阅读说明：本技术 一种电化学发光生物传感器、制备方法及其在碱基切除修复酶检测中的应用 (Electrochemical luminescence biosensor, preparation method and application thereof in detection of base excision repair enzyme ) 是由 张春阳 崔琳 赵敏慧 于 2019-10-17 设计创作，主要内容包括：本发明提供一种电化学发光生物传感器、制备方法及其在碱基切除修复酶检测中的应用。所述电化学发光生物传感器包括β-CD/GO/GCE电极和FeMOF/AuNPs@luminol-Hairpin探针；其中,所述电极由氧化石墨烯和环糊精修饰至玻碳电极上制得；所述探针包括：FeMOF,以及修饰在FeMOF上的AuNPs@luminol,AuNPs@luminol上还修饰有发夹结构探针,所述发夹结构探针的茎区设计待测碱基切除修复酶的目标碱基。本发明电化学发光生物传感器可三倍信号放大检测碱基切除修复酶,因此具有极高的灵敏度,可用于碱基切除修复酶抑制剂/激活剂的筛选、生物样品分析等生物医学研究领域。(The invention provides an electrochemiluminescence biosensor, a preparation method and application thereof in detection of base excision repair enzyme. The electrochemical luminescence biosensor comprises a beta-CD/GO/GCE electrode and a FeMOF/AuNPs @ luminol-Hairpin probe; the electrode is prepared by modifying graphene oxide and cyclodextrin onto a glassy carbon electrode; the probe includes: the kit comprises FeMOF and AuNPs @ lumineol modified on the FeMOF, wherein a hairpin structure probe is further modified on the AuNPs @ lumineol, and a stem region of the hairpin structure probe is designed to be a target base of a base excision repair enzyme to be detected. The electrochemical luminescence biosensor can detect the base excision repair enzyme by three times signal amplification, has extremely high sensitivity, and can be used in the biomedical research fields of screening of base excision repair enzyme inhibitors/activators, analysis of biological samples and the like.)

1. An electrochemiluminescence biosensor for detecting a base excision repair enzyme, which is characterized by comprising a beta-CD/GO/GCE electrode and a FeMOF/AuNPs @ luminol-Hairpin probe;

the beta-CD/GO/GCE electrode is prepared by modifying graphene oxide and cyclodextrin onto a glassy carbon electrode;

the FeMOF/AuNPs @ luminol-Hairpin probe comprises: the kit comprises FeMOF and AuNPs @ lumineol modified on the FeMOF, wherein a hairpin structure probe is further modified on the AuNPs @ lumineol, and a stem region of the hairpin structure probe is designed with one or more target bases of a base excision repair enzyme to be detected.

2. The electrochemiluminescence biosensor of claim 1,

the FeMOF is Fe-MIL-88NH₂(ii) a Or the like, or, alternatively,

the AuNPs @ luminol diameter is 20 nm; the reagent is prepared by reducing chloroauric acid with luminol; or the like, or, alternatively,

the stem region of the hairpin structure probe is modified with ferrocene at the 3 'end and modified with sulfydryl at the 5' end.

3. The electrochemiluminescence biosensor of claim 1, wherein the base excision repair enzyme is a DNA glycosylase comprising alkyl adenine DNA glycosylase, formamidopyrimidine DNA glycosylase, uracil-DNA glycosylase, and thymine-DNA glycosylase;

preferably, when the base excision repair enzyme to be detected is uracil-DNA glucoamylase, the nucleotide sequence of the hairpin structure probe is shown as SEQ ID NO. 1.

4. The electrochemiluminescence biosensor of claim 1, further comprising K₄Fe(CN)₆And HCl.

5. The method for preparing an electrochemiluminescence biosensor as set forth in any one of claims 1 to 4, wherein the method comprises:

preparing a beta-CD/GO/GCE electrode: dripping the GO solution onto the surface of the GCE to obtain GO/GCE; after drying, dropwise adding the beta-CD solution onto GO/GCE to obtain a beta-CD/GO/GCE electrode;

preparation of FeMOF/AuNPs @ luminel-Hairpin probe: mixing FeMOF and AuNPs @ luminol to obtain FeMOF/AuNPs @ luminol, mixing and incubating the FeMOF/AuNPs @ luminol and a hairpin structure probe solution, and centrifuging to obtain the probe.

6. The method according to claim 5, wherein the concentration of the β -CD solution is 1 to 5mM (preferably 2 mM).

7. The method of claim 5, wherein the AuNPs @ luminol is prepared by a method comprising: heating the chloroauric acid solution to a boiling point, adding luminol, stirring vigorously, and continuously boiling until the color of the solution changes from yellow to wine red to obtain the product;

preferably, the mass fraction of the chloroauric acid solution is 0.005-0.02% (preferably 0.01%, w/w), and the concentration of luminol is 0.005-0.02M (preferably 0.01M);

preferably, the mixed incubation treatment condition is a stirring treatment under a low temperature condition for 10 to 16 hours (preferably 12 hours).

8. Use of an electrochemiluminescence biosensor as defined in any of claims 1-4 for detecting a base excision repair enzyme.

9. A method for detecting a base excision repair enzyme based on the electrochemiluminescence biosensor as set forth in any one of claims 1 to 4, the method comprising: adding a FeMOF/AuNPs @ lumineol-Hairpin probe into a sample to be detected to obtain a mixed solution, adding a beta-CD/GO/GCE electrode into the mixed solution for incubation treatment, and transferring the electrode to a medium-voltage (K) containing solution₄Fe(CN)₆Carrying out secondary incubation treatment (the treatment time is 30-60 minutes, preferably 45 minutes) in the mixed solution of HCl and the solution, and then carrying out electrochemiluminescence detection;

preferably, the incubation treatment conditions are: treating at 30-40 deg.C (preferably 37 deg.C) for 40-120 min (preferably 80 min);

preferably, the conditions for performing electrochemiluminescence detection comprise: in the presence of H₂O₂(5mM) in phosphate buffer (0.1M, pH 10).

10. Use of the electrochemiluminescence biosensor according to any one of claims 1 to 4 and/or the detection method according to claim 9 for screening drugs related to base excision repair enzymes, enzyme analysis of biological samples;

preferably, the base excision repair enzyme is a DNA glycosylase including alkyl adenine DNA glycosylase, formamidopyrimidine DNA glycosylase, uracil-DNA glycosylase, and thymine-DNA glycosylase;

the base excision repair enzyme related drugs comprise a base excision repair enzyme inhibitor and a base excision repair enzyme activator;

the biological sample comprises ex vivo blood, body fluid, cells (HeLa cells) or tissue.

Technical Field

The invention belongs to the technical field of electrochemical luminescence detection, and particularly relates to an electrochemical luminescence biosensor, a preparation method and application thereof in detection of base excision repair enzyme.

Background

The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Metal Organic Frameworks (MOFs) consist of metal ions or clusters linked by organic linking groups and have excellent physical and chemical properties. MOFs have the characteristics of large pore volume, large surface area, multiple topological structures, adjustable pore diameter, good surface chemical property and the like, and have wide application prospects in the aspects of adsorption separation, sensing, catalysis, drug delivery, imaging and the like. Prussian blue is considered to be a metal-organic coordination network (MOCN) material in which iron ions are linked by CN groups (- (Fe-CN-Fe) -), which can provide convenient assembly and precise interactive active sites for functional interfaces for sensing and biomedical applications. MOFs can serve as labels for the synthesis of prussian blue. In addition, due to the high electrochemical/electrocatalytic performance and the low oxidation-reduction potential, the prussian blue is often used as a high-efficiency medium and a nano-enzyme of an electrochemical sensor.

Electrochemiluminescence (ECL) is an optical emission process of electrochemical excitation caused by energy relaxation of an excited species. ECL combines the advantages of luminescence and electrochemical technologies and is becoming an increasingly popular biosensing technology for the detection of metal ions and small molecules, ECL immunoassays, ECL gene sensors and ECL cell sensors. The ECL not only inherits the characteristics of high sensitivity and wide dynamic range of chemiluminescence, but also has the advantages of simple, stable and convenient electrochemical method and the like. There are three major types of luminophores widely used in ECL research including ruthenium (II) complexes, Luminol (Luminol) and Quantum Dots (QDs). Wherein the luminol has good chemical stability and lower oxidation potential. In addition, various strategies such as the use of horseradish peroxidase (HRP) and dnase to enhance luminol ECL signal. However, peroxidase enzymes are costly to prepare, purify, and store, are subject to variability under harsh conditions, and have inhibited catalytic activity in certain complex media (e.g., wastewater). Prussian blue, as a mimetic peroxidase, catalyzes the oxidation of luminol by dissolved oxygen, producing an enhanced chemiluminescent signal.

Due to the specificity and biological orthogonality of recognition motif, the supermolecule non-covalent interaction between host and guest molecules has wide application in the fields of catalysis, electrochemical luminescence, electrochemical sensors and the like. Cyclodextrins are specific oligosaccharides with 6, 7, 8 glucose units. The unique cage structure enables CDs and derivatives thereof to have good recognition and encapsulation capabilities on guest molecules, and can be used as host molecules in biosensors. Ferrocene (Fc) is a redox molecule that can be used to form complexes with β -CD to improve solubility, electrical stability and bioavailability. In addition, electrochemically oxidized Fc (Fc)⁺) Can catalyze H₂O₂To form OH^·Thus in luminol-H₂O₂Enhanced ECL signal generation in the system with simultaneous Fc (Fc)⁺) The stability advantage is evident at different temperatures and pH values.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an electrochemiluminescence biosensor based on a subject-object recognition technology, which can be used for detecting base excision repair enzyme by three-time signal amplification, so that the electrochemiluminescence biosensor has extremely high sensitivity and has wide application value in the biomedical research fields of screening of inhibitors/activators of the base excision repair enzyme, analysis of biological samples and the like.

In order to achieve the technical purpose, the technical scheme of the invention is as follows:

in a first aspect of the invention, an electrochemiluminescence biosensor for detecting a base excision repair enzyme is provided, and the electrochemiluminescence biosensor comprises a beta-CD/GO/GCE electrode, wherein the beta-CD/GO/GCE electrode is prepared by modifying Graphene Oxide (GO) and cyclodextrin (beta-CD) onto a Glassy Carbon Electrode (GCE).

Further, the electrochemical luminescence biosensor also comprises a FeMOF/AuNPs @ luminol-Hairpin probe, wherein the FeMOF/AuNPs @ luminol-Hairpin probe comprises:

iron-based metal organic framework (FeMOF) nanometer particle to and the AuNPs @ lumineol of modification on FeMOF, still modified on the AuNPs @ lumineol has hairpin structure probe, stem region design in the hairpin structure probe has the target base of the base excision repair enzyme that awaits measuring, target base number can set up according to actual conditions, like 1, 2, 4, 6 etc..

Wherein the base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG), and the like.

Further, the electrochemiluminescence biosensor further comprises K₄Fe(CN)₆And HCl.

In a second aspect of the present invention, there is provided a method for preparing the above electrochemiluminescence biosensor for detecting a base excision repair enzyme, the method comprising:

(1) preparing a beta-CD/GO/GCE electrode: dripping the GO solution onto the surface of the GCE to obtain GO/GCE; after drying, dropwise adding the beta-CD solution onto GO/GCE to obtain a beta-CD/GO/GCE electrode;

(2) preparation of FeMOF/AuNPs @ luminel-Hairpin probe: mixing FeMOF and AuNPs @ luminol to obtain FeMOF/AuNPs @ luminol, mixing and incubating the FeMOF/AuNPs @ luminol and a hairpin structure probe solution, and centrifuging to obtain the probe.

In a third aspect of the invention, there is provided the use of the above-described electrochemiluminescence biosensor for detecting a base excision repair enzyme.

In a fourth aspect of the present invention, there is provided a method for detecting a base excision repair enzyme based on the above-described electrochemiluminescence biosensor, the method comprising: adding a FeMOF/AuNPs @ lumineol-Hairpin probe into a sample to be detected to obtain a mixed solution, adding a beta-CD/GO/GCE electrode into the mixed solution for incubation treatment, and transferring the electrode to a medium-voltage (K) containing solution₄Fe(CN)₆And performing secondary incubation treatment in HCl solution, and performing electrochemiluminescence detection.

In a fifth aspect of the present invention, there is provided the use of the above-mentioned electrochemiluminescence biosensor and/or detection method in drug screening and enzyme analysis of biological samples related to the enzyme for repairing base excision.

The base excision repair enzyme related drugs include but are not limited to base excision repair enzyme inhibitors and base excision repair enzyme activators;

the biological sample comprises ex vivo blood, body fluid, cells or tissue, such as HeLa cells. Tests prove that the biosensor provided by the invention has better analysis capability on real complex biological samples, can be used for quantitative detection on the activity of cell base excision repair enzyme (such as UDG), and has great application potential in the fields of biomedical basic research, clinical diagnosis and the like.

The invention has the beneficial effects that:

1. use of metal organic framework (FeMOF): the metal organic framework (FeMOF) used in the invention can adsorb luminol gold nanoparticles loaded with a large amount of luminol molecules, and can be used as a label for synthesizing Prussian blue to obviously amplify an electrochemical luminescence signal.

2. High sensitivity: the present invention employs electrochemically oxidized Fc (Fc)⁺) Can catalyze H₂O₂To form OH^·Thus in luminol-H₂O₂The enhanced ECL signal is generated in the system, and the Prussian blue is used for catalyzing luminol, so that the operation procedure is greatly simplified, the UDG can be sensitively detected, and the detection limit is 2.468 multiplied by 10^-4U is per liter.

3. A wide range of potential applications: the electrochemiluminescence biosensor designed by the invention can be used for screening of UDG inhibitors and analysis of biological samples, and has wide potential application in biomedical research.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic diagram of a method for detecting UDG based on host-object recognition and triple signal amplification by an ECL biosensor according to the present invention;

FIG. 2 is a representation of different nanoparticles of the present invention, A is an X-ray diffraction pattern (XRD) of synthetic Fe-MOF, B is an ultraviolet absorption spectrum of luminol (a) nanogold (B), AuNPs @ luminol (C), C is a Transmission Electron Microscope (TEM) image of synthetic AuNPs @ luminol particles, D is a Scanning Electron Microscope (SEM) image of synthetic Fe-MOF, E is a Scanning Electron Microscope (SEM) image of FeMOF/AuNPs @ luminol, and F is a Scanning Electron Microscope (SEM) image of a Prussian blue film prepared on the surface of FeMOF/AuNPs @ luminol;

FIG. 3 is a representation of the experimental feasibility analysis of the invention, A being Fe (CN) at 5 mmoles per liter with 0.1 moles per liter KCl₆ ^3-/4-In the Electrochemical Impedance Spectroscopy (EIS) of different modified electrodes, a is a bare Glassy Carbon Electrode (GCE), b is GO/GCE, c is beta-CD/GO/GCE, d is FeMOF/AuNPs @ luminol-hairpin probe +1U mL^-1UDG + beta-CD/GO/GCE, B is the Electrochemiluminescence (ECL) curve of different modified electrodes in PBS containing 0.1 mol/L of 5 mmol/L, a is AuNPs @ luminol-hairpin probe, B is AuNPs @ luminol-hairpin probe +1U mL^-1UDG, c is FeMOF/AuNPs @ luminel-hairpin probe +1U mL^-1UDG, d is FeMOF/AuNPs @ luminel-hairpin probe +1UmL^-1UDG + prussian blue;

FIG. 4 is an optimization chart of experimental conditions of the present invention, wherein A is the optimization of the concentration of beta-CD, and B is Fe (CN)₆ ^4-Optimizing the concentration, wherein C is the incubation time of one-step reaction of the UDG and the sensor, D is the time of forming Prussian blue, and an error bar represents the standard deviation of three independent experiments;

FIG. 5 is a graph representing the results of the sensitivity test of the present invention, wherein A is an electrochemiluminescence intensity (ECL) curve of a biosensor incubated with different concentrations of UDG (from a to j: 0, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1U per ml), B is a linear relationship between the ECL intensity and the logarithm of the UDG concentration in the range of 0.0005 to 1U per ml, and the detection conditions are as follows: containing 5 millimoles of H per liter₂O₂0.1 moles per liter of phosphoric acid buffer solution at pH10, error bars represent standard deviation of three independent experiments;

FIG. 6 shows the difference in the effect of different concentrations of UGI on the relative activity of UDG in accordance with the present invention. The concentration of UDG was maintained at 1U per ml, with error bars representing the standard deviation of three independent experiments;

FIG. 7 is a graph representing the results of selectivity and stability experiments of the present invention, panel A showing 0.01 mg/ml BSA, 1U per liter hAAG, 0.01 mg/ml IgG, respectively, and error bars representing the standard deviation of three independent experiments; b is the stability of the ECL biosensor under the continuous cyclic potential scanning of 12 periods at 0-0.5V;

FIG. 8 is a graph of the linear correlation between ECL intensity and the logarithm of HeLa cell number from 5 to 10000 cells of the present invention, with error bars representing the standard deviation of three independent experiments.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.

The traditional detection method of the base excision repair enzyme generally wastes time and labor, and has low detection sensitivity. In order to solve the technical problems, the invention provides an electrochemiluminescence biosensor based on a subject-object recognition technology, which can be used for three times of signal amplification and high-sensitivity detection of base excision repair enzymes.

In an exemplary embodiment of the invention, an electrochemiluminescence biosensor for detecting a base excision repair enzyme is provided, and the electrochemiluminescence biosensor comprises a beta-CD/GO/GCE electrode, wherein the beta-CD/GO/GCE electrode is prepared by modifying Graphene Oxide (GO) and cyclodextrin (beta-CD) onto a Glassy Carbon Electrode (GCE).

In yet another embodiment of the present invention, the electrochemiluminescence biosensor further comprises a FeMOF/AuNPs @ luminol-Hairpin probe, the FeMOF/AuNPs @ luminol-Hairpin probe comprising: iron-based metal organic framework (FeMOF) nanometer particle to and the AuNPs @ lumineol of modification on FeMOF, still modified on the AuNPs @ lumineol has hairpin structure probe, the stem region design of hairpin structure probe has the target base of the basic group excision repair enzyme that awaits measuring, the target base sets up according to actual conditions, can be 1 to a plurality ofly, like 1, 2, 4, 6 etc..

In yet another embodiment of the present invention, the iron-based metal organic framework (FeMOF) nanoparticles are Fe-MIL-88NH₂(CCDC: 647646); the Fe-MIL-88NH₂The catalyst has amino functional groups and good electrocatalytic activity.

In yet another embodiment of the present invention, the AuNPs @ luminol has a diameter of 20 nm; is prepared by reducing chloroauric acid with luminol.

In another embodiment of the present invention, the stem region of the hairpin structure probe is modified with ferrocene (Fc) at the 3 'end and thiol at the 5' end, such that the hairpin structure probe is connected to AuNPs @ lumineol via gold-sulfur bond.

In yet another embodiment of the invention, the base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), and thymine-DNA glycosylase (TDG).

In another embodiment of the present invention, when the base excision repair enzyme to be detected is uracil-DNA saccharifying enzyme (UDG), the nucleotide sequence of the hairpin probe can be:

5'-HS-UUUGUCUGUGAA GGA GGT AGA TCA CAG ACA AA-(CH₂)₆-Fc-3' (SEQ ID NO. 1). Wherein, italicized letters represent bases that undergo complementary pairing in the hairpin probe stem region.

In yet another embodiment of the present invention, the electrochemiluminescence biosensor further comprises K₄Fe(CN)₆And HCl.

In another embodiment of the present invention, there is provided a method for preparing the above-described electrochemiluminescence biosensor for detecting a base excision repair enzyme, the method comprising:

(1) preparing a beta-CD/GO/GCE electrode: dripping the GO solution onto the surface of the GCE to obtain GO/GCE; after drying, dropwise adding the beta-CD solution onto GO/GCE to obtain a beta-CD/GO/GCE electrode;

(2) preparation of FeMOF/AuNPs @ luminel-Hairpin probe: mixing FeMOF and AuNPs @ luminol to obtain FeMOF/AuNPs @ luminol, mixing and incubating the FeMOF/AuNPs @ luminol and a hairpin structure probe solution, and centrifuging to obtain the probe.

In another embodiment of the present invention, the concentration of the beta-CD solution is 1 to 5mM (preferably 2 mM); experiments prove that when the concentration of the beta-CD solution is 2mM, the luminous intensity is highest, and the detection effect is optimal.

In another embodiment of the present invention, the method for preparing AuNPs @ luminol is substantially the same as that for preparing AuNPs by a conventional sodium citrate reduction method, except that the sodium citrate is replaced by luminol.

Specifically, the preparation method comprises the following steps: heating the chloroauric acid solution to a boiling point, adding luminol, stirring vigorously, and boiling continuously until the color of the solution changes from yellow to wine red.

Wherein the mass fraction of the chloroauric acid solution is 0.005-0.02% (preferably 0.01%, w/w), and the concentration of luminol is 0.005-0.02M (preferably 0.01M);

in another embodiment of the present invention, the stem region of the hairpin structure probe is designed with one or more target bases of the base excision repair enzyme to be detected, and the number of the target bases can be set according to actual situations, such as 1, 2, 4, 6, and the like.

In another embodiment of the present invention, the stem region of the hairpin structure probe is modified with ferrocene (Fc) at the 3 'end and thiol at the 5' end, such that the hairpin structure probe is connected to AuNPs @ lumineol via gold-sulfur bond.

In yet another embodiment of the invention, the base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), and thymine-DNA glycosylase (TDG).

In another embodiment of the present invention, when the base excision repair enzyme to be detected is uracil-DNA saccharifying enzyme (UDG), the nucleotide sequence of the hairpin probe can be:

5'-HS-UUUGUCUGUGAA GGA GGT AGA TCA CAG ACA AA-(CH₂)₆-Fc-3' (SEQ ID NO. 1). Wherein, italicized letters represent bases that undergo complementary pairing in the hairpin probe stem region.

In another embodiment of the present invention, the mixed incubation treatment is performed under stirring at a low temperature (4 ℃) for 10 to 16 hours (preferably 12 hours).

In still another embodiment of the present invention, there is provided a use of the above-described electrochemiluminescence biosensor for detecting a base excision repair enzyme.

Wherein the base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG).

In another embodiment of the present invention, there is provided a method for detecting a base excision repair enzyme based on the above-described electrochemiluminescence biosensor, the method comprising: adding a FeMOF/AuNPs @ lumineol-Hairpin probe into a sample to be detected to obtain a mixed solution, adding a beta-CD/GO/GCE electrode into the mixed solution for incubation treatment, and transferring the electrode to a medium-voltage (K) containing solution₄Fe(CN)₆Performing a secondary incubation treatment (treatment time is 30-60 minutes, preferably 45 minutes) in a mixed solution of (0.5mM) and HCl (10mM), and then performing electrochemiluminescence detection.

Wherein the incubation treatment conditions are as follows: treating at 30-40 deg.C (preferably 37 deg.C) for 40-120 min (preferably 80 min).

Conditions for performing electrochemiluminescence detection include: containing H₂O₂(5mM) phosphate buffer (0.1M, pH 10).

In another embodiment of the present invention, there is provided the use of the above-mentioned electrochemiluminescence biosensor and/or detection method in drug screening and enzyme analysis of biological samples related to the enzyme for repairing base excision.

The base excision repair enzyme is a DNA glycosylase, including but not limited to alkyl adenine DNA glycosylase (AAG), formamidopyrimidine DNA glycosylase (FPG), uracil-DNA glycosylase (UDG), thymine-DNA glycosylase (TDG); further preferably uracil-DNA saccharifying enzyme (UDG).

The base excision repair enzyme related drugs include but are not limited to base excision repair enzyme inhibitors and base excision repair enzyme activators;

the biological sample comprises ex vivo blood, body fluid, cells or tissue, such as HeLa cells. Tests prove that the biosensor provided by the invention has better analysis capability on real complex biological samples, can be used for quantitative detection on the activity of cell base excision repair enzyme (such as UDG), and has great application potential in the fields of biomedical basic research, clinical diagnosis and the like.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments. In the following examples, the hairpin probe sequence from 5 'to 3' is:

5'-HS-UUUGUCUGUGAA GGA GGT AGA TCA CAG ACA AA-(CH₂)₆-Fc-3'。