CN108918622B - Photoelectrochemical biosensor for detecting 5-hydroxymethylcytosine deoxyribonucleotide and preparation method thereof - Google Patents
Photoelectrochemical biosensor for detecting 5-hydroxymethylcytosine deoxyribonucleotide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a photoelectrochemical biosensor for detecting 5-hydroxymethyl cytosine deoxyribonucleotide and a preparation method thereof, wherein the photoelectrochemical biosensor comprises the following components: the electrode comprises tungsten sulfide, polydopamine, mercaptophenylboronic acid, 5hmC, phos-tag-biotin and streptavidin which are sequentially modified on the surface of the electrode. The invention realizes the high sensitivity and high specificity detection of 5hmC by utilizing the good photoelectric activity and biocompatibility of tungsten sulfide, the excellent electron donor property and biocompatibility of polydopamine, the specific recognition and combination performance of phos-tag-biotin to phosphate groups and the specific reaction of hydroxymethyl and sulfydryl on 5hmC catalyzed by M.HhaI methyltransferase. The method can well eliminate the interference of 5mC on 5hmC detection. The detection method is simple, the miniaturization of the instrument is realized, the operation is easy, and the detection of 5hmC can be realized only by simply processing the surface of the electrode.
Description
Technical Field
The invention relates to the technical field of photoelectrochemical analysis, in particular to a photoelectrochemical biosensor for detecting 5-hydroxymethyl cytosine deoxyribonucleotide and a preparation method thereof.
Background
5-hydroxymethylcytosine deoxyribonucleotide (5hmC), first discovered in the phage in 1952, was modified by glycosyltransferase mediated glycosylation, making the phage genome resistant to degradation by host restriction enzymes after entering the host. However, this finding did not give sufficient attention at that time. In 2009, researchers found that 5hmC is abundantly expressed in human and mouse brains and embryonic stem cells, and the concept of hydroxymethylation once again entered the visual field of people and gained attention. Currently 5hmC is the "6 th base" following the 5mC, which is called the "5 th base". Although the function of 5hmC is not completely understood at present, it may also be an important epigenetic marker, possibly associated with demethylation processes or transcriptional regulation. To understand the biological function of 5hmC, it is necessary to have reliable data on the quantification and localization of 5hmC in genomic DNA, but this work is still facing huge technical challenges today, since standard biochemical methods do not easily distinguish between 5mC and 5 hmC.
At present, methods for detecting 5hmC mainly comprise thin-layer chromatography, liquid chromatography-mass spectrometry, high performance liquid chromatography-mass spectrometry, capillary electrophoresis-mass spectrometry, single-molecule real-time sequencing and the like. These methods all require expensive instruments and cumbersome operation steps, require specialized operators, and the like. Moreover, 5hmC is easily interfered by 5mC when detected by the method. Therefore, it is necessary and urgent to establish a simple, rapid, highly sensitive and highly selective method for detecting 5 hmC.
Photoelectrochemical analysis is an emerging analytical technique with the advantages of electrochemical analysis and photochemical analysis. Which excites the electro-optically active material with light to produce photo-generated electrons and holes. The photo-generated electrons are captured by the electrodes to generate an electric current. The excitation light source and the detection signal are completely different in two forms, so that the interference of background signals can be effectively reduced, and the sensitivity of analysis and detection is greatly improved. The tungsten sulfide has good photoelectric activity and biocompatibility, and can generate photoelectrons under the excitation of visible light to form stable photocurrent. At present, no report of detecting 5hmC by a photoelectrochemical analysis method based on tungsten sulfide exists.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a photoelectrochemical biosensor for detecting 5-hydroxymethyl cytosine deoxyribonucleotide and a preparation method thereof, and high specificity and high sensitivity detection of 5hmC are realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a photoelectrochemical biosensor for detecting 5-hydroxymethylcytosine deoxyribonucleotides, comprising: the electrode comprises tungsten sulfide, polydopamine, mercaptophenylboronic acid, 5hmC, biotin-functionalized phos-tag-biotin and streptavidin which are sequentially modified on the surface of the electrode.
Preferably, the electrode is an ITO electrode.
In a second aspect of the present invention, there is provided a method for preparing the above photoelectrochemical biosensor, comprising the steps of:
(1) pretreating the electrode;
(2) modifying the surface of the electrode treated in the step (1) with tungsten sulfide;
(3) modifying polydopamine on the surface of the electrode treated in the step (2);
(4) modifying the mercapto phenylboronic acid on the surface of the electrode treated in the step (3);
(5) modifying 5hmC to the surface of the electrode treated in the step (4) under the catalysis of M.HhaI;
(6) modifying the phos-tag-biotin to the surface of the electrode treated in the step (5) by utilizing the specific binding effect of the phos-tag-biotin on the phosphate radical on the 5 hmC;
(7) and (4) modifying the streptavidin on the surface of the electrode treated in the step (6) by utilizing the specific binding action between the biotin and the streptavidin, thus preparing the photoelectrochemical biosensor.
Preferably, in the step (1), the electrode pretreatment method comprises: the electrode is firstly cleaned by mixed liquor of ethanol and sodium hydroxide, then cleaned by acetone and secondary water and dried.
Preferably, in the step (2), the method for modifying the tungsten sulfide on the surface of the electrode treated in the step (1) comprises the following steps: preparing tungsten sulfide dispersion liquid from tungsten sulfide by using deionized water, dropwise adding the tungsten sulfide dispersion liquid to the surface of the pretreated electrode, drying, then cleaning for 3-5 times, and drying by using nitrogen.
More preferably, the concentration of the tungsten sulfide dispersion is 0.1 to 5mg/m L.
Preferably, in the step (3), the method for modifying polydopamine on the surface of the electrode treated in the step (2) comprises the following steps: and (3) preparing polydopamine dispersion liquid from polydopamine by using deionized water, dropwise adding the polydopamine dispersion liquid onto the surface of the electrode treated in the step (2), drying, cleaning for 3-5 times, and drying by using nitrogen.
More preferably, the concentration of the polydopamine dispersion is 0.5-5mg/m L.
Preferably, in the step (4), the method for modifying the mercaptophenylboronic acid on the surface of the electrode treated in the step (3) is as follows: dropwise adding a mercapto-phenylboronic acid solution to the surface of the electrode treated in the step (3), and reacting for 1.5-4h at room temperature under a humid condition; then cleaning for 3-5 times, and drying by nitrogen.
More preferably, the concentration of the mercaptophenylboronic acid solution is 2.3 to 6.8 mM.
The application of the photoelectrochemical biosensor in the detection of 5hmC is also the protection scope of the invention.
In a third aspect of the present invention, there is provided a method for detecting 5hmC using the above-mentioned photoelectrochemical biosensor, comprising the steps of:
the photoelectrochemical biosensor is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a Pt wire is used as an auxiliary electrode to form a three-electrode system for electrochemical signal detection, a detection solution is a Tris-HCl buffer solution (pH is 5.4-8.5) containing 0.05-0.28M KCl, the relation between the current and the concentration of 5-hydroxymethylcytosine nucleotide is established, and the content of the 5-hydroxymethylcytosine nucleotide is detected.
Preferably, the detection method used is a current-time method, using a potential of-0.5-0.2V.
Preferably, the concentration of the Tris-HCl buffer solution is 5-40 mM.
The detection method is a non-disease diagnosis method. In the aspect of non-disease diagnosis, related targeted drugs can be found by detecting the content of 5hmC, and a new method is provided for the development of new drugs.
The invention has the beneficial effects that:
(1) the invention utilizes the good photoelectric activity of tungsten sulfide and the excellent electron donor property of polydopamine to realize the amplification of photoelectric signals and improve the detection sensitivity of 5 hmC.
(2) The covalent reaction between hydroxymethyl and sulfydryl on the 5hmC is catalyzed by M.HhaI, so that the specificity of the 5hmC detection is improved.
(3) The detection method is simple, realizes the miniaturization of the instrument, is easy to operate, and can realize the detection of 5hmC only by simply processing the surface of the ITO electrode.
(4) The invention is based on the specific binding effect of the aptamer and 5hmC, and has high detection selectivity.
Drawings
FIG. 1: schematic diagram of 5hmC detection of the present invention.
FIG. 2: photoelectrochemical response curves of different concentrations of 5 hmC; curves a-i represent 5hmC at concentrations of 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01nM, respectively.
FIG. 3: a linear fit of the log photocurrent to the 5hmC concentration.
FIG. 4: 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: 3-15mM Tris-HCl and 20-60mM KCl, pH 7.4.
The m.hhal buffer used in the present invention had the following composition: 20-60mM Tris-HCl, 5-30mM EDTA, pH 6.0-8.5.
The composition of the phos-tag-biotin solid buffer used in the present invention was: 5-30mM Tris-HCl, 0.05-0.35M NaCl, 0.1-0.5% Tween-20, 0.1-0.6mM Zn (NO)3)2，pH 6.0-8.5。
The streptavidin immobilization buffer used in the present invention comprises the following components: 4-18mM Tris-HCl and 8-44mM NaCl, pH 6.0-8.5.
The detection solution used in the invention is: 5-40mM Tris-HCl, 0.05-0.28M KCl, pH 5.4-8.5.
As described in the background section, the prior art 5hmC detection method has certain disadvantages, for example, the method based on DNA hybridization detects 5hmC in DNA chain, but the method is limited by DNA detection probe sequence and length, and cannot detect 5hmC in long-chain DNA, and the method cannot detect 5hmC content in genomic DNA; the PCR method is adopted to detect 5hmC, and has the problems of expensive instrument, complex primer design, need of fluorescent labeling and the like. Based on the structure, the photoelectrochemistry biosensor for detecting 5hmC is constructed, the photoelectrochemistry biosensor can be used for detecting single 5hmC, the application range is wide, and the detection of 5hmC in genome DNA can be realized.
The schematic diagram of the construction and detection of the photoelectrochemical biosensor of the present invention is shown in FIG. 1. And modifying the tungsten sulfide on the surface of the electrode by using the ITO electrode as a matrix electrode and utilizing the electrostatic adsorption force between oxygen-containing groups on the surface of the ITO electrode and the tungsten sulfide. And modifying the polydopamine on the surface of the electrode by utilizing the physical adsorption effect between the tungsten sulfide and the polydopamine. Then, modifying the mercaptophenylboronic acid on the surface of the electrode by utilizing the specific reaction of the o-diol structure on the surface of the polydopamine and the phenylboronic acid on the mercaptophenylboronic acid. Under the catalytic action of M.HhaI, the hydroxymethyl group on the 5hmC and the sulfhydryl group on the mercaptophenylboronic acid are subjected to covalent reaction, and the 5hmC is captured on the surface of the electrode. At this point, the phosphate on the 5hmC is away from the electrode surface. And modifying the phos-tag-biotin on the surface of the electrode by utilizing the binding action of the phos-tag-biotin and phosphate radical. And finally, modifying the streptavidin on the surface of the electrode by utilizing the binding action between the biotin and the streptavidin to obtain the prepared sensor. The polydopamine is an excellent electron donor material, and can obviously promote the photoelectric response of tungsten sulfide, thereby improving the detection sensitivity. When the streptavidin is modified on the surface of the electrode, the photoelectric current is obviously reduced, mainly because the large molecular structure of the streptavidin hinders the migration of photo-generated electrons. Since the amount of streptavidin modification is determined by the concentration of 5hmC, 5hmC can be detected by utilizing the linear relationship between the concentration of 5hmC and the photocurrent.
In one embodiment of the present invention, the construction process of the photoelectrochemical biosensor is as follows:
(1) the preparation of polydopamine comprises weighing 0.5-1g of dopamine, dissolving in 300-600M L deionized water, heating to 45-80 ℃ under magnetic stirring, adding 2-4M L NaOH solution with concentration of 0.5-2M, standing for reaction for 3-8 hours, centrifuging at 2000-5000rpm, collecting suspension, centrifuging at 10000-15000rpm, collecting precipitate, washing the precipitate with deionized water for 3-5 times, and vacuum drying at 60 ℃.
(2) The preparation of the tungsten sulfide dispersion liquid comprises weighing 5-250mg of tungsten sulfide, adding into 50m L deionized water, and ultrasonically dispersing for 1-3 hours.
(3) The preparation of the polydopamine dispersion liquid comprises the steps of weighing 10-100mg of polydopamine, adding the polydopamine into 20m L of deionized water, and carrying out ultrasonic dispersion for 1-3 hours.
(4) ITO electrode pretreatment, namely dividing ITO conductive glass into 5 × 1cm2Then ultrasonically cleaning for 30-60 minutes by using an ethanol/NaOH mixed solution (the ratio is 1:1-1:6), finally respectively cleaning for 30-60 minutes by using acetone and secondary water, and airing at room temperature for later use.
(5) Fixing tungsten sulfide, namely dripping 25-76 mu L tungsten sulfide dispersion liquid on the surface of a pretreated ITO electrode, irradiating and drying by an infrared lamp, then cleaning the electrode by cleaning liquid for 3-5 times, drying by nitrogen, and marking the prepared electrode as WS2/ITO。
(6) Immobilization of Polydopamine by dropping 16-55 μ L Polydopamine Dispersion onto WS2ITO electrode surface, RedAnd (5) drying by external lamp irradiation. Then, the electrode was washed 3 to 5 times with a washing solution. And (5) drying by nitrogen. The prepared electrode was labeled PDA/WS2/ITO。
(7) Immobilization of p-mercaptophenylboronic acid A p-mercaptophenylboronic acid solution (prepared from a 10mM tris-HCl solution (pH 8.0)) having a concentration of 23 to 54. mu. L of 2.3 to 6.8mM was added dropwise to PDA/WS2The reaction is carried out for 1.5 to 4 hours on the surface of an ITO electrode under the conditions of room temperature and humidity. Then, the electrode was washed 3 to 5 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as MPBA/PDA/WS2/ITO。
(8) Modification of 5hmC 12-47 μ L M.HhaI buffer containing 50-200unit/m L M.HhaI and varying concentrations of 5hmC was added dropwise to MPBA/PDA/WS2The reaction is carried out for 1.5-4 hours at 25-45 ℃ on the surface of an ITO electrode under the humid condition. Then, the electrode was washed 3 to 5 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as 5hmC/MPBA/PDA/WS2/ITO。
(9) Phos-tag-biotin immobilisation 20-60 μ L Phos-tag-biotin immobilisation buffer containing 10-40 μ M Phos-tag-biotin was added dropwise to 5hmC/MPBA/PDA/WS2The reaction is carried out for 0.5 to 3 hours on the surface of the ITO electrode under the room temperature and the humid condition. Then, the electrode was washed 3 to 5 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as Phos-tag/5hmC/MPBA/PDA/WS2/ITO。
(10) Immobilization of streptavidin 15-62 μ L streptavidin immobilization buffer containing 0.14-0.85mg/m L streptavidin was added dropwise to Phos-tag/5hmC/MPBA/PDA/WS2The reaction is carried out on the surface of the ITO electrode for 0.5 to 3.5 hours at the temperature of between 25 and 55 ℃ under the humid condition. Then, the electrode was washed 3 to 5 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as SA/Phos-tag/5hmC/MPBA/PDA/WS2/ITO。
In the construction process of the photoelectrochemical sensor, the steps supplement each other, the sequence is strictly limited, each step serves for the next fixed modification, and the lack of the previous step can cause the failure of the subsequent modification. The material fixed and modified on the surface of the ITO electrode can be a commercially available product or can be prepared by itself, as long as the performance meets the use requirement, and the invention is not particularly limited.
In another embodiment of the present invention, the process for detecting 5hmC using the above-described photoelectrochemical biosensor is given by:
(1) SA/Phos-tag/5hmC/MPBA/PDA/WS prepared at different concentrations of 5hmC2The ITO electrode is used as a working electrode, the saturated calomel electrode is used as a reference electrode, the Pt filament is used as an auxiliary electrode to form a three-electrode system for carrying out photoelectrochemical signal detection, a light source is visible light, the applied potential is-0.8-0.2V, and photocurrent is recorded in detection liquid (5-40 mM Tris-HCl, 0.05-0.28M KCl, and the pH value is 5.4-8.5).
(2) Establishing a relation between the current and the concentration of the 5-hydroxymethylcytosine nucleotide, and detecting the content of 5hmC in the sample to be detected by using the relation.
With the increase of the concentration of 5hmC, the amount of streptavidin on the surface of the electrode is increased, and the photoelectric signal is gradually reduced. According to the linear relation between the concentration of 5hmC and the photocurrent, the detection of 5hmC can be realized.
The detection range of the photoelectric chemical biosensor for 5hmC is 0.01-100nM, and the detection limit is 4.12 pM.
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.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available.
Example 1: preparation of polydopamine
0.54g of dopamine is weighed and dissolved in 300M L deionized water, the mixture is heated to 50 ℃ under magnetic stirring, then 2M L M NaOH solution with the concentration of 1M is added, the mixture is kept stand and reacted for 5 hours, the mixture is centrifuged at 4000rpm, suspension is collected, the suspension is centrifuged at 12000rpm, precipitate is collected, the precipitate is washed 3 times by the deionized water, and the mixture is dried in vacuum at 60 ℃.
Example 2: preparation of tungsten sulfide dispersion
12mg of tungsten sulfide was weighed, added to 3m L of deionized water, and ultrasonically dispersed for 1 to 3 hours.
Example 3: preparation of Polydopamine Dispersion
12mg of polydopamine was weighed, added to 6m L of deionized water and dispersed ultrasonically for 1-3 hours.
Example 4: ITO electrode pretreatment
The ITO conductive glass is divided into 5 × 1cm2Then ultrasonically cleaning for 45 minutes by using an ethanol/NaOH mixed solution (the ratio is 1:1), finally respectively cleaning for 45 minutes by using acetone and secondary water, and airing at room temperature for later use.
Example 5: fixation of tungsten sulfide
Dripping 40 mu L tungsten sulfide dispersion liquid on the surface of a pretreated ITO electrode, irradiating and drying by an infrared lamp, then cleaning the electrode by cleaning liquid for 3 times, blowing dry by nitrogen, and marking the prepared electrode as WS2/ITO。
Example 6: immobilization of polydopamine
40 μ L polydidopamine dispersion was added dropwise to WS2Drying the ITO electrode surface by infrared lamp irradiation. Then, the electrode was washed 3 times with a washing solution. And (5) drying by nitrogen. The prepared electrode was labeled PDA/WS2/ITO。
Example 7: immobilization of p-mercaptophenylboronic acid
A20. mu. L mM p-mercaptophenylboronic acid solution was added dropwise to PDA/WS2The reaction was carried out for 2 hours at room temperature and under moist conditions on the ITO electrode surface. Then, the electrode was washed 3 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as MPBA/PDA/WS2/ITO。
Example 8: modification of 5hmC
20 μ L M.HhaI buffer containing 100unit/m L M.HhaI and various concentrations of 5hmC was added dropwise to MPBA/PDA/WS2The reaction was carried out at 37 ℃ for 2 hours under moist conditions on the ITO electrode surface. Then, the electrode was washed 3 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as 5hmC/MPBA/PDA/WS2/ITO。
Example 9: phos-tag-biotin immobilisation
Mu. L Phos-tag-biotin containing 20. mu.M Phos-tag-biotin was added dropwise to 5hmC/MPBA/PDA/WS2ITO electrode surface, room temperature andthe reaction was carried out under moist conditions for 2 hours. Then, the electrode was washed 3 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as Phos-tag/5hmC/MPBA/PDA/WS2/ITO。
Example 10: immobilization of streptavidin
Mu. L streptavidin immobilization buffer containing 0.2mg/m L of streptavidin was added dropwise to Phos-tag/5hmC/MPBA/PDA/WS2The reaction was carried out at 37 ℃ for 2 hours under moist conditions on the ITO electrode surface. Then, the electrode was washed 3 times with a washing solution. And (5) drying by nitrogen. The prepared electrode is marked as SA/Phos-tag/5hmC/MPBA/PDA/WS2/ITO。
Example 11: photoelectrochemical detection
An electrochemical workstation is taken as a signal acquisition instrument, a 500W xenon lamp is taken as a visible light source (a lens for filtering ultraviolet is additionally arranged), and SA/Phos-tag/5hmC/MPBA/PDA/WS2The ITO electrode is a working electrode, the saturated calomel electrode is a reference electrode, the platinum column electrode is a counter electrode, 10mM Tris-HCl (pH 7.4) buffer solution containing 0.1M KCl is used as detection solution, the voltage of minus 0.3V is used as working voltage, the detection research of the object to be detected is carried out by adopting the i-t technology, the relation between the photocurrent and the concentration of 5hmC is established, the linear range is 0.01-100nM, the correction curve is I (nA) ═ 138.22log c (nM) +535.44(R ═ 0.9953), and the detection limit is 4.12pM (figure 2 and figure 3).
Example 12: test for Selectivity
To investigate the specificity of the constructed sensors, 6 deoxyribonucleotides were selected as contrast agents, such as deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), 5-methylcytosine deoxyribose triphosphate (5m-dCTP), N-6 methyladenine deoxyribose triphosphate (m 6-dATP). And the change value of the photocurrent (delta I-I) of the sensor constructed by different contrast agents1-I2，I1Is MPBA/PDA/WS2Current value of/ITO, I2Is MPBA/PDA/WS2The photocurrent values of the electrodes after the electrodes of ITO treated by different nucleotides are treated by phos-tag-biotin and streptavidin, and the concentrations of the contrast reagent and 5hmC are both 1nM) are compared. The results show thatThe current value change of the sensor constructed by the participation of the contrast agent is obviously lower by 5hmC, which indicates that the constructed sensor has good specificity (figure 4).
Example 13: stability test
The same method is adopted to prepare 10 pieces of SA/Phos-tag/5hmC/MPBA/PDA/WS2the/ITO electrode (5hmC concentration of 1nM) detects the photocurrent in the detection solution. The relative standard deviation of the photocurrent obtained was 4.22%, indicating good reproducibility of the method. SA/Phos-tag/5hmC/MPBA/PDA/WS2The ITO sensor is stored for 2 weeks at 4 ℃, and the photoelectrochemical signal is detected in the detection liquid, so that 92.17% of the original response of the photocurrent response is obtained, which indicates that the method has 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 (7)
1. A method for preparing a photoelectrochemical biosensor for detecting 5-hydroxymethylcytosine deoxyribonucleotide, the photoelectrochemical biosensor comprising: the electrode is characterized by comprising tungsten sulfide, polydopamine, mercaptophenylboronic acid, 5hmC, biotin functionalized phos-tag and streptavidin which are sequentially modified on the surface of the electrode; the preparation method comprises the following steps:
(1) pretreating the electrode;
(2) modifying the surface of the electrode treated in the step (1) with tungsten sulfide;
(3) modifying polydopamine on the surface of the electrode treated in the step (2);
(4) modifying the mercapto phenylboronic acid on the surface of the electrode treated in the step (3);
(5) modifying 5hmC to the surface of the electrode treated in the step (4) under the catalysis of M.HhaI;
(6) modifying the phos-tag-biotin to the surface of the electrode treated in the step (5) by utilizing the specific binding effect of the phos-tag-biotin on the phosphate radical on the 5 hmC;
(7) and (4) modifying the streptavidin on the surface of the electrode treated in the step (6) by utilizing the specific binding action between the biotin and the streptavidin, thus preparing the photoelectrochemical biosensor.
2. The method according to claim 1, wherein in the step (1), the electrode is pretreated by: the electrode is firstly cleaned by mixed liquor of ethanol and sodium hydroxide, then cleaned by acetone and secondary water and dried.
3. The preparation method according to claim 1, wherein in the step (2), the tungsten sulfide is modified on the surface of the electrode treated in the step (1) by a method comprising: preparing tungsten sulfide dispersion liquid from tungsten sulfide by using deionized water, dropwise adding the tungsten sulfide dispersion liquid to the surface of the pretreated electrode, drying, then cleaning for 3-5 times, and drying by using nitrogen;
the concentration of the tungsten sulfide dispersion is 0.1-5mg/m L.
4. The preparation method according to claim 1, wherein in the step (3), the polydopamine is modified on the surface of the electrode treated in the step (2) by a method comprising the following steps: preparing polydopamine into polydopamine dispersion liquid by using deionized water, dropwise adding the polydopamine dispersion liquid onto the surface of the electrode treated in the step (2), drying, cleaning for 3-5 times, and drying by using nitrogen;
the concentration of the polydopamine dispersion liquid is 0.5-5mg/m L.
5. The method according to claim 1, wherein in the step (4), the method for modifying the mercaptophenylboronic acid on the surface of the electrode treated in the step (3) comprises the following steps: dropwise adding a mercapto-phenylboronic acid solution to the surface of the electrode treated in the step (3), and reacting for 1.5-4h at room temperature under a humid condition; then cleaning for 3-5 times, and drying by nitrogen;
the concentration of the mercapto phenyl boric acid solution is 2.3-6.8 mM.
6. A method for detecting 5hmC using a photoelectrochemical biosensor prepared by the method of any one of claims 1 to 5, comprising the steps of:
the photoelectrochemical biosensor prepared by the method of any one of claims 1 to 5 is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a Pt filament is used as an auxiliary electrode to form a three-electrode system for electrochemical signal detection, a detection solution is a Tris-HCl buffer solution containing 0.05-0.28M KCl, the pH value is 5.4-8.5, and the relation between the current and the concentration of 5-hydroxymethylcytosine nucleotide is established to detect the content of 5-hydroxymethylcytosine nucleotide.
7. The method of claim 6, wherein: the detection method is a current-time method, and the applied potential is-0.5-0.2V.
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| CN112858417B (en) * | 2021-02-24 | 2021-12-28 | 山东农业大学 | Method for detecting m6A by using photoelectrochemical sensor based on bismuth sulfide-silver bromide heterojunction |
| CN112858419B (en) * | 2021-02-26 | 2021-11-23 | 山东农业大学 | Method for detecting 5-hydroxymethylcytosine by constructing photoelectrochemical sensor |
| CN114295699B (en) * | 2021-12-30 | 2022-10-21 | 山东农业大学 | Photoelectrochemical biosensor for detecting 5-formyl cytosine deoxyribonucleotide and preparation method and application thereof |
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