ for 12-15 h.
9. The method for preparing the photoelectrochemical immunosensor according to claim 5, wherein the blocking in the step (3) is performed at 25 to 37 ℃ for 0.5 to 1 hour.
10. The use of the photoelectrochemical immunosensor of claim 1 for the detection of HE4 for the quantitative detection of HE 4.
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to a photoelectrochemical immunosensor for detecting HE4 and a preparation method and application thereof.
Background
Ovarian cancer is one of the common malignant tumors of female reproductive organs, and the incidence rate is second to cervical cancer and uterine corpus cancer. Early diagnosis of ovarian cancer is helpful for timely treatment of patients to improve the survival rate of patients, but because the ovary is deeply in the pelvic cavity, has small volume, lacks typical symptoms and is difficult to find early, the early diagnosis is a big problem. During the development of tumors, tumor tissues or cells release specific proteins into the circulation, and the level of these specific proteins in blood indicates the stage of tumor development, so these specific proteins are often called tumor markers in biomedicine. Early detection of tumor markers is a key factor affecting the recovery of cancer patients. The HE4 is a novel ovarian cancer specific tumor marker, and research shows that the specificity and sensitivity of HE4 are higher than those of a common marker CA125 especially in an early stage, so that the detection of HE4 with high specificity and high sensitivity is extremely important in the aspect of medical research and detection of ovarian cancer.
At present, enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), radioimmunoassay (IRMA) and the like are commonly used for detecting HE4, but most of the methods are expensive instruments, complex operation and time consumption. Photoelectrochemical analysis is an analysis method for detection based on photoelectric signal conversion of photoelectrochemical active substances, has low cost, simple equipment, easy miniaturization, low background signal and lower detection limit, is widely applied to DNA analysis, cell detection and enzyme sensing, and certainly can not only utilize the advantages of photoelectrochemical analysis but also improve the sensitivity and specificity of detection when being used for detection of markers by photoelectrochemical analysis. In addition, the photoelectric active material plays an important role in photoelectric immunoassay, and the MOF is used as an organic-inorganic hybrid material, has strong porosity, large specific surface area and structural and functional diversity, and can be used for marking biomolecules. Meanwhile, the porphyrin-based MOF is a good photoelectric active substance, and the light absorption efficiency is high. In addition, the size of MOFs can be manipulated by changing the conditions of MOF synthesis, which is more suitable for signal transduction of sensors than the microsized MOFs.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a photoelectrochemical immunosensor based on a stable and high photoelectricity active substance nPCN-224 for detecting an ovarian cancer marker HE4, the photoelectrochemical immunosensor is simple and convenient to operate, high in sensitivity, good in stability and good in selectivity, can realize the detection of trace HE4 antigen, and provides an effective tool for quantitatively detecting
The invention also aims to provide a preparation method of the photoelectrochemical immunosensor for detecting
It is another object of the present invention to provide the use of the photoelectrochemical immunosensor for the detection of
Abbreviations for technical terms in the present invention are as follows:
multi-walled carbon nanotubes: MWCNTs; dopamine hydrochloride: DA; poly-dopamine: a PDA; gold nanoparticles: AuNPs; meso-tetra (p-carboxyphenyl) porphyrin: h2TCPP; benzoic acid: BA; human epididymis protein 4:
Capture antibody in raw material in the present invention: nb2 and labeled antibody: nb3 was synthesized according to the high hly selective and Sensitive Electrochemical evaluation of Cry1C Using Nanobody and pi-pi stabilized Graphene Oxide/Thionine Assembly (anal. chem.2016,88, 9830-9836). In addition, other starting materials are commercially available. Wherein the multi-wall carbon nano-tube MWCNTs are purchased from Xiancheng nano-material science and technology limited company (carboxylated MWCNTs); dopamine DA hydrochloride is purchased from Bailingwei science and technology Co., Ltd; the epididymis protein HE4 antigen is purchased from Beijing Yiqian Shenzhou science and technology Co., Ltd; h2TCPP was purchased from Sigma-Aldrich Sigma Aldrich Co.
The technical scheme is as follows: in order to achieve the purpose, the photoelectrochemical immunosensor for detecting HE4 is characterized in that Nb2 is covalently bonded to a substrate electrode modified by MWCNTs-PDA-AuNPs, HE4 antigen is specifically bonded with Nb2, and Nb3@ nPCN-224 is specifically bonded with HE4 antigen.
Preferably, the substrate electrode is an indium tin oxide semiconductor electrode.
Wherein the MWCNTs-PDA-AuNPs are added from DAAdding into MWCNTs dispersion (solvent is water), stirring to react to obtain MWCNTs-PDA dispersion, and adding HAuCl4And Na3C6H5O7·2H2And adding O into the MWCNTs-PDA dispersion liquid to react to generate the MWCNTs-PDA-AuNPs dispersion liquid.
Preferably, the MWCNTs-PDA-AuNPs are prepared by the following method:
weighing 5-20mg of carboxylated MWCNTs, dispersing in 8-10mL of 1 xTris-HCl buffer solution, adding 0.5-2mL of 10-40mg/mL of DA solution (solvent is 1 xTris-HCl buffer solution), reacting for 18-30H in the dark, centrifuging, washing to obtain MWCNTs-PDA, and dispersing in 20-30mL of H2Adjusting the pH value to 4.0-6.0 in O, then adding 300-400 mu L of 0.05-0.2M chloroauric acid aqueous solution, then slowly adding 4-6mL0.05-0.2M sodium citrate aqueous solution prepared in situ, reacting for 1-3h, and centrifugally washing to obtain MWCNT-PDA-AuNPs.
Wherein the Nb3@ nPCN-224 is formed by H2TCPP、ZrOCl2·8H2Dissolving O and BA in DMF, heating (hydrothermal method) to react to obtain nPCN-224, activating by using a mixed solution of NHS and EDC, and labeling the nPCN-224 by using Nb3 to obtain Nb3@ nPCN-224.
Preferably, the Nb3@ nPCN-224 is prepared by the following method:
(1) synthesis of nPCN-224: mixing 5-20mg of H2TCPP、20-40mg ZrOCl2·8H2Dissolving O and 250-300mg BA in 5-20mL DMF, heating for reaction to obtain a purple solid nPCN-224, centrifuging and washing, and dissolving in DMF to form a dispersion liquid for later use;
(2) synthesis of Nb3@ nPCN-224: adding 100-300 mu L of 50-200 mu g/mL Nb3 PBS solution into the activated dispersion of the nPCN-224, incubating and culturing, then adding BSA solution for blocking, centrifuging, washing by the PBS solution, and finally dispersing in the PBS solution for preservation, namely the Nb3@ nPCN-224.
In the step (1), the heating temperature is 80 to 100 ℃, preferably 90 ℃. The heating time is 2-4h, preferably 3 h.
In step (2), the PBS solution is a 0.01M PBS solution with pH 7.4. The incubation temperature is 3.8-4.2 deg.C, preferably 4 deg.C. The incubation time is 10-14h, preferably 12 h. BSA in a mass fraction of 0.5 to 5%, preferably 1%, in a 0.01M pH 7.4PBS solution. The blocking temperature is from 25 to 37 ℃ and preferably 37 ℃. The sealing time is 20-40min, preferably 30 min.
The preparation method of the photoelectrochemical immunosensor for detecting HE4 comprises the following steps:
(1) fixing a substrate material: dripping MWCNTs-PDA-AuNPs dispersion liquid on the surface of ITO, drying, cleaning with ultrapure water, and drying;
(2) anchoring recognition molecule: dripping mixed solution of EDC/NHS on the surface of the substrate electrode obtained in the step (1), washing by PBS solution after standing at room temperature, dripping the recognition molecule capture antibody Nb2 on the substrate electrode after washing, and washing by PBS solution after incubation;
(3) non-specific site blocking: dripping BSA solution on the surface of the electrode obtained in the step (2), sealing for 0.5-1h at the temperature of 25-37 ℃, and then washing with PBS solution;
(4) incubation of target molecule HE 4: dropwise adding HE4 antigen solution onto the electrode obtained in the step (3), reacting at 25-37 ℃ for 30-70min, and washing with PBS solution to remove the antigen which is not specifically bound;
(5) construction of photoelectrochemical immunosensor: and (3) dropwise adding the Nb3@ nPCN-224 dispersion liquid onto the surface of the electrode obtained in the step (4) for specific reaction, incubating for 30-70min at 25-37 ℃, cleaning with a PBS solution, and airing to obtain the photoelectrochemical immunosensor for detecting
In the step (1), the concentration of MWCNTs-PDA-AuNPs is 0.1-1mg/mL, and the dispersant is H2O is preferably 0.75 mg/mL. MWCNTs-PDA-AuNPs are in excess. The excess is more than the amount actually acting by the dropwise addition, so that washing is required after the reaction is completed.
In step (2), the NHS concentration is 5 to 20mg/mL, preferably 10mg/mL, the solvent is 0.01M, and the pH is 6PBS solution. EDC concentration is 10-30mg/mL, preferably 20mg/mL, solvent is 0.01M, pH 6PBS solution. The concentration of Nb2 is 1-40 μ g/mL, preferably 10 μ g/mL, the solvent is 0.01M, pH 7.4PBS solution. The concentration of the PBS solution was 0.01M and the pH was 7.4. EDC/NHS and Nb2 were in excess.
In step (3), the mass fraction of BSA is 0.5 to 5%, preferably 1%, and the solvent is 0.01M PBS solution with pH 7.4. The BSA solution was in excess.
In step (4), the concentration of the PBS solution was 0.01M, and the pH was 7.4.
In step (5), the concentration of the Nb3@ nPCN-224 dispersion is 0.5 to 2.0mg/mL, preferably 1.0 mg/mL. The Nb3@ nPCN-224 dispersion was in excess. The concentration of the PBS solution was 0.01M and the pH was 7.4.
Preferably, the incubation in step (2) is at 3.8-4.2 ℃ for 12-15 h. In the step (2), the sealing is performed for 0.5-1h at the temperature of 25-37 ℃.
The photoelectrochemical immunosensor for detecting HE4 provided by the invention is applied to quantitative detection of
Specifically, the photoelectrochemical immunosensor for detecting HE4 provides an effective tool for quantitatively detecting HE4, and is applied to preparation of a tool for quantitatively detecting
The working principle is as follows: the porphyrin derivatives are common photoelectrochemical active substances, and have strong light absorption and emission capabilities and good photoelectric conversion efficiency. Meanwhile, due to the porous 3D structure of the MOF, the porphyrin-based MOF can improve the stability of photoelectric signals and the photoelectric conversion efficiency. The invention utilizes H2TCPP is used as a precursor for synthesizing MOF, nPCN-224 with a nano-scale size is synthesized by a hydrothermal method, and then a signal-on photoelectrochemical immunosensor based on the nPCN-224 is constructed by utilizing the characteristics of excellent photoelectric property and capability of being further coupled with biomolecules to detect an ovarian
The MWCNTs-PDA-AuNPs compound with good conductivity and stability is used as a substrate material to be loaded on the surface of an electrode to improve the performance of the sensor, the AuNPs on the surface of the MWCNTs-PDA-AuNPs compound are used for connecting a recognition molecule Nb2 to form a capture layer to be specifically combined with HE4, and then the HE4 is specifically combined with the Nb3@ nPCN-224 compound to form the sandwich type photoelectric immunosensor.
The invention provides a method for detecting an ovarian cancer marker HE4 by using a photo-active substance nPCN-224. Firstly, diluting a single crystal synthesis system to generate more MOF monomers (the synthesized nPCN-224 of the invention) to reduce the MOF size to synthesize the nPCN-224 with nano-scale size, wherein the photoelectric activity of the nPCN-224 is higher than that of a precursor H thereof2The photoelectric activity of TCPP is about 3.5 times stronger, and H can be utilized2The carboxyl at the end of TCPP is further coupled with a labeled antibody Nb3 to form a complex Nb3@ nPCN-224, and the complex can be used as a photoelectrochemical probe of signal-on type immunosensing.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the invention constructs a signal-on type double-anti sandwich photoelectrochemical immunosensor by using a photoelectric active substance nPCN-224, wherein after Nb3@ nPCN-224 and HE4 are combined, the photocurrent is obviously enhanced, and the photoelectric current is used for detecting an ovarian
The invention utilizes MWCNTs-PDA-AuNPs as an electrode substrate to be covalently connected with HE4 first antibody Nb2 and H2TCPPP is used as a precursor to synthesize nano-scale nPCN-224 as a photoelectric active substance by a hydrothermal method, and the nano-scale nPCN-224 is compounded with a second antibody Nb3 of HE4 to form Nb3@ nPCN-224 to be used as a photoelectrochemical probe, so that a signal-on type photoelectrochemical double-antibody sandwich immunosensor is constructed to detect an ovarian
Drawings
FIG. 1 is a photo-electric activity assay of nPCN-224.
FIG. 2 is a diagram of photo-detection current versus time during the assembly of the immunosensor.
FIG. 3 is a graph showing the change in surface impedance of an electrode during the assembly of an immunosensor.
Fig. 4 is a graph of the linear relationship between HE4 concentration and the rate of change of photocurrent intensity.
Detailed Description
The invention will be further described with reference to specific embodiments and the accompanying drawings.