CN104328192B - A Highly Sensitive Electrochemical Immunoassay Method for Ribozyme Amplification - Google Patents
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- 108091092562 ribozyme Proteins 0.000 title claims abstract description 21
- 238000003018 immunoassay Methods 0.000 title abstract description 28
- 230000003321 amplification Effects 0.000 title 1
- 238000003199 nucleic acid amplification method Methods 0.000 title 1
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 31
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 31
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims abstract description 27
- 229960000907 methylthioninium chloride Drugs 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 22
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- 239000000243 solution Substances 0.000 claims abstract description 8
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000010931 gold Substances 0.000 claims abstract description 6
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- AKIZPWSPNKVOMT-UHFFFAOYSA-N 1-sulfanylhexan-1-ol Chemical compound CCCCCC(O)S AKIZPWSPNKVOMT-UHFFFAOYSA-N 0.000 description 1
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
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- 238000012544 monitoring process Methods 0.000 description 1
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
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- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
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- C12Q1/6804—Nucleic acid analysis using immunogens
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Abstract
本发明涉及一种核酶放大的高灵敏电化学免疫分析方法。通过Au‑S键合作用，在金电极表面自组装亚甲基蓝标记的发夹DNA，构成电化学免疫传感界面。以Mg2+、DNA1‑抗体1、DNA2‑抗体2、DNA3‑抗体3的混合溶液为检测液，在目标蛋白质存在时，抗体1、抗体2、抗体3同时识别目标蛋白质形成免疫复合物，使得DNA1、DNA2与DNA3相互靠近进行邻位杂交，形成Mg2+依赖性核酶，进而催化裂解传感界面上的发夹DNA，导致亚甲基蓝脱离电极表面，氧化电流减小。通过检测亚甲基蓝电流的变化，实现对目标蛋白质浓度的测定。该免疫分析方法利用三个DNA‑抗体偶合物邻位形成核酶放大检测信号，提高了检测灵敏度和选择性，同时可对目标蛋白质进行快速、一步化检测，具有很好的临床应用价值。
The invention relates to a highly sensitive electrochemical immunoassay method amplified by ribozymes. Through Au-S bonding, methylene blue-labeled hairpin DNA was self-assembled on the surface of the gold electrode to form an electrochemical immunosensing interface. Using the mixed solution of Mg 2+ , DNA1-antibody 1, DNA2-antibody 2, and DNA3-antibody 3 as the detection solution, when the target protein exists, the antibody 1, the antibody 2, and the antibody 3 simultaneously recognize the target protein to form an immune complex, so that the DNA1, DNA2 and DNA3 approached each other for proximity hybridization to form Mg 2+ -dependent ribozymes, which catalyzed the cleavage of hairpin DNA on the sensing interface, resulting in methylene blue detaching from the electrode surface and reducing the oxidation current. By detecting the change of methylene blue current, the determination of the target protein concentration is realized. The immunoassay method utilizes three DNA-antibody conjugates to form adjacent ribozymes to amplify the detection signal, thereby improving the detection sensitivity and selectivity, and at the same time, the target protein can be detected rapidly and in one step, which has good clinical application value.
Description
One, the technical field
The invention relates to a ribozyme amplified high-sensitivity electrochemical immunoassay method. The hairpin DNA of the marked electrochemical active molecule methylene blue is fixed on the surface of an electrode through an Au-S bond to construct an electrochemical immunosensing interface. Induction of proximity effects and Mg formation by immunological recognition of target proteins2+And (3) the ribozyme is depended on, so that hairpin DNA is catalytically cracked, methylene blue molecules are separated from the surface of the sensor, and the target protein is simply, quickly and highly sensitively measured by detecting the reduction of the oxidation current of the methylene blue.
Second, background Art
Immunoassay has been widely used in the fields of environmental monitoring, clinical diagnosis, food safety, etc., as an analysis method with high selectivity and high sensitivity. Immunoassays can be classified into heterogeneous immunoassays and homogeneous immunoassays, and the latter immunoassays are widely used in clinical tests because they are simple to operate, do not require multiple washing steps, incubation steps, and are easy to automate. Compared with the traditional immunoassay methods such as radiation, fluorescence, chemiluminescence, electrochemiluminescence, surface plasmon resonance and the like, the electrochemical immunoassay has the advantages of cheap instrument, simple and convenient operation, high sensitivity and the like. Based on electrochemical immunoassay methods, various electrochemical immunosensors have been developed, with amperometric immunosensors being the most widely studied and used. However, most of the electrochemical immunoassays developed today use heterogeneous immunoassays, require multi-step washing, are time-consuming and easily contaminate samples, and for better practical applications, the development of new simple, rapid and sensitive electrochemical immunoassays is required.
In recent years, nucleic acid-immunoassay methods developed in conjunction with nucleic acid analysis, such as proximity immunoassay, have attracted the interest of many scholars. The proximity immunoassay utilizes a pair of nucleic acid-antibody conjugates and an antigen (analyte) to perform sandwich immunological recognition to form an immune complex, so that nucleic acids on the nucleic acid-antibody conjugates are close to each other to induce proximity effect, further hybridization or connection is performed to generate new detection nucleic acid, and finally the concentration of the target protein is indirectly determined through detection of the nucleic acid. The method for the ortho-position immunoassay can be carried out homogeneously, and has the advantages of simplicity, high sensitivity, good specificity, less sample consumption and the like. However, this method basically employs PCR technology to detect the produced detection nucleic acid, which is costly and takes a long time. The electrochemical detection and the ortho-immunoassay method are combined, so that the detection time can be shortened, and the analysis cost can be reduced
The invention combines electrochemical detection and ortho-position immunoassay, and the ribozyme circulating signal is amplified through the ortho-position of immunoreaction, thereby improving the sensitivity and selectivity of detection and establishing a simple, rapid and high-sensitivity electrochemical immunoassay method.
Third, the invention
The content of the invention is as follows: constructing electrochemical immunosensing interface with hairpin DNA labeled by electrochemical active molecule, inducing ortho effect by immunoreaction to form Mg2+The dependent ribozyme further catalyzes and cleaves the hairpin DNA, so that the electrochemical active molecule is separated from the surface of the electrode, the oxidation current signal of the electrochemical active molecule is reduced, and the electrochemical immunosensing detection is carried out on the target protein through the change of the oxidation current of the electrochemical active molecule.
The invention is realized by the following technical scheme:
the two ends of the gold electrode surface are respectively marked with hairpin DNA of Sulfhydryl (SH) and Methylene Blue (MB) by self-assembly to form an electrochemical immunosensing interface. In the presence of the target protein, the DNA 1-antibody 1, the DNA 2-antibody 2 and the DNA 3-antibody 3 form immune complexes with the target protein through sandwich immune reaction, and generate an ortho effect to promote the DNA3 to simultaneously hybridize with the DNA1 and the DNA2 to form Mg2+The dependent ribozyme, in turn, catalyzes the cleavage of methylene blue labeled hairpin DNA, causing methylene blue to break away from the immunosensing interface (fig. 1). The concentration of the target protein was measured by detecting the change in the oxidation current of methylene blue.
The hairpin DNA has a hairpin structure formed by complete complementarity of 1-5 bases from the 3 'end and 1-5 bases from the 5' end; DNA3 is completely complementary to DNA1 and DNA2 by 8 bases; hairpin DNA was completely complementary to both DNA1 and DNA2 by 9 bases (FIG. 2).
The above DNA1 and DNA2 contain ACAACGA and GGCTAGCT sequences, respectively, which can be adjacent to Mg2+Coordinated to form Mg2+A dependent ribozyme.
The hairpin DNA contains a gu sequence capable of being Mg-substituted2+Dependent ribozymes recognize and cleave.
The analysis method comprises the following detection steps:
(1) adding target protein to the solution containing Mg2+Mixing the mixed solution of DNA 1-antibody 1, DNA 2-antibody 2 and DNA 3-antibody 3, then dripping the mixed solution on an electrochemical immunosensing interface, and incubating for 40 minutes at room temperature;
(2) after a sensing interface is washed by a washing liquid, a sensor is inserted into an electrolyte solution, and oxidation current of methylene blue is detected by a differential pulse voltammetry method;
(3) and (3) obtaining the percentage of reduction of the oxidation current of the methylene blue, plotting the logarithm of the ratio and the concentration of the target protein to obtain a standard curve, and obtaining the concentration of the target protein through the standard curve.
The principle of the detection system for detecting the target protein is as follows:
the principle of the ribozyme amplified high-sensitivity electrochemical immunoassay method is shown in figure 1: through immune reaction, the target protein is simultaneously recognized by the antibody 1, the antibody 2 and the antibody 3 and a target protein/DNA 1-antibody 1/DNA 2-antibody 2/DNA 3-antibody 3 complex is generated, so that the DNA1, the DNA2 and the DNA3 are close to each other to generate ortho-position hybridization, and Mg is formed2+The ribozyme can recognize the enzyme cutting site on the hairpin DNA and catalytically cleave the enzyme, so that the methylene blue labeled on the hairpin DNA is separated from the immunosensing interface. Detecting the oxidation current of methylene blue by differential pulse voltammetry; the oxidation current of methylene blue is inversely related to the concentration of the target protein. And (4) plotting the percentage of reduction of the methylene blue oxidation current to the logarithm of the concentration of the target protein to obtain a standard curve, and measuring the concentration of the target protein in the solution to be measured through the standard curve.
Compared with the prior art, the invention has the following characteristics:
the invention constructs an electrochemical immunosensing interface through a nucleic acid chain, and combines an ortho-position effect and Mg induced by immunoreaction2+The oxidation current response of the dependent ribozyme and the electrochemical active molecule on the surface of the electrode constructs a simple, rapid and high-sensitivity electrochemical immunoassay method. Compared with the existing electrochemical immunoassay method, the method has the following characteristics:
(1) the electrochemical immunosensing interface is constructed by using the nucleic acid chain, the preparation is simple, the repeatability is good, and meanwhile, the detection of the target protein corresponding to the antibody can be realized by combining other DNA-antibody conjugates, so that the method has better universality.
(2) The detection principle of the target protein is designed by utilizing the immunoreaction induced proximity effect, the analysis process is simplified, one-step detection can be realized, and the determination of a single sample can be completed within 40 minutes.
(3) Formation of Mg by using the ortho effect2+The detection signal is amplified by the dependent ribozyme, the detection linear range is widened, the sensitivity is improved, and the detection limit of the selected model protein is 1.5 pg/mL.
(4) No additional substrate solution is required.
Description of the drawings
FIG. 1 is a schematic diagram of a ribozyme-amplified highly sensitive electrochemical immunoassay.
FIG. 2 ortho-induced Mg2+Schematic representation of the generation of dependent ribozymes and their catalytic cleavage.
Fifth, detailed description of the invention
Example 1: with the attached figure 1, the preparation of the high-sensitivity universal nucleic acid electrochemical immunosensing interface
Polishing and cleaning a gold electrode with the diameter of 2mm, dripping 6 mu L of hairpin DNA with 0.5 mu M of thiol (SH) and Methylene Blue (MB) labeled at two ends on the treated gold electrode, reacting for 2 hours at room temperature in a dark place, cleaning the electrode with 0.01M phosphate buffer containing 0.1M NaCl, and drying by nitrogen. Then, 6. mu.L of 1mM mercaptohexanol was dropped onto the electrode surface, and the reaction was carried out at room temperature for 40 minutes, followed by washing with phosphate buffer and then blowing with nitrogen. Preparing the nucleic acid electrochemical immunosensing interface, and storing at 4 ℃ for later use.
Example 2: the application of the ribozyme-amplified high-sensitivity electrochemical immunoassay method is illustrated by taking carcinoembryonic antigen (CEA) as an example with reference to the attached figure 1.
(1) Preparing a sample incubation solution: the CEA standard solution (or CEA-containing sample solution) and DNA 1-antibody 1, DNA 2-antibody 2, and DNA 3-antibody 3 were mixed with 30mM MgCl2The mixture was mixed with the 0.01M phosphate buffer solution of (1), wherein the concentrations of DNA 1-antibody 1, DNA 2-antibody 2 and DNA 3-antibody 3 were all 50 nM.
(2) 6 μ L of sample incubation was added dropwise to the electrochemical immunosensing interface, incubated for 40 minutes at room temperature, and then rinsed with rinse solution.
(3) The electrode after washing was inserted into a detection electrolyte, and differential pulse voltammetry was carried out at a potential range of-0.4V to 0V with a pulse amplitude of 50mV and a pulse width of 200ms with this electrode as a working electrode, an Ag/AgCl electrode as a reference electrode, and a platinum wire as a counter electrode. And (3) solving the percentage of reduction of the oxidation current according to the recorded oxidation current response of the methylene blue, drawing a graph by using the ratio to the logarithm of the CEA concentration to obtain a working curve of the CEA detection, and obtaining the concentration of the CEA in the sample solution through the working curve.
Claims (2)
1. A ribozyme amplified high-sensitivity electrochemical immune analysis method is characterized in that the analysis method takes a gold electrode of hairpin DNA with a self-assembled methylene blue mark on the surface as an electrochemical immune sensing interface and contains Mg2+The mixed solution of DNA 1-antibody 1, DNA 2-antibody 2 and DNA 3-antibody 3 is a detection solution, when the target protein exists, the antibody 1, the antibody 2 and the antibody 3 simultaneously recognize the target protein to form an immune complex, so that the DNA1, the DNA2 and the DNA3 are close to each other and undergo ortho-hybridization to form Mg2+The dependent ribozyme further catalyzes and cleaves hairpin DNA on the surface of the electrode, so that methylene blue is separated and the oxidation current of the methylene blue is reduced, and finally the concentration of the target protein is determined by detecting the oxidation current change of the methylene blue; the hairpin DNA is completely complementary-paired from 1 to 5 bases from the 5 'end and from 1 to 5 bases from the 3' end, and contains a functional group capable of being Mg-bonded2+Dependent ribozyme recognition gu sequence, hairpin DNA both ends respectively modify sulfydryl and methylene blue, through Au-S bonding effect modification on gold electrode surface; the DNA 1-antibody 1 is a conjugate of a monoclonal antibody 1 of a target protein and DNA1, the DNA1 contains a specific ACAACGA sequence, and 9 and 8 bases are completely complementary and paired with hairpin DNA and DNA3 respectively; the DNA 2-antibody 2 is a conjugate of a monoclonal antibody 2 of a target protein and DNA2, the DNA2 contains a specific GGCTAGCT sequence, and the DNA is completely complementary with 9 and 8 bases of hairpin DNA and DNA3 respectively; the DNA 3-antibody 3 is a conjugate of monoclonal antibody 3 of a target protein and DNA3, and is added into Mg2+When present, DNA3 hybridizes with DNA1 and DNA2 at the ortho-position to form Mg2+A dependent ribozyme.
2. The assay method of claim 1, characterized by the following detection steps:
(1) adding a sample containing target protein into a detection solution, mixing, and then dripping the mixture on an electrochemical immunosensing interface for incubation;
(2) after a sensing interface is washed by a washing liquid, a sensor is inserted into an electrolyte solution, and electrochemical detection is carried out by differential pulse voltammetry to obtain oxidation current of methylene blue;
(3) and (3) obtaining the percentage of reduction of the oxidation current of the methylene blue, plotting the logarithm of the ratio and the concentration of the target protein to obtain a standard curve, and measuring the concentration of the target protein in the sample solution through the standard curve.
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| CN105092850B (en) * | 2015-06-04 | 2017-01-11 | 塔里木大学 | Electrochemical immunosensor for simultaneous detection of azocyclotin and chlorpyrifos, and preparation method and application thereof |
| CN106525920B (en) * | 2015-09-11 | 2019-08-09 | 南京理工大学 | Method for detecting carcinoembryonic antigen based on terminal elongase electrochemical nucleic acid aptamer sensor |
| CN105648033B (en) * | 2016-02-23 | 2019-06-04 | 青岛科技大学 | A method for the determination of trypsin in pancreatic tissue samples of exercise mice |
| CN105911289B (en) * | 2016-04-13 | 2018-04-10 | 南京大学 | A kind of electrochemical sensor and its preparation and application based on dynamic sandwich structure |
| CN106980022B (en) * | 2017-04-07 | 2023-07-07 | 南京大学 | Homogeneous immunoassay method based on target protein induced DNase circulation generation |
| CN108613934B (en) * | 2018-03-19 | 2021-01-05 | 安徽师范大学 | A DNA sensor and preparation method thereof, and a method for detecting short-chain DNA |
| CN110836921B (en) * | 2019-12-02 | 2020-05-08 | 青岛大学 | Preparation method of nano-electrochemical aptamer sensor for detecting stress-induced phosphoproteins |
| CN113866239A (en) * | 2021-09-24 | 2021-12-31 | 中国人民解放军陆军军医大学 | A DNA methylation specific detection method for electrochemical immunoassay |
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