CN110426428B - Detection of N by Ti-MOF-based photoelectrochemical sensor1Method for producing (E) -methyladenine - Google Patents

Detection of N by Ti-MOF-based photoelectrochemical sensor1Method for producing (E) -methyladenine Download PDF

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CN110426428B
CN110426428B CN201910692480.5A CN201910692480A CN110426428B CN 110426428 B CN110426428 B CN 110426428B CN 201910692480 A CN201910692480 A CN 201910692480A CN 110426428 B CN110426428 B CN 110426428B
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周云雷
王月
殷焕顺
隋程吉
李菲
陈燕
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Shandong Agricultural University
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Abstract

The invention discloses a method for detecting N1The construction and detection method of-methyladenine photoelectrochemical sensor are implemented by sequentially modifying the surface of a base electrodeDecorating a heterojunction of graphite-phase carbon nitride and bismuth vanadate, 4.0-generation polyamide-amine, p-carboxyphenylboronic acid, m1Antibody A, m1ATP、TiO2@NH2MIL-125(Ti) to prepare a photoelectric electrochemical sensor. The invention utilizes the good photoelectric activity and biocompatibility of the heterojunction of graphite-phase carbon nitride and bismuth vanadate, the specific recognition performance of an antibody on an antigen, and TiO2@NH2MIL-125(Ti) as signal amplification unit, implementing the pair N1High sensitivity and high specificity detection of methyladenine. The invention uses Ti-MOF to recognize N for the first time1-methyladenine, simple detection method, miniaturization of instrument, easy operation, and realization of N-p-channel polymorphism1-detection of methyladenine.

Description

Detection of N by Ti-MOF-based photoelectrochemical sensor1Method for producing (E) -methyladenine
Technical Field
The invention belongs to the field of photoelectrochemical analysis, and particularly relates to a method for detecting m1An ATP photoelectrochemical sensor and a preparation method thereof.
Background
RNA methylation is an important epigenetic event that plays an important role in post-transcriptional regulation of gene expression, directly affecting protein production. Methylation of RNA occurs primarily at the exocyclic amine groups, at some specific nitrogen and carbon positions of purines and pyrimidines, and at the oxygen atom at the 2' -OH position.
N1-methyladenine (N)1-methyladenosine，m1A) Is a ubiquitous methylation modification in noncoding RNA (ncRNA) and messenger RNA (mRNA) occurring on thousands of different gene transcripts from yeast to mammalian eukaryotic cells due to m1A at Watson Crick interface N1Methyl in position, m1A interferes with normal base pairing. In addition, m1A also plays an important role in tRNA tertiary structure, ribosome biogenesis and translation. However, for m1A detection, the single base resolution of the current sequencing technology has not been reached, which hinders m1A, and therefore little is known about its biological function, and m1A, it is not clear which methyltransferase catalyzes m in mRNA1And (A) forming. Thus, m1The detection of A has important significance for researching biological influence, action mechanism and the like. So far, some m has been continuously studied1The detection method of A mainly includes thin layer chromatography, gas chromatography, column liquid chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis and the like. However, these methods have the disadvantages of requiring expensive large-scale instruments, cumbersome sample pretreatment, requiring professional operators, low detection sensitivity, and poor specificity. Thus, a simple, fast, highly sensitive and highly selective method for m is established1The detection of A is crucial.
Photoelectrochemical (PEC) sensing is a relatively new, dynamically developed technology for performing various biological assays (e.g., nucleic acids, proteins, and cells) with greater sensitivity and simplicity than conventional electrochemical methods. In addition, the photoelectrochemistry analysis method also has the advantages of simple device, easy operation of equipment, high response speed, easy miniaturization and the like. The advantages of photoelectrochemical analysis make it attract a lot of attention in the fields of biological analysis and environmental detection. The optical active material is the most important part in the photoelectrochemical sensor, and how to establish the action relationship between the optical active material and the substance to be detected so as to improve the sensitivity of the photoelectric activity of the optical active material is the difficulty of photoelectrochemical detection. At present, the detection of N by a photoelectrochemical immunosensor based on Ti-MOF is not seen1A method for producing (E) -methyladenine.
Disclosure of Invention
In view of the prior art, the invention aims to provide a Ti-MOF-based photoelectrochemical immunosensor for N detection1Preparation method of-methyladenine by utilizing good photoelectric activity and biocompatibility of heterojunction of graphite-phase carbon nitride and bismuth vanadate, and antibodySpecific recognition property for antigen, and TiO2@NH2MIL-125(Ti) is used as a signal amplification unit, and high-sensitivity and high-specificity detection of N1-methyladenine is realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a method for detecting N is provided1A method for preparing a photoelectrochemical biosensor for methyladenine, comprising the steps of:
(1) taking an ITO electrode as a basic electrode, and pretreating the basic electrode; oxygen-containing group and BiVO on ITO electrode surface4-g-C3N4BiVO (BiVO) by electrostatic adsorption force4-g-C3N4Modifying the surface of the treated electrode;
(2) using BiVO4-g-C3N4And (2) performing physical adsorption with PAMAM, and modifying the PAMAM on the surface of the electrode treated in the step (1);
(3) modifying the CPBA on the surface of the electrode treated in the step (2) through the interaction between the carboxyl of the CPBA and the amino of the PAMAM;
(4) using boronic acid groups of CPBA with m1Glycoside reaction in antibody A, and1modifying the surface of the electrode treated in the step (3) with an antibody A;
(5) recognition of m by specific recognition of antigen and antibody1ATP, transformation of m1ATP is modified on the surface of the electrode treated in the step (4);
(6) by using TiO2@NH2Ti of MIL-125(Ti)4+Identification m1The phosphate radical in ATP is simultaneously amplified to obtain TiO2@NH2And (5) modifying the electrode surface treated in the step (5) with MIL-125 (Ti).
Preferably, the pretreatment method of the base electrode comprises the following steps: the basic electrode is firstly cleaned by mixed liquor of ethanol and sodium hydroxide for 30 minutes in an ultrasonic mode, then cleaned by deionized water, cleaned by acetone for 30 minutes in an ultrasonic mode, finally cleaned by deionized water and dried in the air. The electrode without pretreatment generally has larger overpotential, thereby causing slow reaction and high energy consumption. In order to exert the advantages of the electrode and improve the activity of the electrode, the surface of the electrode needs to be pretreated. The electrode overpotential can be reduced by adopting the electrode pretreatment method, so that the activity of the electrode is effectively improved.
Preferably, BiVO is added4-g-C3N4The method for modifying the surface of the treated electrode comprises the following steps: BiVO (bismuth oxide) is added4-g-C3N4BiVO is prepared by deionized water4-g-C3N4Dispersing BiVO in water4-g-C3N4And dropwise adding the dispersed liquid onto the surface of the pretreated electrode, drying under the irradiation of an infrared lamp, then cleaning for 3-5 times, and drying by nitrogen.
More preferably, the BiVO4-g-C3N4The concentration of the dispersion is 0.1-8 mg/mL.
Preferably, the method for modifying the PAMAM on the surface of the electrode treated in the step (1) comprises the following steps: dropwise adding PAMAMA solution to the modified BiVO4-g-C3N4The surface of the electrode is dried under the irradiation of an infrared lamp, then cleaned for 3-5 times and dried by nitrogen.
Preferably, CPBA is modified on the surface of the electrode treated in step (2): and (3) dropwise adding the CPBA solution to the surface of the electrode modified with the PAMAM, reacting for 1.5-4h at room temperature under a humid condition, then cleaning for 3-5 times, and drying by nitrogen.
Preferably, m is1Modifying the surface of the electrode treated in the step (3) with the antibody A: m is to be1And dropwise adding the antibody solution A onto the surface of the electrode modified with CPBA, reacting for 5-15h at room temperature under a humid condition, and then cleaning for 2-6 times.
Preferably, m is1ATP is modified on the surface of the electrode treated in the step (4): m is to be1Dropping ATP solution to modified m1And (3) reacting the surface of the electrode of the antibody A for 0.5-4h at 25-45 ℃ under a humid condition, then cleaning for 3-5 times, and drying by nitrogen.
Preferably, TiO is added2@NH2The method for modifying the electrode surface treated in the step (5) by using MIL-125(Ti) comprises the following steps: will contain 0.01-10mg/ml TiO2@NH2-MIL-125(Ti) solution is added dropwise to the modification m1Reacting the electrode surface of ATP for 0.5-3h at room temperature under humid condition, then cleaning for 3-5 times, and drying by nitrogen.
In a second aspect of the invention, a method of detecting N is provided1-a photoelectrochemical biosensor for methyladenine.
The photoelectrochemistry biosensor detects m1The use of ATP is also within the scope of the present invention.
In a third aspect of the present invention, there is provided a method for detecting m using the above-mentioned photoelectrochemical sensor1A method of ATP comprising the steps of:
an electrochemical workstation is used as a signal acquisition instrument, a 500W xenon lamp is used as a visible light source, and TiO of a photoelectrochemical sensor is used2@NH2-MIL-125(Ti)/m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4The ITO electrode is a working electrode, the saturated calomel electrode is a reference electrode, the platinum column electrode is a counter electrode, 0.1M phosphate (pH 7.4) buffer solution containing 0.1M ascorbic acid is used as detection solution, and current and M are established1Relationship between ATP concentration, for m1And detecting the ATP content.
The invention has the beneficial effects that:
(1) the detection method is simple, and m can be performed only by performing simple modification on the surface of the ITO electrode for 6 times1And (4) detecting ATP.
(2) The photoelectrochemical sensor realizes the miniaturization of instruments, is easy to operate and has low detection cost.
(3) The detection has very high detection sensitivity, and the detection limit can reach 16.667 pM; furthermore, the invention is based on m1ATP and its antibody specific recognition function, has very high detection selectivity.
Drawings
FIG. 1: the photoelectrochemical sensor of the present invention is schematically assembled.
FIG. 2: photocurrent and m1Linear fit curve of ATP concentration.
FIG. 3: histogram of changes in photoelectrochemical response under different nucleotide conditions.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The "room temperature" in the present invention is in the range of 20 to 30 ℃.
"humid conditions" in the present invention are humidity greater than 90%; preferably, the humidity is 95-99%.
The cleaning solution used in the invention comprises the following components: 0.01-0.1M Tris-HCl and 20-60mM KCl, pH 7.4.
M used in the present invention1The composition of the antibody buffer A is as follows: 0.01-0.1M PBS, pH 6.0-8.5.
The detection solution used in the invention is: 0.01-0.1MNaH2PO4And 0.05-0.2MAA, pH 5.4-8.5.
BiVO for heterojunction of graphite-phase carbon nitride and bismuth vanadate in the invention4-g-C3N4The 4.0 generation polyamidoamines are shown by PAMAM and the p-carboxyphenylboronic acid is shown by CPBA. The test materials used in the present invention, which are not specifically described, are conventional in the art and commercially available.
As described in the background section, m1A also plays an important role in tRNA tertiary structure, and in ribosome biogenesis and translation. Thus for m1The detection and analysis of the A content are helpful for the research of diagnosing diseases in the aspects of immune system, endocrine system, nervous system and the like, and the research of biological development processes such as stem cell differentiation, genome senescence, meiosis, circadian rhythm and the like. But due to m1The conventional detection method for ATP has the disadvantages of the need for radioactive element labeling and the need for sophisticated instruments, and therefore, a new detection method needs to be developed.
The inventors of the present application based on g-C in earlier studies3N4-CdS QDs、Ru@SiO2Ru @ UiO-66 and BiVO4@TiO2The materials are equal to prepare 4 PEC biosensorsDevice, realizes the p-methylation of RNA and m6ATP is detected quickly and sensitively. The optical active material is the most important part in the photoelectrochemical sensor, and how to establish the action relationship between the optical active material and the substance to be detected so as to improve the sensitivity of the photoelectric activity of the optical active material is the difficulty of photoelectrochemical detection. Therefore, it is of great importance to develop new photoactive materials and to establish the functional relationship between the new photoactive materials and the substances to be detected.
The invention provides a photoelectric chemical immunosensor for detecting N based on Ti-MOF for the first time1-methyladenine, effecting the reaction of1High specificity and high sensitivity detection of ATP. The invention adopts BiVO with proper energy band structure position4And g-C3N4，BiVO4Photoexcited electrons in the conduction band can easily migrate to g-C3N4Adjacent photogenerated holes in the valence band, thereby inhibiting g-C3N4The recombination rate of the photo-generated electron-hole pairs enables efficient electron-hole transfer and separation, thus BiVO4-g-C3N4The composite material has good photoelectric properties. And NH2MIL-125(Ti) has adjustable porosity, can contain functional objects and has unique synergistic effect, and NH is adopted in the invention2TiO obtained by encapsulating amorphous titanium dioxide with MIL-125(Ti)2@NH2MIL-125(Ti) to improve the carrier transfer of photon generation and reduce the recombination of electron-hole pairs, thereby achieving the purpose of enhancing photocurrent. According to the photocurrent and m1Linear relation of ATP concentration, can realize m1And (4) detecting ATP.
Detection m of the invention1The construction and detection principle of the photoelectric chemical sensor for ATP is shown in FIG. 1, an ITO electrode is used as a substrate electrode, and oxygen-containing groups and BiVO on the surface of the ITO electrode are utilized4-g-C3N4BiVO (BiVO) by electrostatic adsorption force4-g-C3N4And modifying the surface of the electrode. Recycle BiVO4-g-C3N4And PAMAM is modified on the surface of the electrode through physical adsorption. Then, byThe interaction between the carboxyl of the CPBA and the amino of the PAMAM modifies the CPBA on the surface of the electrode. Followed by the use of a boronic acid group with m1The glycosidic reaction in antibody a links the antibody. Recognition of m by specific recognition of antigen and antibody1ATP. Finally, by means of TiO2@NH2Ti of MIL-125(Ti)4+Identification m1The phosphate in ATP is simultaneously amplified. TiO 22@NH2MIL-125(Ti) can obviously improve the photoelectric response of the sensor, thereby improving the detection sensitivity. Thus, using m1The linear relation between the ATP concentration and the photocurrent can be realized by1And (4) detecting ATP.
In one embodiment of the present invention, a method for constructing a photoelectrochemical sensor is given as follows:
(1)BiVO4-g-C3N4preparation of the dispersion: weighing 3-24 mg BiVO4-g-C3N4Adding the mixture into deionized water, and ultrasonically dispersing for 1-3 hours to prepare BiVO with the concentration of 0.1-8mg/mL4-g-C3N4And (3) dispersing the mixture.
Wherein BiVO4-g-C3N4The preparation method comprises the following steps:
(a) preparation of g-C3N4: putting melamine into a porcelain boat, calcining the melamine in a tube furnace at 550 ℃ for 3-5 h, cooling the melamine to room temperature, taking out a sample, grinding the sample, and weighing 500mg g-C3N4Adding into 200mL isopropanol, ultrasonic treating for 8 hr, centrifuging at 10000rpm for 10min, and drying in 60 deg.C oven to obtain stripped g-C3N4。
(b) Preparation of flaky BiVO4：1.225g Bi(NO3)3·5H2O is dissolved in 5mL of 4.0mol/L HNO3；0.29gNH4VO3Dissolve in 5 mL2.0mol/LNaOH. Respectively weighing 0.125g of Sodium Dodecyl Benzene Sulfonate (SDBS) and adding the two solutions into the two solutions, violently stirring for 0.5h, mixing the two solutions, adjusting the pH value to 7.0 by using 2.0mol/L NaOH, stirring for 0.5h, putting the solutions into a reaction kettle, putting the reaction kettle into a 200 ℃ oven, reacting for 3-5 h, cooling to room temperature, centrifuging at 10000rpm for 10min, and mixing the two solutionsThe solid was washed three times with deionized water and dried at 100 ℃ for 4h to give a yellow solid which was ground in a mortar for further use.
(c) Preparation of BiVO4-g-C3N4The composite material comprises the following components: according to the mass ratio of 3: 1 separately weighing BiVO4And g-C after peeling3N4Grinding in a mortar to mix uniformly, then putting in a porcelain boat, calcining at 400 ℃ by introducing nitrogen gas in a tube furnace for 0.5-2 h, cooling to room temperature, taking out a sample, putting in the mortar to grind to obtain BiVO4-g-C3N4。
(2) Pretreating an ITO electrode: cutting ITO conductive glass into 5 × 1cm2And then ultrasonically cleaning the mixture for 30 to 60 minutes by using an ethanol/NaOH mixed solution (the volume ratio is 1: 1-1: 6), finally respectively cleaning the mixture for 30 to 60 minutes by using acetone and secondary water, and airing the mixture at room temperature for later use.
Wherein the TiO is2@NH2-MIL-125(Ti) is prepared by the following method:
(a) preparation of NH2-MIL-125 (Ti): adding 2-aminoterephthalic acid (1.36g,7.5mmol) and tetrabutyl titanate (1.8mL, 5.3mmol) into a solution containing 9mLN, N-Dimethylformamide (DMF) and 1mL of methanol, stirring for 60min, then placing into a 50mL reaction kettle, reacting for 24-72 h at 150 ℃, cooling to room temperature, centrifuging at 10000rpm for 10min, washing the solid with DMF and methanol for three times respectively, drying at 60 ℃ to obtain a yellow solid, placing the solid into a mortar, and grinding into a powder for later use.
(b) Preparation of TiO2@NH2-MIL-125 (Ti): 0.40g of NH are weighed2Adding MIL-125(Ti) into 40mL of methanol, performing ultrasonic treatment for 20min, stirring for 0.5-2 h, slowly dropwise adding 500 mu L of tetrabutyl titanate, rapidly heating to 85 ℃, refluxing for 12h, cooling to room temperature, centrifuging for 10min at 10000rpm, washing for 3 times with methanol, drying in a 60 ℃ oven, taking out and grinding to obtain TiO2@NH2-MIL-125(Ti)。
(3)BiVO4-g-C3N4Fixing: subjecting 25-76 mu LBiVO4-g-C3N4Dropping the dispersion liquid to the pretreated ITO electrodeAnd (5) drying the surface by irradiating an infrared lamp. Then, the electrode is cleaned with a cleaning solution 3 to 5 times. And (5) drying by nitrogen. The prepared electrode is marked as BiVO4-g-C3N4/ITO。
(4) Modification of PAMAM: dropwise adding 16-55 mu L PAMAM solution to BiVO4-g-C3N4Drying the ITO electrode surface by infrared lamp irradiation. Then, the electrode is cleaned with a cleaning solution 3 to 5 times. And (5) drying by nitrogen. The prepared electrode is marked as PAMAM/BiVO4-g-C3N4/ITO。
(5) CPBA fixation: dripping 23-54 μ L of CPBA solution with concentration of 2.3-6.8 mM to PAMAM/BiVO4-g-C3N4And/on the surface of the ITO electrode, reacting for 1.5-4 hours at room temperature under a humid condition. Then, the electrode is cleaned with a cleaning solution 3 to 5 times. And (5) drying by nitrogen. The prepared electrode is marked as CPBA/PAMAM/BiVO4-g-C3N4/ITO。
(6)m1Immobilization of antibody a: 5 to 25 μ L of a surfactant containing 5 μ g/mL m1Buffer dropwise addition of A antibody to CPBA/PAMAM/BiVO4-g-C3N4The surface of the ITO electrode is reacted for 5-15 hours at room temperature under a humid condition, and is cleaned for 2-6 times by using a cleaning solution. The electrode mark is Ab/CPBA/PAMAM/BiVO4-g-C3N4/ITO。
(7)m1Modification of ATP: 12-47 μ L of m with different concentrations1Dropwise adding ATP solution to Ab/CPBA/PAMAM/BiVO4-g-C3N4And/on the surface of the ITO electrode, reacting for 0.5-4 hours at 25-45 ℃ under a humid condition. Then, the electrode is cleaned with a cleaning solution 3 to 5 times. And (5) drying by nitrogen. The prepared electrode is marked as m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4/ITO。
(8)TiO2@NH2Modification of MIL-125 (Ti): the content of the compound is 0.01-10mg/ml TiO2@NH2Dropwise addition of MIL-125(Ti) solution to m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4The surface of the ITO electrode is reacted for 0.5-3h at room temperature under a humid condition, then cleaned for 3-5 times, and dried by nitrogen. The prepared electrode is marked as TiO2@NH2-MIL-125(Ti)/m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4/ITO。
In the construction process of the photoelectrochemistry biosensor, the steps supplement each other, the sequence is strictly limited, each step serves for the next fixed modification, the last step is lacked, the subsequent modification can fail, and the steps have a synergistic promotion effect. The invention utilizes BiVO4-g-C3N4Good photoelectric activity and biocompatibility, specific recognition performance of antibody to antigen, and utilization of TiO2@NH2Ti of MIL-125(Ti)4+Identification m1The phosphate in ATP is simultaneously amplified. To m1High sensitivity and high specificity detection of ATP.
In another embodiment of the present invention, there is provided the detection of m using the above-mentioned photoelectrochemical sensor1A method of ATP comprising the steps of:
(1) m in different concentrations1Dropping ATP to Ab/CPBA/PAMAM/BiVO4-g-C3N4The surface of the modified ITO electrode is incubated, and m is identified by utilizing the specific recognition function of the antigen and the antibody1ATP, recycled TiO2@NH2-MIL-125(Ti) for signal amplification;
(2) by establishing photocurrent sum m1Relation curve of ATP concentration to m1ATP is detected.
The detection method is a non-disease diagnosis method. In the aspect of non-disease diagnosis, m can be detected1The ATP content is used for finding related targeted drugs, and a new method is provided for the development of new drugs.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
Example 1: detecting m1Construction of photoelectrochemical sensor for ATP
(1)BiVO4-g-C3N4Preparation of the dispersion: weighing BiVO4-g-C3N4Adding into deionized water, and ultrasonically dispersing for 2 hours to prepare BiVO with the concentration of 2.5mg/mL4-g-C3N4And (3) dispersing the mixture.
(2) Pretreating an ITO electrode: cutting ITO conductive glass into 5 × 1cm2Then ultrasonically cleaning for 60 minutes by using an ethanol/NaOH mixed solution (the volume ratio is 1:3), finally respectively cleaning for 30 minutes by using acetone and secondary water, and airing at room temperature for later use.
(3)BiVO4-g-C3N4Fixing: 50 mu LBiVO4-g-C3N4And dropwise adding the dispersion liquid to the surface of the pretreated ITO electrode, and irradiating and drying by using an infrared lamp. Then, the electrode was washed 4 times with the washing solution. And (5) drying by nitrogen. The prepared electrode is marked as BiVO4-g-C3N4/ITO。
(4) Modification of PAMAM: dropwise adding 16-55ml of PAMAM solution to BiVO4-g-C3N4Drying the ITO electrode surface by infrared lamp irradiation. Then, the electrode was washed 4 times with the washing solution. And (5) drying by nitrogen. The prepared electrode is marked as PAMAM/BiVO4-g-C3N4/ITO。
(5) P-carboxyphenylboronic acid immobilization: 30 μ L of CPBA solution at a concentration of 5.0mM was added dropwise to PAMAM/BiVO4-g-C3N4The reaction was carried out for 2.5 hours at room temperature under moist conditions on the ITO electrode surface. Then, the electrode was washed 4 times with the washing solution. And (5) drying by nitrogen. The prepared electrode is marked as CPBA/PAMAM/BiVO4-g-C3N4/ITO。
(6)m1Immobilization of antibody a: 15 μ L of antibody solution containing 5 μ g/mL was added dropwise to CPBA/PAMAM/BiVO4-g-C3N4The surface of the ITO electrode is reacted for 12 hours under the room temperature and the humid condition, and is washed for 5 times by using a washing liquid. The electrodes are labeled: Ab/CPBA/PAMAM/BiVO4-g-C3N4/ITO。
(7)m1Modification of ATP: 30 μ L of the suspension containing m at various concentrations1Dropwise adding ATP solution to Ab/CPBA/PAMAM/BiVO4-g-C3N4ITO electrode surface, reaction at 35 ℃ under humid conditions2.5 hours. Then, the electrode was cleaned 4 times with a cleaning solution and blown dry with nitrogen. The prepared electrode is marked as m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4/ITO。
(8)TiO2@NH2Modification of MIL-125 (Ti): 30 μ L of the suspension containing 4mg/ml of TiO2@NH2Dropwise addition of MIL-125(Ti) solution to m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4The surface of the ITO electrode is reacted for 6 hours at room temperature under the humid condition, and then cleaned for 4 times and dried by nitrogen. The prepared electrode is marked as TiO2@NH2-MIL-125(Ti)/m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4/ITO。
Example 2: photoelectrochemical detection
An electrochemical workstation is used as a signal acquisition instrument, a 500W xenon lamp is used as a visible light source (a lens for filtering ultraviolet is additionally arranged), and TiO2@NH2-MIL-125(Ti)/m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4The ITO electrode is a working electrode, the saturated calomel electrode is a reference electrode, the platinum column electrode is a counter electrode and contains 0.1MNaH2PO4And buffer solution of 0.1MAA (pH 7.4) as detection solution, and working voltage of-0.3V as working voltage, and performing detection research on the substance to be detected by using i-t technology to establish photocurrent and m1The linear range of the relationship between ATP concentrations was 0.05-35nM, the calibration curve was i (na) ═ 145.63log c (nM) +688.94(R ═ 0.9958), and the detection limit was 16.7pM (fig. 2).
Example 3: test for Selectivity
To investigate the specificity of the constructed sensors, AMP, CMP, GMP, UMP, 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC), and 5-methyluracil (5mU) were selected as comparison reagents. And the change value of the photocurrent (delta I-I) of the sensor constructed by different contrast agents1-I2，I1Is Ab/CPBA/PAMAM/BiVO4-g-C3N4The electrode of ITO after being treated by different nucleotides is continuously treated by TiO2@NH2Of electrodes after MIL-125(Ti) treatmentPhotocurrent value, I2Is Ab/CPBA/PAMAM/BiVO4-g-C3N4Current value of ITO, comparison reagent and m1ATP concentrations were all 1nM for comparison. The results show (FIG. 3) that the current value change of the sensor is obviously lower than m when the contrast agent participates in the construction1The current value of the sensor constructed by the ATP participating in the reaction shows that the constructed sensor has good specificity.
Example 4: stability test
Photochemical sensor (m) prepared using example 11ATP concentration was 1nM), and photocurrent was detected in the detection solution. The standard relative deviation of the photocurrent obtained was 0.18%, indicating that the method had good stability.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (8)

1. Detect N1-a method for preparing a photoelectrochemical sensor of methyladenine, characterized by comprising the following steps:
(1) taking an ITO electrode as a basic electrode, and pretreating the basic electrode; utilizing oxygen-containing group on the surface of ITO electrode and BiVO4-g-C3N4BiVO (BiVO) by electrostatic adsorption force4-g-C3N4Modifying the surface of the treated electrode; BiVO (bismuth oxide) is added4-g-C3N4The method for modifying the surface of the treated electrode comprises the following steps: preparing BiVO from heterojunction of graphite-phase carbon nitride and bismuth vanadate by using deionized water4-g-C3N4Dispersing BiVO in water4-g-C3N4Dropwise adding the dispersed liquid onto the surface of the pretreated electrode, drying under the irradiation of an infrared lamp, then cleaning for 3-5 times, and drying by nitrogen; the BiVO4-g-C3N4The concentration of the dispersion is 0.1-8 mg/ml;
(2) using BiVO4-g-C3N4Physical adsorption with polyamide-amine PAMAM, and modifying the PAMAM on the surface of the electrode treated in the step (1);
(3) modifying the CPBA on the surface of the electrode treated in the step (2) through the interaction between the carboxyl of the p-carboxyphenylboronic acid CPBA and the amino of the PAMAM;
(4) using boronic acid groups of CPBA with m1Glycoside reaction in antibody A, and1modifying the surface of the electrode treated in the step (3) with an antibody A;
(5) recognition of m by specific recognition of antigen and antibody1ATP, transformation of m1ATP is modified on the surface of the electrode treated in the step (4);
(6) by using TiO2@NH2Ti of MIL-125(Ti)4+Identification m1The phosphate radical in ATP is simultaneously amplified to obtain TiO2@NH2Modifying MIL-125(Ti) on the surface of the electrode treated in the step (5); adding TiO into the mixture2@NH2The method for modifying the electrode surface treated in the step (5) by using MIL-125(Ti) comprises the following steps: will contain 0.01-10mg/ml TiO2@NH2-MIL-125(Ti) solution is added dropwise to the modification m1Reacting the electrode surface of ATP for 0.5-3h at room temperature under humid condition, then cleaning for 3-5 times, and drying by nitrogen.
2. The method for preparing the PAMAM of claim 1, wherein in the step (2), the PAMAM is modified on the surface of the electrode treated in the step (1) by the following steps: dropwise adding PAMAM solution to the modified BiVO4-g-C3N4The surface of the electrode is dried under the irradiation of an infrared lamp, then cleaned for 3-5 times and dried by nitrogen.
3. The method for preparing the electrode according to claim 1, wherein in the step (3), the CPBA is modified on the surface of the electrode treated in the step (2) by a method comprising: and (3) dropwise adding the CPBA solution to the surface of the electrode modified with the PAMAM, reacting for 1.5-4h at room temperature under a humid condition, then cleaning for 3-5 times, and drying by nitrogen.
4. According to the claimsThe process according to claim 1, wherein in the step (4), m is1The method for modifying the antibody A on the surface of the electrode treated in the step (3) comprises the following steps: m is to be1And dropwise adding the antibody solution A onto the surface of the electrode modified with CPBA, reacting for 5-15h at room temperature under a humid condition, and then cleaning for 2-6 times.
5. The production method according to claim 1, wherein in the step (5), m is1The method for modifying the ATP on the surface of the electrode treated in the step (4) comprises the following steps: m is to be1Dropping ATP solution to modified m1And (3) reacting the surface of the electrode of the antibody A for 0.5-4h at 20-45 ℃ under a humid condition, then cleaning for 3-5 times, and drying by nitrogen.
6. Assay N prepared by the method of any one of claims 1 to 51-a photoelectrochemical sensor of methyladenine.
7. Assay N according to claim 61Photoelectrochemical sensor for detecting m1Use in ATP.
8. Detection N using claim 61Photoelectrochemical sensor detection of-methyladenine m1A method of ATP comprising the steps of:
an electrochemical workstation is used as a signal acquisition instrument, a 500W xenon lamp is used as a visible light source, and TiO of a photoelectrochemical sensor is used2@NH2-MIL-125(Ti)/m1ATP/Ab/CPBA/PAMAM/BiVO4-g-C3N4The ITO electrode is a working electrode, the saturated calomel electrode is a reference electrode, the platinum column electrode is a counter electrode, 0.1M phosphate buffer solution containing 0.1M ascorbic acid is used as detection solution, the pH value of the phosphate buffer solution is 7.4, and current and M are established1Relationship between ATP concentration, for m1And detecting the ATP content.
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