Photoelectrochemical immunosensor for detecting activity of DNA methylase and preparation method and application thereof
阅读说明：本技术 一种检测dna甲基化酶活性的光电化学免疫传感器及其制备方法和应用 (Photoelectrochemical immunosensor for detecting activity of DNA methylase and preparation method and application thereof ) 是由 沈艳飞 陈开洋 薛怀佳 于 2019-08-27 设计创作，主要内容包括：本发明公开了一种检测DNA甲基化酶活性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Sb<Sub>2</Sub>Se<Sub>3</Sub>-GO-CS修饰基底电极,hDNA共价结合到修饰后的基底电极上,Dam MTase甲基化和DPn I剪切后在电极上得到ssDNA,S1-AuNPs与ssDNA特异性结合得到。本发明传感器可以增加了溶液的分散性,进一步共价偶联生物分子,通过酰胺键共价结合大量hDNA,再将hDNA用Dam MTase甲基化通过DPn I的剪切作用释放出ssDNA。本发明传感器具有良好的稳定性与灵敏性,操作简便,反应迅速,能够实现对DNA甲基化酶活性的定量检测,将为疾病的早期诊断与治疗提供新的平台。(The invention discloses a photoelectrochemical immunosensor for detecting DNA methylase activity and a preparation method and application thereof, wherein the photoelectrochemical immunosensor is prepared from Sb 2 Se 3 The GO-CS modified substrate electrode is characterized in that hDNA is covalently bonded to the modified substrate electrode, ssDNA is obtained on the electrode after Dam MTase methylation and DPn I shearing, and S1-AuNPs are specifically bonded with the ssDNA. The sensor can increase the dispersibility of the solution, further covalently couples biomolecules, covalently combines a large amount of hDNA through amido bonds, and then methylates the hDNA by Dam MTase to release ssDNA through the shearing action of DPn I. The sensor of the invention has good stability and sensitivityThe method is simple and convenient to operate and rapid in reaction, can realize quantitative detection of the activity of the DNA methylase, and provides a new platform for early diagnosis and treatment of diseases.)
1. A photoelectrochemical immunosensor for detecting DNA methylase activity is characterized in that Sb is used as a material2Se3The GO-CS modified substrate electrode is characterized in that hDNA is covalently bonded to the modified substrate electrode, ssDNA is obtained on the electrode after Dam MTase methylation and DPn I shearing, and S1-AuNPs are specifically bonded with the ssDNA.
2. The photoelectrochemical immunosensor of claim 1, wherein the substrate electrode is an indium tin oxide semiconductor electrode; the sequence of the hDNA is as follows: 5' -CAGAGATCCATATACGTTTTTCGTATATGGATCTCTGAAAAA (CH)2)6-NH2-3'; the sequence of the ssDNA is 5' -TCTCTGAAAAA (CH)2)6-NH2-3’。
3. The photoelectrochemical immunosensor of claim 1, wherein the Sb is2Se3The mass concentration of CS in GO-CS is 0.01 wt% -0.5 wt%.
4. The photoelectrochemical immunosensor of any one of claims 1-3, wherein the Sb is2Se3GO from aqueous GO solution with Sb2Se3Mixing the aqueous solution and performing ultrasonic treatment to obtain the product; sb2Se3The volume ratio of aqueous solution to GO aqueous solution is preferably 1: (0.01-1).
5. The photoelectrochemical immunosensor of claim 4, wherein the Sb is2Se3From a precursor solution of Se with SbCl3Mixing the 2-ME solution, heating for reaction, washing and drying to obtain the product.
6. The photoelectrochemical immunosensor of claim 1, wherein the S1-AuNPs is obtained by stirring AuNPs and single-stranded DNA S1 for reaction; the AuNPs are prepared from HAuCl4Heating the aqueous solution to boiling, adding a sodium citrate solution, continuing to react, and cooling to room temperature to obtain the compound S1, wherein the sequence of S1 is 5' -TTTTTCAGAGA (CH)2)6-SH-3’。
7. The method for preparing the photoelectrochemical immunosensor for the detection of DNA methylase activity according to claim 1, comprising the steps of:
(1) fixing a signal layer: sb2Se3Dripping the mixed solution of GO and CS on the surface of a substrate electrode, drying at room temperature, drying again, washing with deionized water, and drying in the air;
(2) anchoring recognition molecule: dropping EDC/NHS mixed solution on the surface of the substrate electrode obtained in the step (1), standing at room temperature for 1-2h, washing with PBS solution, dropping the recognition molecule hDNA on the substrate electrode after washing, incubating for 2-3h at 25-37 ℃, and then washing with PBS solution;
(3) non-specific site blocking: dripping MCH solution on the surface of the substrate electrode obtained in the step (2), sealing for 0.5-1h, and then washing with PBS solution;
(4) methylation reaction: dropwisely coating the Dam Mtase solution on the surface of the substrate electrode obtained in the step (3), incubating for 1-2h at 25-37 ℃, and then washing with a PBS solution;
(5) action of a cleavage enzyme: dripping the Dpn I solution on the surface of the substrate electrode obtained in the step (4), reacting for 1-2h at 25-37 ℃, and then washing with a PBS solution;
(6) construction of photoelectrochemical immunosensor: and (4) dropwise adding an S1-AuNPs solution to the surface of the substrate electrode obtained in the step (5) to perform a specific reaction, incubating for 1-2h at 25-37 ℃, then washing with a PBS solution, and airing to obtain the photoelectrochemical immunosensor.
8. The method of claim 7, wherein the concentration of NHS in step (2) is 5-20mg/mL, the concentration of EDC is 10-30mg/mL, and the concentration of hDNA is 0.1-0.5. mu.M.
9. The method for preparing the photoelectrochemical immunosensor according to claim 7, wherein the concentration of MCH in step (3) is 1 to 4 mM.
10. The use of the photoelectrochemical immunosensor of claim 1 for the detection of DNA methylase activity for the quantitative detection of DNA methylase activity.
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to a photoelectrochemical immunosensor for detecting DNA methylase activity and a preparation method and application thereof.
Background
Tumor formation is mainly influenced by genetic and epigenetic modifications. DNA methylation belongs to epigenetic modification, and plays a role in regulating and controlling individual life processes such as growth and development and the like under the catalysis of DNA methylation transferase (Dam MTase). In recent years, studies have shown that DNA methylation abnormality, that is, dysregulation of MTase activity, is associated with various diseases such as tumor and cancer. The intensive study on the activity of Dam MTase can provide valuable guidance for clinical diagnosis and tumor treatment. Conventional methods for MTase assay include radiolabeling of DNA substrates, high performance liquid chromatography and gel electrophoresis, among others. However, these detection methods require a long assay procedure, use of expensive instruments and unsafe isotope labeling, etc., thereby limiting their practical applications. Therefore, it is still a challenge to develop a method capable of rapidly detecting Dam MTase activity with high sensitivity and conciseness.
Sb2Se3The p-type semiconductor material with narrow band gap (1.0-1.3eV) has excellent photoelectric property and thermoelectric property. Recently Sb2Se3Have become promising non-toxic and low cost light absorbers for solar energy conversion devices. In the prior art, after the Sb is successfully applied to a thin-film solar cell, Sb is developed2Se3Photocathodes are used for PEC water splitting. In addition, there are reports that the modified TiO compounds are used2Sb of Pt and2Se3the photocathode composed of the nano needles can be used for PEC hydrogen production. Due to Sb2Se3The history of development of photocathodes is relatively short, and with respect to Sb2Se3There are few reports of PEC sensors as photocathodes for detecting biomolecules. In view of its great potential, further investigation is urgently needed. Recently, fluorescence resonance energy transfer between semiconductor nanocrystals and metal nanoparticles has proven to be an advanced and viable bioassay scheme.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides Sb based on a high photoelectric active substance2Se3The photoelectrochemical immunosensor is used for detecting the activity of the DNA methylase, has simple and convenient operation steps, quick reaction and high sensitivity, and can realize the detection of the activity of the DNA methylaseAnd (6) carrying out quantitative detection.
The invention also aims to provide a preparation method of the photoelectrochemical immunosensor.
It is another object of the present invention to provide the use of the photoelectrochemical immunosensor.
Abbreviations for technical terms in the present invention are as follows:
antimony selenide: sb2Se3(ii) a And (3) graphene oxide: GO; hairpin DNA: hDNA; dam methyltransferase: dam Mtase (purchased from NEB Corp.); restriction endonucleases: DPn I (purchased from NEB); single-stranded DNA: ssDNA; and (3) chitosan: CS; antimony trichloride: SbCl3(ii) a 2-methoxyethanol: 2-ME; thioglycolic acid: TGA; ethanolamine: EA; 2-mercaptoethanol: MCH; indium tin oxide semiconductor electrode: ITO; exciton energy transfer: an EET.
Both hDNA and S1 were synthesized by biological engineering (Shanghai) in the present invention.
The technical scheme is as follows: in order to achieve the above objects, the present invention provides a photoelectrochemical immunosensor for detection of DNA methylase activity, which comprises Sb2Se3-GO-CS modified substrate electrode, hDNA (5' -CAGAGATCCATATACGTTTTTCGTATATGGATCTCTGAAAAA (CH)2)6-NH2-3 ') (SEQ ID NO.1) covalently bound to the modified substrate electrode, Dam MTase methylation and DPn I cleavage to give ssDNA (5' -TCTCTGAAAAA (CH)2)6-NH2-3’)(SEQ IDNO.2)，S1(5’-TTTTTCAGAGA(CH2)6-SH-3') (SEQ ID NO.3) -AuNPs are specifically bound to ssDNA.
Preferably, the substrate electrode is an indium tin oxide semiconductor electrode.
Wherein, the Sb is2Se3The mass concentration of CS in GO-CS is 0.01 wt% -0.5 wt%, preferably 0.05 wt%; the solvent of the CS solution is acetic acid.
Wherein, said Sb2Se3the-GO is prepared from 0.8mg/mL GO aqueous solution and 0.8mg/mL Sb2Se3Mixing the water solutions, and performing ultrasonic treatment for 2-3h to obtain the compound; sb2Se3The volume ratio of aqueous solution to GO aqueous solution is 1: (0.01-1), preferably 1: 0.75; the ultrasonic power is 60-100Hz, preferably 80 Hz.
Wherein, the Sb is2Se3From a precursor solution of Se with SbCl3Mixing the 2-ME solution, heating for reaction, washing and drying to obtain the product. Specifically, 0.3-0.5g Se is weighed into a mixed solution of TGA and EA, and then the solution is mixed with 0.05-0.2M SbCl3Mixing the two solutions, heating to react at 60-90 deg.C for 12-14h, washing and drying.
Wherein, the S1-AuNPs are obtained by stirring and reacting AuNPs and S1; the AuNPs are prepared from HAuCl4Heating the aqueous solution to boiling, adding a sodium citrate solution, continuing to react, and cooling to room temperature to obtain the sodium citrate. Specifically, the S1-AuNPs are prepared by the following method: transferring 5-10 μ L of 50-200 μ M S1 into 0.5-2mL AuNPs, and magnetically stirring at 3.8-4.2 deg.C for reaction for 10-14 h. The AuNPs are prepared by the following method: taking 30-40mL of 0.005-0.02% HAuCl4Heating the solution in a round-bottom flask to boil, adding 35.0-40.0mM sodium citrate solution, reacting for 10-20min, and cooling to room temperature to obtain the final product.
The preparation method of the photoelectrochemical immunosensor for detecting the activity of the DNA methylase comprises the following steps:
(1) fixing a signal layer: sb2Se3Dripping the mixed solution of GO and CS on the surface of a substrate electrode, drying at room temperature, drying again at 50-100 ℃, washing with deionized water, and drying;
(2) anchoring recognition molecule: dropping EDC/NHS mixed solution on the surface of the substrate electrode obtained in the step (1), standing at room temperature for 1-2h, washing with PBS solution, dropping the recognition molecule hDNA on the substrate electrode after washing, incubating for 2-3h at 25-37 ℃, and then washing with PBS solution;
(3) non-specific site blocking: dripping MCH solution on the surface of the substrate electrode obtained in the step (2), sealing for 0.5-1h, and then washing with PBS solution;
(4) methylation reaction: dropwisely coating the Dam Mtase solution on the surface of the substrate electrode obtained in the step (3), incubating for 1-2h at 25-37 ℃, and then washing with a PBS solution;
(5) action of a cleavage enzyme: dripping the Dpn I solution on the surface of the substrate electrode obtained in the step (4), reacting for 1-2h at 25-37 ℃, and then washing with a PBS solution;
(6) construction of photoelectrochemical immunosensor: and (4) dropwise adding an S1-AuNPs solution to the surface of the substrate electrode obtained in the step (5) to perform a specific reaction, incubating for 1-2h at 25-37 ℃, then washing with a PBS solution, and airing to obtain the photoelectrochemical immunosensor.
Wherein, in the step (1), Sb2Se3The GO-CS solution is 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.
Wherein, in the step (2), the concentration of NHS is 5-20mg/mL, preferably 10mg/mL, the solvent is 0.01M, and the pH is 6 PBS. EDC concentration is 10-30mg/mL, preferably 20mg/mL, solvent is 0.01M, pH 6 PBS. The concentration of hDNA was 0.1-0.5 μ M, preferably 0.4 μ M, the solvent was 0.01M, and the pH was 7.4 PBS. The concentration of the washing PBS solution was 0.01M, pH 7.4. EDC/NHS and hDNA were in excess.
Wherein, the concentration of MCH in the step (3) is 1-4 mM. Preferably 2mM, and the solvent is H2And O. The concentration of the washing PBS solution was 0.01M, pH 7.4. The MCH solution is in excess.
Wherein the concentration of Dam Mtase in the step (4) is 5-20 U.mL-1Preferably 10 U.mL-1The solvent is Dam methylase buffer solution. The concentration of the washing PBS solution was 0.01M, pH 7.4. Dam Mtase is in excess.
Wherein the concentration of Dpn I in the step (5) is 5-20 U.mL-1Preferably 10 U.mL-1And the solvent is CutSmart buffer. The concentration of the PBS solution was 0.01M and the pH was 7.4. Dpn I is in excess.
In step (6), the S1-AuNPs solution is in excess. The concentration of the PBS solution was 0.01M and the pH was 7.4.
The photoelectrochemical immunosensor for detecting the activity of the DNA methylase is applied to the quantitative detection of the activity of the DNA methylase.
The starting materials and reagents used in the present invention, including enzymatic reagents, are commercially available.
The working principle is as follows: sb2Se3Is a p-type semiconductor material with narrow band gap (1.0-1.3eV), has excellent photoelectric property and thermoelectric property, and is based on Sb2Se3Competition with Au NPs for the effects of light absorption and Exciton Energy Transfer (EET) has developed a sandwich-type "signal-off" PEC biosensor for sensitive detection of DNA MTase. Due to Sb2Se3Has overlap with the Au NPs plasma band, and the strong EET function between the Au NPs plasma band and the Au NPs plasma band can reduce Sb2Se3The photocurrent signal of (a).
In the invention, Sb2Se3-the GO-CS complex as a substrate for a PEC sensor. On the one hand, Chitosan (CS) can form a membrane with better permeability to enable Sb2Se3-GO is immobilized on the electrode surface. On the other hand, GO has abundant carboxyl active sites on the surface, can be further covalently coupled with biomolecules, simultaneously increases the dispersibility of the solution, is covalently bonded with a large amount of hairpin DNA (hDNA) through amido bonds, and releases single-stranded DNA (ssDNA) through the shearing action of restriction endonuclease (DPn I) after the hDNA is methylated by Dam MTase.
When Au NPs functionalized DNA (S1) exists, S1-AuNPs can be hybridized with ssDNA left on the electrode after shearing by the shearing enzyme, and the formed immune complex draws the distance between the AuNPs and the electrode substrate and induces Sb2Se3EET interaction with AuNPs, thereby effectively quenching Sb2Se3-GO PEC signal, which in turn reduces photocurrent. In addition, the photocurrent can be further reduced due to increased steric hindrance by the immunocomplex formed by hybridization. The constructed 'signal-off' PEC immunosensor has good stability and sensitivity, and provides a new platform for early diagnosis and treatment of diseases.
The present invention designs hDNA sequence and its functional group (-NH) for combining with carboxyl on the substrate material2) (ii) a Then obtaining ssDNA sequence through methylation and shearing; meanwhile, S1 designed in the invention is designed to be specifically combined with ssDNA according to the base complementary pairing principle, wherein the functional group-SH in S1 is used for connecting with Au. The hDNA is obtained after methylation and shearingssDNA, which can specifically bind to S1 to form a sandwich-type immunosensor.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the invention utilizes a quenching agent S1-AuNPs to counter Sb on an electrode base2Se3The sandwich type photoelectrochemistry immunosensor is constructed by the principle that GO has good quenching effect and is used for detecting the activity of DNA methylase. Sb in the invention2Se3Is a photoelectric active material with high efficiency, low toxicity, low price and stability, can improve the biocompatibility after being compounded with GO, and is based on Sb2Se3Compared with the traditional optical method and other methods, the immunosensor constructed by GO has the characteristics of simple and convenient operation, low technical requirement, quick response, low price, easy miniaturization and the like, and can play an important role in medical diagnosis.
Drawings
FIG. 1 is a quencher response graph of a photoelectrochemical immunosensor;
FIG. 2 is a graph of current versus time for detection of photovoltaic activity;
FIG. 3 is a graph showing the change in surface current of an electrode during the assembly of an immunosensor;
FIG. 4 is a graph of the linear relationship between Dam Mtase concentration and photocurrent intensity.
Detailed Description
The invention will be further described with reference to specific embodiments and the accompanying drawings.