CN110702910A

CN110702910A - Photoelectrochemical immunosensor for detecting activity of DNA methylase and preparation method and application thereof

Info

Publication number: CN110702910A
Application number: CN201910796666.5A
Authority: CN
Inventors: 沈艳飞; 陈开洋; 薛怀佳
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2019-08-27
Filing date: 2019-08-27
Publication date: 2020-01-17
Anticipated expiration: 2039-08-27
Also published as: CN110702910B

Abstract

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 Sb₂Se₃The 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.

Description

Photoelectrochemical immunosensor for detecting activity of DNA methylase and preparation method and application thereof

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.

Example 1

Photoelectric active material Sb₂Se₃Preparation of-GO

(1)Sb₂Se₃The synthesis of (2): accurately weighing 0.405g of selenium powder by using an analytical balance, and dissolving the selenium powder in a mixed solution of 0.46mL of TGA and 7.54mL of EA to obtain a precursor solution of Se; then, the Se precursor solution was mixed with 12mL of a solution containing 0.1M SbCl₃The 2-ME solution was co-transferred to a round bottom flask and stirred at 80 ℃ for 12 h. Then centrifugally washing the mixture by using ethanol and ultrapure water for multiple times in sequence, and drying the mixture.

(2)Sb₂Se₃-GO complexSynthesis of the Compound: 100 μ L of 0.8mg/mL Sb₂Se₃Mixing the water solution and 75 mu L0.8mg/mLGO water solution for 2h of ultrasound with the ultrasound power of 80Hz to obtain Sb₂Se₃-a GO complex.

(3) Synthesis of AuNPs: 34mL of 0.1mg/mL HAuCl was taken₄After the solution was stabilized at 100 ℃ in a 100mL round-bottomed flask, 850. mu.L of a 38.8mM sodium citrate solution was added, the reaction was continued for 15min, cooled to room temperature, and stored in a refrigerator at 4 ℃.

(4) S1-Synthesis of AuNPs: 1mL of AuNPs obtained in step (3) was placed in a glass bottle, 5. mu.L of 100. mu.M single-stranded DNA (S1) was added thereto, and the mixture was reacted at 4 ℃ for 12 hours with magnetic stirring and stored in a refrigerator at 4 ℃ until use.

Example 2

Preparation method of photoelectrochemical immunosensor

(1) Signal layer anchoring

To 0.16cm²To the ITO was added dropwise 10. mu.L of Sb₂Se₃-mixed solution of GO (prepared in example 1) and CS (0.05 wt%), overnight at room temperature, then dried at 80 ℃ for 1h, then washed with deionized water and air dried to obtain Sb₂Se₃GO/ITO substrate electrodes.

(2) Anchored recognition molecules

10. mu.L EDC (20 mg. mL) was added dropwise^-1) And NHS (10 mg. mL)^-1) The mixed solution of (3) was applied to the substrate electrode obtained in step (2) to activate the carboxyl groups on the GO surface, then 10 μ L of 0.4 μ M hDNA solution was added dropwise to the electrode, incubated at 37 ℃ for 2 hours to anchor and support the electrode material sufficiently, and then the unbound hDNA was washed with 0.01M PBS (pH 7.4) solution.

(3) Non-specific site blocking: to cover non-specific sites on the electrode surface that can bind to hDNA in addition to the antibody, 10. mu.L of 2mM MCH solution (solvent H) was added dropwise to the substrate electrode surface obtained in step (2)₂O), incubated at 37 ℃ for 40min, and then washed with 0.01M PBS (pH 7.4) to remove excess MCH solution.

(4) Methylation reaction

10 μ L of Dam Mtase (10U. mL) was added dropwise^-1) Incubating the solution at 37 ℃ on the substrate electrode obtained in the step (3)After incubation for 2h, excess Dam MTase solution was removed by washing with 0.01M PBS (pH 7.4).

(5) Action of a cleavage enzyme

10. mu.L of Dpn I (10U. mL) was added dropwise^-1) The solution was reacted with the substrate electrode obtained in step (4) at 37 ℃ for 2 hours to release ssDNA, and then washed with 0.01M PBS (pH 7.4) to remove excess DpnI solution.

(6) Construction of photoelectrochemical immunosensor

And (3) dropwise adding 10 mu L S1-AuNPs solution (prepared in example 1) to the substrate electrode obtained in the step (5), incubating at 37 ℃ for 2h, washing with 0.01M PBS (pH 7.4) solution to remove unbound S1-AuNPs, and airing to obtain the photoelectrochemical immunosensor.

And (3) putting the prepared sensor into 0.1M ascorbic acid phosphate buffer solution to detect the photocurrent changes of the electrode after the action of the shear enzyme and the combination of S1-AuNPs, and analyzing the result.

Example 3

Detection of the photoelectric Activity of a quencher

(1) The instrument comprises the following steps: shanghai Chenghua electrochemical workstation (chi660e software), xenon lamp light source, universal meter

(2) Materials and reagents:

ITO: indium tin oxide semiconductor electrode with specification of 0.4cm × 0.4cm

Electrolyte solution: 0.1M ascorbic acid phosphate buffer

Reagent: S1-AuNPs, S1

(3) The method comprises the following steps:

electrode treatment: and (3) placing the ITO electrode in acetone, ethanol and ultrapure water for 20min respectively, drying by using nitrogen, testing the front side and the back side by using a universal meter, and marking for later use.

Preparing an electrode: preparation of electrode Dpn I/Dam/hDNA/Sb according to Steps (1) to (5) of example 2₂Se₃GO/ITO, followed by incubation of 10 μ L S1-AuNPs (prepared in example 1) with 10 μ L of 100 μ M S1, 37 ℃, 2h, respectively, on the above electrodes, followed by washing with 0.01M PBS (pH 7.4) solution to remove unbound S1-AuNPs and S1, and finally detecting the electrode photocurrent intensity.

And (3) testing: xenon lamp light source system for providing illuminationObserving the difference between the intensity of the current on the surface of the electrode under illumination and the intensity of the current without illumination by using a three-electrode system of an electrochemical workstation, and comparing the current on the surface of the electrode with the current on the substrate Sb₂Se₃Quenching effect of the photoelectric intensity of GO.

(4) As a result:

the quenching effect of S1-AuNPs is shown in FIG. 1, from which it can be seen that the Δ I/I of S1-AuNPs modified electrode₀Delta I/I significantly higher than S1₀AuNPs can be used as quenchers of the sensor, and the constructed 'signal-off' immunosensor is feasible. This example demonstrates that selected AuNPs are paired with Sb₂Se₃The photoelectricity of (2) has excellent quenching effect.

Example 4

Monitoring of surface photocurrent of electrode in photoelectric chemical immunosensor assembling process

(1) The instrument comprises the following steps: same as example 3

(2) Materials and reagents:

ITO: same as example 3

Electrolyte solution: same as example 3

Reagent: sb₂Se₃GO (prepared in example 1), 0.05 wt% CS, 20 mg. mL^-1EDC、10mg·mL^-1NHS, 0.4. mu.M hDNA, 2mM MCH, 10U/mL Dam MTase, 10U/mL Dpn I, S1-AuNPs (prepared in example 1)

(3) The method comprises the following steps:

electrode treatment: same as example 3

Assembling a sensor: the assembly of the photoelectric immunosensor was performed as in example 2.

And (3) testing: a xenon lamp light source system provides illumination, an electrochemical workstation three-electrode system is utilized, the difference between the intensity of the electrode surface current under illumination and the intensity of the electrode surface current without illumination is detected and observed in 0.1M ascorbic acid phosphate buffer solution in sequence, and the feasibility of the constructed immunosensor is explored.

(4) As a result:

FIG. 2 is a current-time diagram for detection of photoelectric activity: a is a base electrode Sb₂Se₃GO/ITO (example 2 step 1), b is hDNA/Sb₂Se₃GO/ITO (example 2)Step 2), c is Dpn I/Dam/hDNA/Sb₂Se₃GO/ITO (example 2, step 5), d is S1-AuNPs/Dpn I/Dam/hDNA/Sb2Se3-GO/ITO (i.e. the photoelectric sensor of the invention, example 2). As can be seen from FIG. 2, when Sb is present₂Se₃After the-GO is fixed on the ITO electrode, a remarkable photocurrent is shown, which indicates Sb₂Se₃GO is an excellent photo-electro-active material for constructing PEC sensors. Then, hDNA is continuously fixed on the electrode, and the photocurrent is obviously reduced, and the transfer between AA and the electron of the electrode is blocked because the hDNA belongs to biological macromolecules. Upon further assembly of Dam MTase and Dpn I, the photocurrent increased slightly, indicating that Dpn I successfully cleaved methylated hDNA to ssDNA, reducing electrode surface steric hindrance. When S1-AuNPs are modified, the photocurrent is remarkably reduced, because DNA hybridization draws the distance between the AuNPs and an electrode substrate, and induces the AuNPs and Sb₂Se₃The EET effect between GO and the increase of the steric hindrance greatly reduce the photocurrent of the sensor, which shows the feasibility of the constructed photoelectric immunosensor.

Example 5

Electrode surface current monitoring during photoelectrochemical immunosensor assembly process

(1) The instrument comprises the following steps: shanghai Chenghua electrochemical workstation (chi660e software)

(2) Materials and reagents:

ITO: same as example 3

Electrolyte solution: 2mM potassium ferricyanide, solvent 0.01M PBS, pH 7.4

Reagent: same as example 4

(3) The method comprises the following steps:

electrode treatment: ITO electrode treatment the same as in example 2

And (3) testing: the normal progress of the sensor assembly was monitored by sequentially detecting CV maps (CV) in 2mM potassium ferricyanide phosphate buffer, comparing the change in the interface current between the electrode surface and the solution at each step.

(4) As a result:

sensor groupAnd (3) monitoring the surface current of the electrode in the assembling process, wherein a curve from a to d is a surface current change curve of the electrode in the layer-by-layer assembling process of the electrode. When modifying the substrate Sb on the electrode₂Se₃After GO, a pair of distinct redox peaks (a-curves) appear. When the hDNA was further modified on the electrode, a significant decrease in peak current was observed (b-curve) because the hDNA itself was very sterically hindered, hindering electron transfer at the electrode surface. Subsequently, after further assembly of MCH, Dam MTase and Dpn I on the electrode (c-curve), the redox peak current was slightly enhanced compared to hDNA, since hDNA is cleaved to ssDNA under the combined action of Dam MTase and Dpn I, the steric hindrance is reduced, and thus the redox peak current is increased. Finally, when the electrode was further incubated with S1-AuNPs (d-curve), the redox peak current increased again, since AuNPs contribute to [ Fe (CN)₆]^3-/4-And the ITO electrode. This example is to demonstrate the successful assembly of the photoelectrochemical immunosensor.

Example 6

Linear relationship between Dam MTase concentration and photocurrent reduction ratio

(1) The instrument comprises the following steps: same as example 3

(2) Materials and reagents:

ITO: same as example 3

Electrolyte solution: same as example 3

Reagent: sb₂Se₃GO (molar ratio 1:0.75), 0.05 wt% CS, 20 mg. mL^-1EDC、10mg·mL^-1NHS、0.4μM hDNA、2mM MCH、0.001-100U/mL Dam MTase、10U/mL Dpn I、S1-AuNPs

(3) The method comprises the following steps:

electrode treatment: the ITO electrode treatment was the same as in example 3.

Assembling a sensor: the assembly of the photoelectric immunosensor was performed as in example 2, except that the Dam MTase solution (0.001-100U. mL) was prepared at different concentrations in the step (4)^-1) And (4) carrying out measurement.

And (3) testing: the xenon lamp light source system provides illumination, the intensity of the surface current of the electrode under illumination is observed by utilizing the electrochemical workstation three-electrode system, the photocurrent change between the two steps after the action of the shear enzyme and the combination of S1-AuNPs is compared, and the relationship between the photocurrent intensity change rate and the Dam MTase concentration is analyzed.

FIG. 4 is a graph of the linear relationship between Dam MTase concentration and photocurrent intensity. As can be seen from fig. 4, the reduction ratio of the photocurrent is linearly related to the Dam MTase concentration, y is 0.0234x +0.375, and the correlation coefficient R is²The detection range is 1mU/mL-100U/mL, which is 0.988. This example demonstrates that the photoelectric immunosensor of the present invention can quantitatively detect DNA methylase activity, and has the advantages of good detection effect, wide detection range, simple operation and rapid reaction.

Example 7

Example 7 was prepared as in example 1, Sb₂Se₃The GO complex preparation differs in that: 0.3g Se was weighed into the mixture of TGA and EA, and the solution was mixed with 0.05M SbCl₃Mixing the 2-ME solution, heating to react for 14h at 60 ℃, washing and drying to obtain Sb₂Se₃(ii) a Aqueous GO solution with Sb₂Se₃Mixing the aqueous solution, and performing ultrasonic treatment for 3h with ultrasonic power of 60Hz and Sb₂Se₃Volume ratio to GO 1: 0.01, Sb can be prepared₂Se₃-a GO complex.

The synthesis of S1-AuNPs differs in that: 30mL of 0.02% HAuCl was taken₄Heating the solution in a round-bottom flask to boil, adding 35.0mM sodium citrate solution, reacting for 10min, cooling to room temperature to obtain AuNPs, and storing in a refrigerator at 4 ℃; transferring 5 mu L of 50 mu M S1 into 0.5mL AuNPs, magnetically stirring and reacting for 14h at 3.8 ℃ to obtain S1-AuNPs, and storing in a refrigerator at 4 ℃ for later use.

Example 8

Example 8 was prepared as in example 1, Sb₂Se₃The GO complex preparation differs in that: 0.5g Se was weighed into the mixture of TGA and EA, and the solution was mixed with a solution containing 0.2M SbCl₃Mixing the 2-ME solution, heating to react for 12h at 90 ℃, washing and drying to obtain Sb₂Se₃(ii) a Aqueous GO solution with Sb₂Se₃Mixing the aqueous solution, wherein the ultrasonic power is 100Hz and Sb is 2h₂Se₃Volume ratio to GO 1: 1, so as to obtain Sb₂Se₃-a GO complex.

The synthesis of S1-AuNPs differs in that: 40mL of 0.005% HAuCl was taken₄Heating the solution in a round-bottom flask to boil, adding 40.0mM sodium citrate solution, reacting for 20min, cooling to room temperature to obtain AuNPs, and storing in a refrigerator at 4 ℃; transferring 10 mu L of 200 mu M S1 into 0.5mL AuNPs, magnetically stirring and reacting for 10h at 4.2 ℃ to obtain S1-AuNPs, and storing in a refrigerator at 4 ℃ for later use.

Example 9

(1) Signal layer anchoring

To 0.16cm²To the ITO was added dropwise 10. mu.L of Sb₂Se₃-mixed solution of GO (prepared in example 7) and CS (0.01 wt%) overnight at room temperature, then dried at 80 ℃ for 1h, then washed with deionized water and air dried to obtain Sb₂Se₃GO/ITO substrate electrodes.

(2) Anchored recognition molecules

10. mu.L EDC (5 mg. mL) was added dropwise^-1) And NHS (10 mg. mL)^-1) The mixed solution of (2) was applied to the substrate electrode obtained in step (1) to activate the carboxyl groups on the GO surface, 10 μ L of 0.1 μ M hDNA solution was added dropwise to the electrode, incubated at 25 ℃ for 2 hours to anchor and support the electrode material sufficiently, and then the unbound hDNA was washed with 0.01M PBS (pH 7.4) solution.

(3) Non-specific site blocking: to cover non-specific sites on the electrode surface that can bind to hDNA in addition to the antibody, 10. mu.L of 1mM MCH solution (solvent H) was added dropwise to the substrate electrode surface obtained in step (2)₂O), incubated at 37 ℃ for 40min, and then washed with 0.01M PBS (pH 7.4) to remove excess MCH solution.

(4) Methylation reaction

10 μ L of Dam Mtase (5U. mL) was added dropwise^-1) The solution was incubated at 25 ℃ for 2 hours on the substrate electrode obtained in step (3), and then washed with 0.01M PBS (pH 7.4) to remove excess Dam MTase solution.

(5) Action of a cleavage enzyme

10. mu.L of Dpn I (5U. mL) was added dropwise^-1) The solution was reacted with the substrate electrode obtained in step (4) at 25 ℃ for 2 hours to release ssDNA, and then washed with 0.01M PBS (pH 7.4) to remove the ssDNAExcess Dpn I solution.

(6) Construction of photoelectrochemical immunosensor

And (3) dropwise adding 10 mu L S1-AuNPs solution (prepared in example 7) to the substrate electrode obtained in the step (5), incubating at 25 ℃ for 2h, washing with 0.01M PBS (pH 7.4) solution to remove unbound S1-AuNPs, and airing to obtain the photoelectrochemical immunosensor.

Example 10

(1) Signal layer anchoring

To 0.16cm²To the ITO was added dropwise 10. mu.L of Sb₂Se₃-mixed solution of GO (prepared in example 8) and CS (0.5 wt.%) overnight at room temperature, then dried at 80 ℃ for 1h, then washed with deionized water and air dried to give Sb₂Se₃GO/ITO substrate electrodes.

(2) Anchored recognition molecules

10. mu.L EDC (20 mg. mL) was added dropwise^-1) And NHS (30 mg. mL)^-1) The mixed solution of (2) was applied to the base electrode obtained in step (1) to activate the carboxyl groups on the GO surface, 10 μ L of 0.5 μ M hDNA solution was added dropwise to the electrode, incubated at 37 ℃ for 1 hour to anchor and support the electrode material sufficiently, and then unbound hDNA was washed with 0.01M PBS (pH 7.4) solution.

(3) Non-specific site blocking: to cover non-specific sites on the electrode surface that can bind to hDNA in addition to the antibody, 10. mu.L of 4mM MCH solution (solvent H) was added dropwise to the substrate electrode surface obtained in step (2)₂O), incubated at 37 ℃ for 40min, and then washed with 0.01M PBS (pH 7.4) to remove excess MCH solution.

(4) Methylation reaction

10 μ L of Dam Mtase (20U. mL) was added dropwise^-1) The solution was incubated at 37 ℃ for 1 hour on the substrate electrode obtained in step (3), and then washed with 0.01M PBS (pH 7.4) to remove excess Dam MTase solution.

(5) Action of a cleavage enzyme

10. mu.L of Dpn I (20U. mL) was added dropwise^-1) The solution was reacted with the substrate electrode obtained in step (4) at 37 ℃ for 1 hour to release ssDNA, and then washed with 0.01M PBS (pH 7.4) to remove excess Dpn I solution.

(6) Construction of photoelectrochemical immunosensor

And (3) dropwise adding 10 mu L S1-AuNPs solution (prepared in example 8) to the substrate electrode obtained in the step (5), incubating at 37 ℃ for 1h, washing with 0.01M PBS (pH 7.4) solution to remove unbound S1-AuNPs, and airing to obtain the photoelectrochemical immunosensor.

Sequence listing

<110> university of southeast

<120> photoelectrochemical immunosensor for detecting DNA methylase activity and preparation method and application thereof

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

cagagatcca tatacgtttt tcgtatatgg atctctgaaa aa 42

<210>2

<211>11

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

tctctgaaaa a 11

<210>3

<211>11

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

tttttcagag a 11

Claims

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;

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.