Photoelectrochemical biosensor for detecting DNA demethylase MBD2 and preparation method thereof

Abstract

The present invention discloses a photoelectrochemical biosensor for detecting DNA demethylase MBD2 and a preparation method thereof, and belongs to the field of photoelectrochemical analysis technology. The photoelectrochemical biosensor of the present invention comprises a base electrode, Bi4O5Br2, nanogold, double-stranded DNA, mercaptohexanol, Zr fixed on the surface of the electrode in sequence. 4+ , aminomethylphosphonic acid, glutaraldehyde, polyaspartic acid and alkaline phosphatase; the double-stranded DNA comprises: a first DNA strand modified with a thiol group and a second DNA strand modified with a methyl group, the sequence of the first DNA strand is shown in SEQ ID NO.1; the sequence of the second DNA strand is shown in SEQ ID NO.2. The present invention realizes high sensitivity and high specificity detection of DNA demethylase MBD2, the detection method is simple, the instrument is miniaturized and easy to operate, and the detection sensitivity is high.

Description

Technical Field

[0001] The invention relates to the technical field of photoelectrochemical analysis, and in particular to a photoelectrochemical biosensor for detecting DNA demethylase MBD2 and a preparation method thereof. Background Art

[0002] DNA demethylation, as the opposite process of DNA methylation, plays a vital role in the epigenetic regulation of genes. The methyl group at the C5 position of cytosine of genomic CpG dinucleotides can be removed by the DNA demethylase MBD2. Maintaining a dynamic DNA methylation state by balancing the methylation and demethylation processes is essential for the normal function of cells. Dysfunction of DNA demethylases is associated with many important diseases, such as cancer, imprinting-related diseases, and mental illness. The analysis and detection of the DNA demethylation process helps to understand the biological processes related to epigenetic modifications and provide a basis for clinical diagnosis and treatment. However, in contrast to the booming development of DNA methyltransferase detection, research on DNA demethylation and DNA demethylase detection is very limited. Therefore, it is very valuable to achieve sensitive detection of DNA demethylation and demethylase activity, and it is of great significance to establish a rapid, inexpensive and sensitive method for detecting DNA demethylase activity.

[0003] At present, the relevant detection methods for DNA demethylase activity mainly include electrochemical methods, surface enhanced Raman spectroscopy and fluorescence methods. Although these methods have achieved the detection of DNA demethylase, there are still some shortcomings, such as the need for sophisticated instruments, cumbersome sample pretreatment, and the need for professional operators, and further improvement is still needed in terms of detection range or sensitivity. Therefore, it is crucial to establish a simple, fast, highly sensitive and highly selective method to detect the activity of DNA demethylase.

[0004] Photoelectrochemical (PEC) biosensors are developed based on rapid photoelectric conversion and efficient biomolecule-specific recognition, and have the advantages of electrochemical analysis and photochemical analysis. They use light to excite photoactive materials to generate photogenerated electrons and holes. The photogenerated electrons are captured by electrodes to generate current. The excitation light source and the detection signal are two completely different forms, which can effectively reduce the interference of background signals, thereby greatly improving the sensitivity of analytical detection, and have the advantages of fast detection speed, easy miniaturization, simple operation, simple instrumentation and low background signal. Bi4O5Br2 is a new type of visible light catalytic material. Due to its low price, good visible light response, easy preparation and adjustable ribbon structure, it has a wide range of applications in the fields of energy and environment. However, there are no reports on photoelectrochemical sensors based on Bi4O5Br2 nanomaterials for detecting the activity of DNA demethylase MBD2. Summary of the invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide a photoelectrochemical biosensor for detecting DNA demethylase MBD2 and a preparation method thereof.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a photoelectrochemical biosensor is provided, comprising a base electrode, Bi4O5Br2, nanogold, double-stranded DNA, mercaptohexanol, Zr immobilized on the surface of the electrode in sequence, 4+ , aminomethylphosphonic acid, glutaraldehyde, polyaspartic acid, and alkaline phosphatase;

[0008] The double-stranded DNA includes: a first DNA strand modified with thiol groups and a second DNA strand modified with methyl groups, the sequence of the first DNA strand is shown as SEQ ID NO.1; the sequence of the second DNA strand is shown as SEQ ID NO.2.

[0009] The methylated double-stranded DNA removes methyl groups under the action of DNA demethylase MBD2 and triggers the cutting of restriction endonuclease HpaII. After this process is completed, subsequent fixation modification is carried out.

[0010] Preferably, the base electrode is an ITO electrode.

[0011] The second aspect of the present invention provides a method for preparing the above-mentioned photoelectrochemical biosensor, comprising the following steps:

[0012] (1) dropping a Bi4O5Br2 dispersion onto the pretreated surface of the base electrode and drying the mixture to obtain a Bi4O5Br2 / electrode;

[0013] (2) dropping the nano-gold solution onto the Bi4O5Br2 / electrode surface and drying to obtain AuNPs / Bi4O5Br2 / electrode;

[0014] (3) Immobilizing double-stranded DNA onto AuNPs / Bi4O5Br2 / electrode, and then adding mercaptohexanol to seal the electrode to obtain dsDNA-CH3 / AuNPs / Bi4O5Br2 / electrode;

[0015] (4) DNA demethylase MBD2 was added dropwise to the dsDNA-CH3 / AuNPs / Bi4O5Br2 / electrode for reaction for 0.5-3h, and then restriction endonuclease HpaII was added dropwise for reaction for 0.5-3h to obtain dsDNA / AuNPs / Bi4O5Br2 / electrode;

[0016] (5) Zr 4+Add it dropwise onto dsDNA / AuNPs / Bi4O5Br2 / electrode, and then add aminomethylphosphonic acid to obtain AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode;

[0017] (6) Add glutaraldehyde dropwise onto the electrode of step (5) to obtain GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode;

[0018] (7) Add polyaspartic acid dropwise onto the electrode of step (6), and then add EDC and NHS dropwise to obtain PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode;

[0019] (8) Add alkaline phosphatase dropwise onto the electrode of step (7) to prepare ALP / PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0020] Preferably, in step (1), the Bi4O5Br2 dispersion is obtained by dispersing Bi4O5Br2 nanomaterials in deionized water, and the concentration of the Bi4O5Br2 dispersion is 0.2-5 mg / ml.

[0021] More preferably, the Bi4O5Br2 nanomaterial is prepared by the following method:

[0022] Dissolve Bi(NO3)3·5H2O and KBr in ethylene glycol, add a mixed solution of water and ammonia under stirring, collect the precipitate after stirring, wash with water and ethanol by centrifugation, dry and collect the solid, and obtain Bi4O5Br2 nanomaterial.

[0023] Preferably, in step (3), the double-stranded DNA is prepared by the following method:

[0024] The first DNA chain modified with thiol and the second DNA chain modified with methyl were mixed evenly in equal amounts, reacted at 95°C for 5-10 minutes, and naturally cooled to room temperature to obtain double-stranded DNA.

[0025] More preferably, the concentration of the double-stranded DNA is 0.5-2.5 μM.

[0026] The third aspect of the present invention provides the use of the above-mentioned photoelectrochemical biosensor in detecting DNA demethylase MBD2.

[0027] A fourth aspect of the present invention provides a method for detecting DNA demethylase MBD2, comprising the following steps:

[0028] The surface of the ITO electrode was modified with Bi4O5Br2, nanogold, double-stranded DNA, mercaptohexanol, a series of concentrations of DNA demethylase MBD2, restriction endonuclease HpaII, Zr 4+ , aminomethylphosphonic acid, glutaraldehyde, polyaspartic acid and alkaline phosphatase to prepare ALP / PASP / GA / AMPA / Zr after reaction with different concentrations of DNA demethylase MBD2 4+ / dsDNA / AuNPs / Bi4O5Br2 / ITO electrode was used as the working electrode, a saturated calomel electrode was used as the reference electrode, and a Pt wire was used as the auxiliary electrode to form a three-electrode system. Photoelectrochemical signal detection was carried out in the electrode detection liquid. The light source was visible light, the applied potential was -0.5-0.5V, the photocurrent intensity was recorded, and the relationship between the photocurrent intensity and the concentration of DNA demethylase MBD2 was established.

[0029] More preferably, the electrode detection solution is a Tris-HCl buffer solution containing 10-100 mM AAP; the pH of the electrode detection solution is 5.5-8.5.

[0030] Beneficial effects of the present invention:

[0031] (1) The present invention utilizes the carboxyl group on polyaspartic acid to expand the number of alkaline phosphatases introduced and increase the loading amount of alkaline phosphatase. Alkaline phosphatase catalyzes the hydrolysis of L-ascorbic acid-2-phosphate trisodium salt to generate ascorbic acid. Ascorbic acid enhances the photoelectric signal of Bi4O5Br2 and improves the detection sensitivity of DNA demethylase MBD2.

[0032] (2) The present invention utilizes the demethylation effect of MBD2 to trigger the endonuclease to cut and recognize double-stranded DNA, and performs subsequent ligation modification to improve the specificity of DNA demethylase MBD2 detection.

[0033] (3) The detection method of the present invention is simple, low-cost, and realizes miniaturization of the instrument. Only a simple treatment of the ITO electrode surface is required to achieve efficient and sensitive detection of the DNA demethylase MBD2. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Schematic diagram of the construction of the photoelectrochemical biosensor of the present invention and the detection principle of DNA demethylase MBD2.

[0035] Figure 2 : Linear fitting curve of photocurrent intensity and logarithmic value of DNA demethylase MBD2 concentration.

[0036] Figure 3: Bar graph showing the changes in photoelectrochemical response under different protein conditions. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0038] Terminology Note:

[0039] The range of "room temperature" in the present invention is 20-30°C.

[0040] The "humid condition" in the present invention means that the humidity is greater than 90%; the preferred humidity is 95-99%.

[0041] As mentioned above, the existing technology has very limited research on DNA demethylation and DNA demethylase detection, and lacks specific and sensitive detection methods for DNA demethylase.

[0042] Based on this, the present invention constructs a photoelectrochemical biosensor for detecting DNA demethylase MBD2, comprising: an electrode, Bi4O5Br2, nanogold, double-stranded DNA, mercaptohexanol, Zr 4+ , aminomethylphosphonic acid, glutaraldehyde, polyaspartic acid and alkaline phosphatase (ALP). Among them, the methylated double-stranded DNA removes methyl groups under the action of DNA demethylase MBD2 and triggers the cutting of restriction endonuclease HpaII. After the process is completed, subsequent fixation modification is carried out.

[0043] The schematic diagram of the construction and detection of the photoelectrochemical biosensor of the present invention is shown in Figure 1 The photoelectrochemical biosensor of the present invention uses an ITO electrode as a base electrode, and Bi4O5Br2 is modified on the electrode surface. Nanogold is modified on the electrode surface by electrostatic adsorption, and methylated double-stranded DNA is modified on the electrode surface by the reaction between nanogold and the thiol modified by the DNA chain. Then, DNA demethylase MBD2 is used to remove the methyl group of the double-stranded DNA and trigger the recognition and cutting of restriction endonuclease HpaII to expose the phosphate group. The specific binding between the phosphate group and zirconium is used to modify Zr 4+Modify the electrode surface, and then use the specific binding between phosphate and zirconium to modify aminomethylphosphonic acid to the electrode, use the amino group of aminomethylphosphonic acid to react with the aldehyde group of glutaraldehyde to modify glutaraldehyde to the electrode surface, use the aldehyde group of glutaraldehyde to react with the amino group of polyaspartic acid to modify polyaspartic acid to the electrode surface, and use the carboxyl group of polyaspartic acid to react with the amino group of alkaline phosphatase to modify alkaline phosphatase to the electrode surface. Alkaline phosphatase catalyzes L-ascorbic acid-2-phosphate trisodium salt to generate ascorbic acid, and ascorbic acid enhances the photoelectric signal of Bi4O5Br2. Therefore, the linear relationship between the concentration of DNA demethylase MBD2 and the photocurrent intensity can be used to detect DNA demethylase MBD2.

[0044] In the above modification process, Zr 4+ The purpose of using 1, 2, 3, 4, 5-aminomethylphosphonic acid and glutaraldehyde is to modify polyaspartic acid onto the electrode, and finally the alkaline phosphatase is modified onto the electrode by using the reaction between the carboxyl group of polyaspartic acid and the amino group of alkaline phosphatase. The steps of the modification process complement each other, and the order is strictly limited. Each step serves the next step of fixed modification. The lack of the previous step may lead to the failure of the subsequent modification.

[0045] In one embodiment of the present invention, the construction process of the photoelectrochemical biosensor is as follows:

[0046] (1) Preparation of Bi4O5Br2: Dissolve 1.0-5.0 g Bi(NO3)3·5H2O and 0.5-2.0 g KBr in 40 mL ethylene glycol, mix under stirring, and stir for 10-60 min to obtain a mixed solution. Then, add deionized water-ammonia solution (V 去离子水 :V 氨水 =2:1-6:1) is added dropwise to the above mixed solution, stirred at room temperature for 2-10 hours, and then the precipitate is collected by centrifugation at a speed of 6000-12000 rpm for 10 minutes, washed by centrifugation with water and anhydrous ethanol for 2-6 times, and the collected solid is dried at 40-60°C to obtain the Bi4O5Br2 nanomaterial.

[0047] (2) Preparation of Bi4O5Br2 dispersion: Weigh 2-10 mg of the Bi4O5Br2 nanomaterial prepared in step (1), add it to 2-10 mL of deionized water, and disperse it by ultrasonication for 0.5-3 hours.

[0048] (3) ITO electrode pretreatment: Split the ITO conductive glass into 5×1 cm 2 , acetone, 1-4M NaOH alcohol solution (V 无水乙醇 :V 二次水=1:1-1:6), treat the ITO electrode with ultrasound for 15-60min with secondary water, then rinse with secondary water, dry naturally and set aside.

[0049] (4) Fixation of Bi4O5Br2: 20-80 μL of Bi4O5Br2 dispersion was added to the pre-treated ITO electrode surface and dried under infrared light. Then, the electrode was cleaned 1-5 times with cleaning solution. Blow dried with nitrogen. The prepared electrode was marked as Bi4O5Br2 / ITO.

[0050] (5) AuNPs fixation: 20-80 μL of the nanogold solution was added dropwise to the Bi4O5Br2 / electrode obtained in step (4) and dried. Then, the electrode was cleaned 1-5 times with a cleaning solution and dried with nitrogen gas. The prepared electrode was AuNPs / Bi4O5Br2 / electrode.

[0051] The present invention mainly utilizes the good biocompatibility and electrical conductivity of nano-gold, and does not specifically limit the source of the nano-gold solution. The nano-gold solution can be an existing commercial product (for example, the nano-gold solution produced by Jicang Nano, which uses nano-gold particles with a particle size of less than 15nm to be dispersed in an aqueous solution to form a neutral solution with a stable and uniform color); or HAuCl4 can be used as a raw material to prepare nano-gold using an existing method, and then dispersed in an aqueous solution.

[0052] (6) dsDNA-CH3 fixation: 10-50 μL 0.5-2.5 μM methylated double-stranded DNA is added dropwise to the electrode obtained in step (5), and the reaction is carried out under room temperature and humid conditions for 2-12 hours. Then, the electrode is cleaned 1-5 times with a cleaning solution. Then 20-80 μL 0.5-10 mM 6-mercapto-1-hexanol (MCH) is added dropwise to the above electrode, and the reaction is carried out under 25-40°C and humid conditions for 0.5-3 hours. Then, the electrode is cleaned 1-5 times with a cleaning solution. The prepared electrode is dsDNA-CH3 / AuNPs / Bi4O5Br2 / electrode.

[0053] The double-stranded DNA comprises: a first DNA strand modified with a thiol group and a second DNA strand modified with a methyl group, the sequence of the first DNA strand is shown in SEQ ID NO.1; the sequence of the second DNA strand is shown in SEQ ID NO.2; specifically as follows:

[0054] 5'-ATCCGGTACTGTGATGGTACC-SH-3' (SEQ ID NO. 1);

[0055] 5'-GGTACCATCACAGTAC m CGGAT-3' (SEQ ID NO. 2).

[0056] The double-stranded DNA is prepared by the following method:

[0057] The first DNA chain modified with thiol and the second DNA chain modified with methyl were mixed evenly in equal amounts, heated at 95° C. for 5-10 minutes, and naturally cooled to room temperature to obtain methylated double-stranded DNA (dsDNA-CH3).

[0058] The first DNA chain modified with thiol groups and the second DNA chain modified with methyl groups are in the form of dry powder and need to be diluted with a buffer when used. The buffer used to dilute the first DNA chain contains: 3-15mM Tris-HCl, 1-5mM EDTA, 1-5M NaCl, 1-5mM TCEP, pH 6.0-8.5; the buffer used to dilute the second DNA chain contains: 3-15mM Tris-HCl, 1-5mM EDTA, 1-5M NaCl, pH 6.0-8.5.

[0059] The purpose of adding 6-mercapto-1-hexanol is to utilize the thiol group of mercaptohexanol to react with the gold nanoparticles other than those that fix dsDNA-CH3, thereby blocking the redundant sites on the electrode and avoiding interference with subsequent reactions.

[0060] (7) DNA demethylation and HpaII recognition and cleavage: 10-50 μL of DNA demethylase MBD2 of different concentrations was added to the electrode obtained in step (6), and the reaction was carried out at 25-40°C and humid conditions for 0.5-3 hours, and then the electrode was cleaned 1-5 times with a cleaning solution. Then 10-50 μL of 10-50 U / mL restriction endonuclease HpaII was added to the above electrode, and the reaction was carried out at 25-40°C and humid conditions for 0.5-3 hours, and then the electrode was cleaned 1-5 times with a cleaning solution. The prepared electrode is dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0061] (8) Zr 4+ Fixation: 20-80 μL 0.5-10 mM Zr 4+ (solution prepared by dissolving ZrOCl2·8H2O solid in water) is added dropwise to the electrode obtained in step (7), and reacted for 0.5-3h at 25-40°C and humid conditions, and then the electrode is cleaned 1-5 times with a cleaning solution. The prepared electrode is Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0062] (9) Fixation of AMPA: 20-80 μL of 0.5-10 mM aminomethylphosphonic acid was added dropwise to the electrode obtained in step (8), and the reaction was carried out at 25-40°C and in a humid environment for 0.5-3 h. Then, the electrode was cleaned 1-5 times with a cleaning solution.4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0063] (10) Fixation with glutaraldehyde: 20-80 μL of 0.5-10% glutaraldehyde is added dropwise to the electrode obtained in step (9), and the reaction is carried out at 25-40°C and in a humid environment for 0.5-3 h. Then, the electrode is cleaned 1-5 times with a cleaning solution. The prepared electrode is GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0064] (11) Fixation of polyaspartic acid: 20-80 μL of 0.5-10 mM polyaspartic acid is added dropwise to the electrode obtained in step (10), and the reaction is carried out at 25-40°C and humid conditions for 0.5-3 h, and then the electrode is cleaned 1-5 times with a cleaning solution. Then 20-60 μL of a mixed solution containing 10-150 mM EDC and 5-50 mM NHS is added dropwise to the above electrode, and the reaction is carried out at 25-40°C and humid conditions for 0.5-3 h, and then the electrode is cleaned 1-5 times with a cleaning solution. The prepared electrode is PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0065] (12) Immobilization of alkaline phosphatase: 10-50 μL of 0.01-1 mg / mL alkaline phosphatase was added dropwise to the electrode obtained in step (12), and reacted at 25-40°C in a humid environment for 0.5-3 h. Then, the electrode was cleaned 1-5 times with a cleaning solution. The prepared electrode is ALP / PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode, that is, a photoelectrochemical biosensor for detecting DNA demethylase MBD2 was prepared.

[0066] In another embodiment of the present invention, a method for detecting DNA demethylase MBD2 using the above-mentioned photoelectrochemical biosensor is provided, and the steps are as follows:

[0067] (1) Preparation of electrode detection solution: Use sterile water to prepare a Tris-HCl buffer solution containing 10-100 mM L-ascorbic acid-2-phosphate trisodium salt (abbreviated as AAP, CAS No. 66170-10-3); the pH of the Tris-HCl buffer solution is 5.5-8.5 and the concentration is 5-500 mM; use it as the electrode detection solution.

[0068] (2) ALP / PASP / GA / AMPA / Zr prepared with different concentrations of DNA demethylase MBD2 4+ / dsDNA / AuNPs / Bi4O5Br2 / ITO electrode was used as the working electrode, saturated calomel electrode was used as the reference electrode, and Pt wire was used as the auxiliary electrode to form a three-electrode system for photoelectrochemical signal detection. The light source was visible light, the applied potential was -0.5-0.5V, and the photocurrent was recorded in the electrode detection solution.

[0069] (3) Establish the relationship between the photocurrent intensity and the concentration of DNA demethylase MBD2, and use this relationship to quantitatively detect the content of DNA demethylase MBD2.

[0070] Figure 1 Middle b is the construction of ALP / PASP / GA / AMPA / Zr after adding DNA demethylase to react on the electrode 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode, and tested the response signal obtained by photocurrent. a is the ALP / PASP / GA / AMPA / Zr reaction constructed on the electrode without adding DNA demethylase 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode, and test the response signal obtained by photocurrent

[0071] The modified ALP connected to the electrode can catalyze the AAP in the electrode detection solution to generate ascorbic acid (AA), because AA can enhance the photocurrent response of the substrate material Bi4O5Br2, and the amount of DNA demethylase MBD2 will be related to the amount of phosphate exposed after HpaII cleavage, which in turn affects the amount of subsequent modification and connection, and finally affects the amount of ALP connected to the electrode surface, which is also related to the amount of AA generated. As the content of DNA demethylase MBD2 increases, the amount of ALP connected to the electrode surface also increases, and the amount of AA generated by ALP-catalyzed hydrolysis of AAP also increases. AA enhances the photoelectric signal of Bi4O5Br2. According to the linear relationship between the concentration of DNA demethylase MBD2 and the photocurrent intensity, the detection of DNA demethylase MBD2 can be achieved.

[0072] The detection range of the photoelectric chemical biosensor for DNA demethylase MBD2 is 0.3-200 ng / mL, and the detection limit is 0.12 ng / mL.

[0073] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0074] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out in accordance with conventional test methods or the operating instructions recommended by the supplier. Among them:

[0075] The DNA demethylase MBD2 detected by the present invention is MBD2 methyl-CpG binding domain protein 2, Gene ID: 8932.

[0076] The cleaning solution used in the present invention has the following components: 3-15 mM Tris-HCl, pH 7.4.

[0077] The DNA demethylase MBD2, restriction endonuclease HpaII and alkaline phosphatase in the present invention are all dissolved in corresponding buffers for use.

[0078] The composition of the buffer for DNA demethylase MBD2 is: 5-30 mM Tris-HCl, 50-100 mM NaCl, pH 6.0-8.5.

[0079] The composition of the HpaII buffer is: 5-30 mM Tris-acetic acid, pH 6.0-8.5.

[0080] The composition of the ALP buffer is: 5-30 mM PBS, pH 7.4.

[0081] Example 1

[0082] (1) Preparation of Bi4O5Br2

[0083] 4.85 g Bi(NO3)3·5H2O and 1.19 g KBr were dissolved in 40 mL ethylene glycol, and the mixture was stirred for 30 min to obtain a mixed solution. Then, a deionized water-ammonia mixed solution (V 去离子水 :V 氨水 =4:1) was added dropwise to the mixed solution, stirred at room temperature for 4 h, and then centrifuged at 8000 rpm for 10 min to collect the precipitate, washed 4 times by centrifugation with water and anhydrous ethanol, and dried at 60 °C to collect the solid. The obtained solid was Bi4O5Br2 nanomaterial.

[0084] (2) Preparation of Bi4O5Br2 dispersion

[0085] Weigh 10 mg of the Bi4O5Br2 nanomaterial prepared in step (1), add it to 5 mL of deionized water, and disperse it by ultrasonic for 1 hour to prepare a Bi4O5Br2 dispersion.

[0086] (3) ITO electrode pretreatment

[0087] Cut the ITO conductive glass into 5×1cm 2 , acetone, 1M NaOH alcohol solution (V 无水乙醇 :V二次水 =1:1), treat the ITO electrode with ultrasound for 20 min with secondary water, then rinse with secondary water, dry naturally and set aside.

[0088] (4) Fixation of Bi4O5Br2

[0089] 40 μL of Bi4O5Br2 dispersion was added dropwise to the surface of the pretreated ITO electrode prepared in step (3) and dried under infrared light. Then, the electrode was cleaned 1-5 times with cleaning solution and dried with nitrogen. The prepared electrode was marked as Bi4O5Br2 / ITO.

[0090] (5) AuNPs fixation: 40 μL of nanogold was added dropwise to the Bi4O5Br2 / electrode obtained in step (4), and dried. Then, the electrode was cleaned 1-5 times with a cleaning solution and dried with nitrogen. The prepared electrode was AuNPs / Bi4O5Br2 / electrode.

[0091] (6) dsDNA-CH3 fixation: 20 μL of 1 μM methylated double-stranded DNA was added to the electrode obtained in step (5), and the reaction was carried out under room temperature and humid conditions for 12 hours. Then, the electrode was washed with a cleaning solution for 1-5 times. Then, 40 μL of 1 mM MCH was added to the above electrode, and the reaction was carried out under 37°C and humid conditions for 1 hour. Then, the electrode was washed with a cleaning solution for 1-5 times. The prepared electrode is dsDNA-CH3 / AuNPs / Bi4O5Br2 / electrode. The double-stranded DNA includes: a first DNA chain modified with a thiol group and a second DNA chain modified with a methyl group, the sequence of the first DNA chain is shown in SEQ ID NO.1; the sequence of the second DNA chain is shown in SEQ ID NO.2.

[0092] (7) DNA demethylation and HpaII recognition and cleavage: 20 μL of DNA demethylase MBD2 of different concentrations was added to the electrode obtained in step (6), and the reaction was carried out at 37°C and humid conditions for 1 hour, and then the electrode was washed with a cleaning solution for 1-5 times. Then 20 μL of 40U / mL restriction endonuclease HpaII was added to the above electrode, and the reaction was carried out at 37°C and humid conditions for 1 hour, and then the electrode was washed with a cleaning solution for 1-5 times. The prepared electrode is dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0093] (8) Zr 4+ Fixation: 40 μL 5 mM Zr 4+ Add dropwise to the electrode obtained in step (7), react at 37°C under humid conditions for 1 hour, then wash the electrode with a cleaning solution 1-5 times. 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0094] (9) Fixation of aminomethylphosphonic acid: 40 μL of 5 mM aminomethylphosphonic acid was added dropwise to the electrode obtained in step (8), and the reaction was carried out at 37°C under humid conditions for 1 h. Then, the electrode was cleaned 1-5 times with a cleaning solution. 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0095] (10) Fixation with glutaraldehyde: 40 μL of 2.5% glutaraldehyde was added dropwise to the electrode obtained in step (9), and the reaction was carried out at 37°C under humid conditions for 1 h. Then, the electrode was cleaned 1-5 times with a cleaning solution. The prepared electrode is GA / AMPA / Zr 4 + / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0096] (11) Fixation of polyaspartic acid: 40 μL of 5 mM polyaspartic acid was added to the electrode obtained in step (10), and the reaction was carried out at 37°C and humid conditions for 1 hour. Then, the electrode was washed 1-5 times with a cleaning solution. Then, 40 μL of a mixed solution containing 100 mM EDC and 25 mM NHS was added to the above electrode, and the reaction was carried out at 37°C and humid conditions for 1.5 hours. Then, the electrode was washed 1-5 times with a cleaning solution. The prepared electrode is PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0097] (12) Immobilization of alkaline phosphatase: 20 μL of 0.02 mg / mL alkaline phosphatase was added dropwise to the electrode obtained in step (11), and reacted for 1 h at 25-40°C in a humid environment. Then, the electrode was cleaned 1-5 times with a cleaning solution. The prepared electrode is ALP / PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

[0098] Example 2: Photoelectrochemical detection

[0099] The electrochemical workstation was used as the signal acquisition instrument, a 500W xenon lamp was used as the visible light source (with a lens to filter out ultraviolet light), and the ALP / PASP / GA / AMPA / Zr prepared in Example 1 was used. 4+ / dsDNA / AuNPs / Bi4O5Br2 / ITO electrode was used as the working electrode, saturated calomel electrode was used as the reference electrode, Pt electrode was used as the counter electrode, Tris-HCl buffer solution containing 100mM AAP, pH 8.5, and concentration 100mM was used as the electrode detection solution, and -0.3V voltage was used as the working voltage. The it technology was used to detect the analyte and establish the relationship between the photocurrent intensity and the DNA demethylase MBD2 ( Figure 2 ). The detection range of DNA demethylase MBD2 concentration is 0.3-200 ng / mL, the detection limit is 0.12 ng / mL, and the detection sensitivity is greatly improved.

[0100] Example 3: Detection selectivity experiment

[0101] Selectivity is an important indicator of the performance of photoelectrochemical sensors. In order to study the specificity of the sensor constructed in the present invention, DNA demethylase MBD2, RNA endonuclease (MazF), histone acetyltransferase (HAT), horseradish peroxidase (HRP), N 6 -methyladenine (m 6 A) Demethylase (FTO) Four different proteins were used as interfering substances to study the selectivity of the sensor. The photocurrent change values ​​(ΔI=I2-I1) of the sensors constructed with different interfering substances were compared. In the current value determination of I1, no MBD2 or other proteins were added to construct the sensor, and the remaining construction steps and detection process were consistent with the method of Example 1. In the current value determination of I2, MBD2 was added to construct a group of sensors, and the other four proteins were added to construct 4 groups of sensors. The remaining construction steps and detection process were consistent with the method of Example 1; the concentrations of other proteins and MBD2 were all 10ng / mL.

[0102] The results showed that the change in the current value of the sensor constructed by the interference substance was significantly lower than that of the DNA demethylase MBD2, indicating that the sensor constructed by the present invention has good specificity ( Figure 3 ).

[0103] Example 4: Stability test

[0104] The method of Example 1 was used to prepare 7 ALP / PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / ITO electrode (DNA demethylase MBD2 10ng / mL), the photocurrent was detected in the test solution. The relative standard deviation of the photocurrent was 2.89%, indicating that the method has good reproducibility. 4+The / dsDNA / AuNPs / Bi4O5Br2 / ITO sensor was measured continuously for 7 cycles, and the photoelectrochemical signal was detected in the detection liquid. The standard deviation of the photocurrent was 2.24%, indicating that the method has good stability.

[0105] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A photoelectrochemical biosensor, characterized in that: It includes a basic electrode, Bi4O5Br2, nanogold, double-stranded DNA, mercaptohexanol, Zr fixed on the surface of the electrode in sequence. 4+ , aminomethylphosphonic acid, glutaraldehyde, polyaspartic acid, and alkaline phosphatase; The double-stranded DNA comprises: a first DNA strand modified with a thiol group and a second DNA strand modified with a methyl group, the sequence of the first DNA strand is shown in SEQ ID NO.1; the sequence of the second DNA strand is shown in SEQ ID NO.2; The photoelectrochemical biosensor is prepared by the following method: (1) dropping a Bi4O5Br2 dispersion onto the pretreated surface of the base electrode and drying the mixture to obtain a Bi4O5Br2 / electrode; (2) dropping the nano-gold solution onto the Bi4O5Br2 / electrode surface and drying to obtain AuNPs / Bi4O5Br2 / electrode; (3) Immobilizing double-stranded DNA onto AuNPs / Bi4O5Br2 / electrode, and then adding mercaptohexanol to seal the electrode to obtain dsDNA-CH3 / AuNPs / Bi4O5Br2 / electrode; (4) DNA demethylase MBD2 was added dropwise to the dsDNA-CH3 / AuNPs / Bi4O5Br2 / electrode for reaction for 0.5-3h, and then restriction endonuclease HpaII was added dropwise for reaction for 0.5-3h to obtain dsDNA / AuNPs / Bi4O5Br2 / electrode; (5) Zr 4+ Add it dropwise onto dsDNA / AuNPs / Bi4O5Br2 / electrode, and then add aminomethylphosphonic acid to obtain AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode; (6) Add glutaraldehyde dropwise onto the electrode of step (5) to obtain GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode; (7) Add polyaspartic acid dropwise onto the electrode of step (6), and then add EDC and NHS dropwise to obtain PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode; (8) Add alkaline phosphatase dropwise onto the electrode of step (7) to prepare ALP / PASP / GA / AMPA / Zr 4+ / dsDNA / AuNPs / Bi4O5Br2 / electrode.

2. The photoelectrochemical biosensor according to claim 1, characterized in that: The base electrode is an ITO electrode.

3. The photoelectrochemical biosensor according to claim 1, characterized in that: In step (1), the Bi4O5Br2 dispersion is obtained by dispersing Bi4O5Br2 nanomaterials in deionized water, and the concentration of the Bi4O5Br2 dispersion is 0.2-5 mg / ml.

4. The photoelectrochemical biosensor according to claim 3, characterized in that: The Bi4O5Br2 nanomaterial is prepared by the following method: Dissolve Bi(NO3)3·5H2O and KBr in ethylene glycol, add a mixed solution of water and ammonia under stirring, collect the precipitate after stirring, wash with water and ethanol by centrifugation, dry and collect the solid, and obtain Bi4O5Br2 nanomaterial.

5. The photoelectrochemical biosensor according to claim 1, characterized in that: In step (3), the double-stranded DNA is prepared by the following method: The first DNA chain modified with thiol and the second DNA chain modified with methyl were mixed evenly in equal amounts, reacted at 95°C for 5-10 minutes, and naturally cooled to room temperature to obtain double-stranded DNA.

6. The photoelectrochemical biosensor according to claim 5, characterized in that: The concentration of the double-stranded DNA is 0.5-2.5 μM.

7. Use of the photoelectrochemical biosensor according to any one of claims 1 to 2 in detecting DNA demethylase MBD2.

8. A method for detecting DNA demethylase MBD2, characterized in that: The following steps are involved: The surface of the ITO electrode was modified with Bi4O5Br2, nanogold, double-stranded DNA, mercaptohexanol, a series of concentrations of DNA demethylase MBD2, restriction endonuclease HpaII, Zr 4+ , aminomethylphosphonic acid, glutaraldehyde, polyaspartic acid and alkaline phosphatase to prepare ALP / PASP / GA / AMPA / Zr after reaction with different concentrations of DNA demethylase MBD2 4+ / dsDNA / AuNPs / Bi4O5Br2 / ITO electrode was used as the working electrode, a saturated calomel electrode was used as the reference electrode, and a Pt wire was used as the auxiliary electrode to form a three-electrode system. Photoelectrochemical signal detection was carried out in the electrode detection liquid. The light source was visible light, and the applied potential was -0.5-0.5V. The photocurrent intensity was recorded, and the relationship between the photocurrent intensity and the concentration of DNA demethylase MBD2 was established. The concentration of DNA demethylase MBD2 was quantitatively detected.

9. The method according to claim 8, characterized in that The electrode detection solution is a Tris-HCl buffer solution containing 10-100 mM AAP; the pH of the electrode detection solution is 5.5-8.5.

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