Electrochemical biosensor for detecting base excision repair enzyme and preparation method and application thereof
阅读说明：本技术 一种检测碱基切除修复酶的电化学生物传感器及其制备方法与应用 (Electrochemical biosensor for detecting base excision repair enzyme and preparation method and application thereof ) 是由 张春阳 崔琳 赵敏慧 于 2019-10-15 设计创作，主要内容包括：本发明提供一种检测碱基切除修复酶的电化学生物传感器及其制备方法与应用。所述生物传感器包括β-CD/FeCN/GCE电极,所述β-CD/FeCN/GCE电极由含铁富氮碳纳米管和环糊精修饰至玻碳电极上制得；所述电化学生物传感器还包括MB-hairpin/AuNPs探针,所述MB-hairpin/AuNPs探针包括金纳米颗粒,以及修饰在AuNP上的硫醇化的MB-发夹结构探针,所述MB-发夹结构探针中的茎区设计有1至多个待测碱基切除修复酶的目标碱基。本发明制备得到电化学生物传感器灵敏度高、背景信号低等优点,在碱基切除修复酶抑制剂的筛选以及对于生物样品的分析等领域具有广泛的应用价值。(The invention provides an electrochemical biosensor for detecting base excision repair enzyme and a preparation method and application thereof. The biosensor comprises a beta-CD/FeCN/GCE electrode, wherein the beta-CD/FeCN/GCE electrode is prepared by modifying an iron-containing nitrogen-rich carbon nanotube and cyclodextrin onto a glassy carbon electrode; the electrochemical biosensor also comprises an MB-hairpin/AuNPs probe, wherein the MB-hairpin/AuNPs probe comprises gold nanoparticles and a thiolated MB-hairpin structure probe modified on AuNP, and a stem region in the MB-hairpin structure probe is designed with 1 to a plurality of target bases of the base excision repair enzyme to be detected. The electrochemical biosensor prepared by the invention has the advantages of high sensitivity, low background signal and the like, and has wide application value in the fields of screening of the base excision repair enzyme inhibitor, analysis of biological samples and the like.)
1. The electrochemical biosensor for detecting the base excision repair enzyme is characterized by comprising a beta-CD/FeCN/GCE electrode, wherein the beta-CD/FeCN/GCE electrode is prepared by modifying an iron-containing nitrogen-rich carbon nanotube and cyclodextrin onto a glassy carbon electrode.
2. The electrochemical biosensor of claim 1, further comprising an MB-hairpin/AuNPs probe comprising gold nanoparticles and a thiolated MB-hairpin probe modified on the AuNP, wherein the stem region of the MB-hairpin probe is designed with 1 to multiple target bases of the base excision repair enzyme to be detected.
3. The electrochemical biosensor of claim 1, wherein the base excision repair enzymes are DNA glycosylases including alkyl adenine DNA glycosylases, formamidopyrimidine DNA glycosylases, uracil-DNA glycosylases, and thymine-DNA glycosylases.
4. The electrochemical biosensor as set forth in claim 2, wherein the stem region of the MB-hairpin probe is modified at the 3 '-end with methylene blue and at the 5' -end with a thiol group;
preferably, when the base excision repair enzyme to be detected is uracil-DNA glucoamylase, the nucleotide sequence of the hairpin structure is shown as SEQ ID NO. 1.
5. The method for preparing an electrochemical biosensor for detecting a base excision repair enzyme according to any one of claims 1 to 4, comprising:
preparation of beta-CD/FeCN/GCE electrode: dropwise adding a FeCN solution onto the surface of the GCE to obtain FeCN/GCE; after drying, dropwise adding the beta-CD solution on FeCN/GCE to obtain a beta-CD/FeCN/GCE electrode;
preparation of MB-hairpin/AuNPs Probe: mixing and incubating the gold nanoparticle solution and the thiolated MB-hairpin structure probe solution, centrifuging, washing and dispersing to obtain the gold nanoparticle probe.
6. The method according to claim 5, wherein the FeCN solution has a concentration of 0.5 to 4mg/mL (preferably 2 mg/mL); the concentration of the beta-CD solution is 0.5-4 mg/mL (preferably 2 mg/mL).
7. The method according to claim 5, wherein the gold nanoparticles have a diameter of 13nm and are prepared by a sodium citrate reduction method; or the like, or, alternatively,
the activating treatment process of the thiolated MB-hairpin structure probe comprises the following steps: reducing the disulfide bond-bonded oligonucleotide with tris (2-carboxyethyl) phosphine hydrochloride for 0.5 to 1.5 hours (preferably 1 hour); or the like, or, alternatively,
the mixing incubation time is 10 to 20 hours (preferably 16 hours).
8. Use of the electrochemical biosensor according to any one of claims 1 to 4 for detecting a base excision repair enzyme.
9. A method for detecting a base excision repair enzyme based on the electrochemical biosensor as set forth in any one of claims 1 to 4, comprising: adding an MB-hairpin/AuNPs probe into a sample to be detected to obtain a mixed solution, adding a beta-CD/FeCN/GCE electrode into the mixed solution for incubation treatment, and performing electrochemical detection;
preferably, the incubation condition is 40 to 120min (more preferably 80min), and the incubation temperature is 30 to 40 ℃ (more preferably 37 ℃).
10. Use of the electrochemical 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 comprising alkyl adenine DNA glycosylase, formamidopyrimidine DNA glycosylase, uracil-DNA glycosylase and thymine-DNA glycosylase;
preferably, the base excision repair enzyme related drug comprises a base excision repair enzyme inhibitor and a base excision repair enzyme activator;
preferably, the biological sample comprises cells of an organism (HeLa cells).
Technical Field
The invention belongs to the technical field of electrochemical detection, and particularly relates to an electrochemical biosensor for detecting base excision repair enzyme, and a preparation method and application thereof.
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.
The natural enzyme has high activity and good specificity, and plays an important role in organism metabolism and various biochemical reactions. However, natural enzymes face significant challenges including high cost of preparation, purification and storage, susceptibility to denaturation under harsh conditions, and inhibition of catalytic activity in certain complex media (e.g., wastewater). The demand for highly efficient specific enzymes has led chemists to design synthetases with similar structures and analogous functions using novel nanotechnology, and a large number of functional nanomaterials continue to emerge. Noble metals (e.g. Pd and Au), metal oxides (e.g. MnO)2，Fe3O4And CeO2) Carbon-based nanomaterials, metal complexes, porphyrins, and polymers have been extensively studied to mimic the structure and function of natural enzymes. Compared with natural enzymes, the nano material has the advantages of low cost, adjustable catalytic activity, good stability, good robustness under severe environment conditions (such as methanol, ethanol and dimethylformamide), easiness in long-term storage and treatment and the like, and is expected to become a research hotspot of artificial enzymes.
The realization of supramolecular host-guest recognition through non-covalent interactions is a new approach to large functional structure assembly. In general, the host (e.g., β -CD) contains a large volume cavity and has outstanding recognition and encapsulation capabilities for guest molecules. Due to the specificity and bio-orthogonality of the recognition motifs, host-guest interactions have found widespread use in bioassays.
Base Excision Repair (BER) is one of the DNA repair mechanisms, uracil-DNA glycosylase (UDG) plays a key role in maintaining genome integrity. UDG is capable of removing uracil damage from DNA by catalyzing the hydrolysis of the n-glycosidic bond between uracil and deoxyribose. Because DNA glycosylase is crucial to DNA damage repair and is associated with individual and population disease susceptibility, UDG has become a promising biomarker and a potential therapeutic target. Traditional methods for detecting DNA glycosylase include gel electrophoresis, radioactive detection and chromatography, but these methods are generally time-consuming and labor-consuming, involve dangerous radioactive substances and complicated procedures. In addition, several new UDG detection methods have been developed, including colorimetric and fluorescent methods, but they involve the preparation of hairpin probes and functional nucleic acid probes with dye labels. Therefore, there is a need to develop a simple and sensitive method for detecting DNA glycosylase.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an electrochemical biosensor based on subject-object action and simulated enzyme electrocatalytic signal amplification, and a preparation method and application thereof.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
in a first aspect of the present invention, an electrochemical biosensor for detecting a base excision repair enzyme is provided, which comprises a beta-CD/FeCN/GCE electrode, wherein the beta-CD/FeCN/GCE electrode is prepared by modifying iron-containing nitrogen-rich carbon nanotubes (FeCN) and cyclodextrin (beta-CD) onto a Glassy Carbon Electrode (GCE).
Further, the electrochemical biosensor further comprises an MB-hairpin/AuNPs probe, wherein the MB-hairpin/AuNPs probe comprises gold nanoparticles (AuNPs) and a thiolated MB-hairpin structure (hairpin) probe modified on the AuNPs, a stem region in the MB-hairpin structure probe is designed with a target base of the repair enzyme for base excision to be detected, and the number of the target base can be set according to actual conditions, such as 1, 2, 4, 6 and the like.
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; preferably uracil-DNA saccharifying enzyme (UDG).
In a second aspect of the present invention, there is provided a method for preparing the electrochemical biosensor for detecting a base excision repair enzyme, the method comprising:
(1) preparation of beta-CD/FeCN/GCE electrode: dropwise adding a FeCN solution onto the surface of the GCE to obtain FeCN/GCE; after drying, dropwise adding the beta-CD solution on FeCN/GCE to obtain a beta-CD/FeCN/GCE electrode;
(2) preparation of MB-hairpin/AuNPs Probe: mixing and incubating gold nanoparticle (AuNP) solution and thiolated MB-hairpin structure probe solution, centrifuging, washing and dispersing to obtain the target product.
In a third aspect of the present invention, there is provided the use of the above electrochemical 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 electrochemical biosensor, the method comprising: and adding the MB-hairpin/AuNPs probe into a sample to be detected to obtain a mixed solution, adding the beta-CD/FeCN/GCE electrode into the mixed solution for incubation treatment, and performing electrochemical detection.
In a fifth aspect of the present invention, there is provided the use of the electrochemical biosensor and/or the detection method described above 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 cells of an organism, 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 mimetic enzymes: although natural enzymes have good activity and specificity, the wide application of the natural enzymes is limited by high cost and changeability, the iron-nitrogen-rich carbon nanotube (FeCN) used by the invention is prepared by one-step self-assembly of low-cost dicyandiamide and iron (II) chloride tetrahydrate, and then is synthesized by simple heat treatment, the method is simple and convenient, the cost is low, and the iron-nitrogen-rich carbon nanotube (FeCN) simulating peroxidase can electrocatalytic methylene blue with electrocatalytic activity to obviously amplify electrochemical signals.
2. High sensitivity: the catalytic oxidation of MB by FeCN is mediated by the catalytic substrate RSH which is relatively stable in O2 oxidation, but not by external H2O2Greatly simplifying the experimental process, and being capable of sensitively detecting the UDG with the detection limit of 7.413 multiplied by 10-5U mL-1。
3. Low background signal: due to the stem-loop structure of hairpin DNA and the steric effect of AuNPs, supramolecular host-guest reactions occur between MB and β -CD, resulting in very low background signals.
4. A wide range of potential applications: the electrocatalytic amplification 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 an electrochemical biosensor for one-step detection of UDG based on host-guest interaction and FeCN mimic enzyme electrocatalytic signal amplification.
FIG. 2 is a representation of different nanoparticles of the present invention, wherein A is an X-ray diffraction pattern (XRD) of a synthesized iron-containing nitrogen-rich carbon nanotube (FeCN), B is an ultraviolet absorption spectrum of the synthesized nanogold and the MB-modified hairpin probe in combination with the nanogold, C is a Transmission Electron Microscope (TEM) image of the synthesized iron-containing nitrogen-rich carbon nanotube (FeCN), and D is a Transmission Electron Microscope (TEM) image of the gold nanoparticle.
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-The Electrochemical Impedance Spectroscopy (EIS) of different modified electrodes in (1), wherein a is a bare Glassy Carbon Electrode (GCE), b is FeCN/GCE, c is beta-CD/FeCN/GCE,d is MB-hairpin/AuNPs +1U mL-1UDG + beta-CD/FeCN/GCE; b is an electrochemical Differential Pulse Voltammetry (DPV) curve containing 0.1 mol/L PBS of different modified electrodes, a is MB-hairpin/AuNPs + beta-CD/FeCN/GCE, B is MB-hairpin/AuNPs +1U mL-1UDG + beta-CD/FeCN/GCE, c is MB-hairpin/AuNPs +1U mL-1UDG + beta-CD/FeCN/GCE +10mM cysteine, d is MB-hairpin/AuNPs +1UmL- 1UDG+FeCN/GCE。
FIG. 4 is a graph of experimental condition optimization of the present invention, where A is the optimization of FeCN concentration, B is the optimization of β -CD concentration, C is the incubation time of one-step reaction of UDG with a sensor, and error bars represent the standard deviation of three independent experiments.
FIG. 5 is a graph representing the results of the sensitivity and selectivity experiments of the present invention, A is the electrochemical Differential Pulse Voltammetry (DPV) curve of biosensors 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 the linear relationship between current and logarithm of UDG concentration in the range of 0.0005 to 1U per ml, detection conditions: 0.1 mol/l phosphate buffered solution at pH 7.4 containing 10 mmol/l cysteine, C is a graph characterizing the results of the selectivity experiment, 0.01 mg/ml BSA, 1U/l hAAG, 1U/l FPG, respectively, and the error bars represent the 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, and the error bars represent the standard deviation of three independent experiments.
FIG. 7 is a graph of the linear correlation between the current of the invention and the logarithm of the number of HeLa cells from 5 to 10000 cells, 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.
As previously mentioned, conventional DNA glycosylase detection methods are often time consuming, laborious, involve hazardous radioactive materials and complicated procedures. In order to solve the technical problems, the invention provides a method for detecting a base excision repair enzyme based on a host-guest action and a mimic enzyme mediated electrocatalytic amplification biosensor.
In an exemplary embodiment of the present invention, an electrochemical biosensor for detecting a base excision repair enzyme is provided, which includes a β -CD/FeCN/GCE electrode fabricated by modifying an iron-containing nitrogen-rich carbon nanotube (FeCN) and cyclodextrin (β -CD) onto a Glassy Carbon Electrode (GCE).
In another embodiment of the present invention, the electrochemical biosensor further includes an MB-hairpin/AuNPs probe, where the MB-hairpin/AuNPs probe includes gold nanoparticles (AuNPs) and a thiolated MB-hairpin structure (hairpin) probe modified on the AuNPs, a stem region in the MB-hairpin structure probe is designed with 1 to multiple target bases of the base excision repair enzyme to be detected, and the number of the target bases may be set according to actual situations, such as 1, 2, 4, 6, and the like.
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, the stem region of the hairpin structure probe is modified with Methylene Blue (MB) at the 3 'end and thiol at the 5' end, such that the hairpin structure is connected to the gold nanoparticle (AuNP) through a gold-sulfur bond.
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 structure can be:
5’-HS-UUUGUCUGUGAA GGA GGT AGA TCA CAG ACA AA-(CH2)6-MB-3' (SEQ ID NO. 1). Wherein, italicized letters represent bases that undergo complementary pairing in the stem region of the hairpin structure.
In another embodiment of the present invention, there is provided a method for preparing the electrochemical biosensor for detecting a base excision repair enzyme, the method comprising:
(1) preparation of beta-CD/FeCN/GCE electrode: dropwise adding a FeCN solution onto the surface of the GCE to obtain FeCN/GCE; and after drying, dropwise adding the beta-CD solution onto FeCN/GCE to obtain the beta-CD/FeCN/GCE electrode.
(2) Preparation of MB-hairpin/AuNPs Probe: mixing and incubating gold nanoparticle (AuNP) solution and thiolated MB-hairpin structure probe solution, centrifuging, washing and dispersing to obtain the target product.
In another embodiment of the invention, the concentration of the FeCN solution is 0.5-4 mg/mL (preferably 2 mg/mL); the concentration of the beta-CD solution is 0.5-4 mg/mL (preferably 2 mg/mL); experiments prove that when the concentration of the FeCN solution is 2mg/ml and the concentration of the beta-CD solution is 2mg/ml, the electrochemical peak current is strongest, and the detection effect is optimal.
The preparation method of the FeCN comprises the following steps: heating dicyandiamide and ferrous salt to 490-510 ℃ in an inert gas atmosphere to self-assemble to form a precursor, heating the precursor to 890-910 ℃ in the inert gas atmosphere, and calcining to obtain the iron-containing nitrogen-rich carbon nanotube FeCN.
Wherein the diameter of the gold nanoparticle (AuNP) is 13nm, and the gold nanoparticle is preferably prepared by a sodium citrate reduction method.
The activating treatment process of the thiolated MB-hairpin structure probe comprises the following steps: the disulfide-bonded oligonucleotide is reduced with tris (2-carboxyethyl) phosphine hydrochloride (TCEP) for 0.5 to 1.5 hours (preferably 1 hour).
The mixing incubation time is 10-20 hours (preferably 16 hours), namely, the thiolated hairpin probe is connected to AuNPs through a gold-sulfur bond.
In still another embodiment of the present invention, there is provided a use of the above electrochemical 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 electrochemical biosensor, the method comprising: and adding the MB-hairpin/AuNPs probe into a sample to be detected to obtain a mixed solution, adding the beta-CD/FeCN/GCE electrode into the mixed solution for incubation treatment, and performing electrochemical detection.
Wherein the incubation treatment condition is 40-120 min (preferably 80min), and the incubation treatment temperature is 30-40 ℃ (preferably 37 ℃).
In another embodiment of the present invention, the electrochemical biosensor and/or the detection method are used for 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 cells of an organism, 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 probes have the sequence from 5 'to 3':
5’-HS-UUUGUCUGUGAA GGA GGT AGA TCA CAG ACA AA-(CH2)6-MB-3’。