CN108918622A - A kind of Photoelectrochemistrbiosensor biosensor and preparation method thereof detecting 5-hydroxymethyl cytosine deoxyribonucleotide - Google Patents
A kind of Photoelectrochemistrbiosensor biosensor and preparation method thereof detecting 5-hydroxymethyl cytosine deoxyribonucleotide Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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Abstract
The invention discloses it is a kind of detect 5-hydroxymethyl cytosine deoxyribonucleotide Photoelectrochemistrbiosensor biosensor and preparation method thereof, the Photoelectrochemistrbiosensor biosensor, including：Electrode is successively modified and successively modifies the tungsten sulfide in electrode surface, poly-dopamine, mercaptophenyl boronic acid, 5hmC, phos-tag-biotin and Streptavidin in electrode surface.The present invention utilizes the good photoelectric activity of tungsten sulfide and bio-compatibility, the excellent electron donor property of poly-dopamine and bio-compatibility, specific recognition and binding performance of the phos-tag-biotin to phosphate group, and on the 5hmC of M.HhaI methyl transferase catalytic methylol and sulfydryl idiosyncrasy, realize and the high sensitivity and high specific of 5hmC detected.The method of the present invention can eliminate the interference that 5mC detects 5hmC well.Detection method of the invention is simple, realizes instrument miniaturization, and easily operated, is only simply handled electrode surface, and the detection to 5hmC can be realized.
Description
Technical field
The present invention relates to photoelectrochemical assay technical fields, and in particular to a kind of detection 5-hydroxymethyl cytosine deoxyribose
Photoelectrochemistrbiosensor biosensor of nucleotide and preparation method thereof.
Background technique
5-hydroxymethyl cytosine deoxyribonucleotide (5hmC) is most found in bacteriophage early in nineteen fifty-two, it can quilt
Glycosyl transferase mediate it is glycosylation modified, to make phage genome that can resist the drop of host's restriction enzyme after entering host
Solution.But this discovery did not caused enough attention at that time.2009, researcher find 5hmC people, mouse brain and
There is abundant expression in embryonic stem cell, methylolated concept just enters again the visual field of people and paid attention to.Mesh
Preceding 5hmC is " the 6th base " after 5mC is referred to as " the 5th base ".Although the function of 5hmC is not yet completely bright at present
, but it may be also an important epigenetics label, and it may be related with demethylation process or transcriptional control.It has wanted
5hmC is solved in biological function, grasping quantitative and positioning authentic data of the 5hmC in genomic DNA is necessary,
But nowadays this work is still faced with huge technological challenge, because the biochemical method of standard is not easy to distinguish 5mC and 5hmC.
Currently, the method for being used to detect 5hmC mainly includes thin-layer chromatography, liquid chromatography-mass spectrometry, efficient liquid
Phase chromatograph-mass spectrometer coupling technology, capillary electrophoresis-laser-induced fluorescence technology, unimolecule are sequenced in real time.These methods require to hold high
Expensive instrument and cumbersome operating procedure, the disadvantages of needing professional operator.Moreover, detecting 5hmC using the above method, very
It is easy the interference by 5mC.Therefore, establish it is simple, quick, with high sensitivity and highly selective method, realize to 5hmC
Detection be very necessary and urgent.
Photoelectrochemical assay is a kind of emerging analytical technology, has the advantages that electrochemical analysis and spectrochemical analysis.Its
Using phot-luminescence electroactive material, light induced electron and hole are generated.And light induced electron is then captured by electrode, generates electric current.Its
Excitation light source and detection signal are two kinds of entirely different forms, can be effectively reduced the interference of background signal in this way, thus
Greatly improve the sensitivity of analysis detection.Tungsten sulfide has good photoelectric activity and bio-compatibility, in excited by visible light
Under, it can produce photoelectron, form stable photoelectric current.There has been no the photoelectrochemical assay method detections based on tungsten sulfide at present
The report of 5hmC.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of detection 5-hydroxymethyl cytosine dezyribonucleosides
The Photoelectrochemistrbiosensor biosensor and preparation method thereof of acid realizes high specific and the high sensitivity detection to 5hmC.
To achieve the above object, the present invention adopts the following technical scheme that：
The first aspect of the present invention provides a kind of photoelectrochemical student for detecting 5-hydroxymethyl cytosine deoxyribonucleotide
Object sensor, including：Electrode successively modifies the tungsten sulfide in electrode surface, poly-dopamine, mercaptophenyl boronic acid, 5hmC, biotin
The phos-tag (phos-tag-biotin) and Streptavidin of functionalization.
Preferably, the electrode is ITO electrode.
The second aspect of the present invention provides the preparation method of above-mentioned Photoelectrochemistrbiosensor biosensor, includes the following steps：
(1) electrode is pre-processed；
(2) tungsten sulfide is modified in step (1) treated electrode surface；
(3) poly-dopamine is modified in step (2) treated electrode surface；
(4) mercaptophenyl boronic acid is modified in step (3) treated electrode surface；
(5) under the catalytic action of M.HhaI, by 5hmC modification to step (4) treated electrode surface；
(6) it is acted on using specific bond of the phos-tag-biotin to phosphate radical on 5hmC, by phos-tag-biotin
Modify step (5) treated electrode surface；
(7) it is acted on using the specific bond between biotin and Streptavidin, by Streptavidin modification to step (6)
Treated electrode surface, that is, be prepared Photoelectrochemistrbiosensor biosensor.
Preferably, in step (1), the pretreated method of electrode is：Electrode is first used to the mixed liquor of ethyl alcohol and sodium hydroxide
Ultrasonic cleaning, then cleaned with acetone and secondary water, it dries.
Preferably, in step (2), it is in the method for step (1) treated electrode surface by tungsten sulfide modification：It will vulcanization
Tungsten spends ion water making into tungsten sulfide dispersion liquid, and tungsten sulfide dispersant liquid drop is added to pretreated electrode surface, dry, then
Cleaning 3-5 times, is dried with nitrogen.
It is furthermore preferred that the concentration of the tungsten sulfide dispersion liquid is 0.1-5mg/mL.
Preferably, in step (3), it is in the method for step (2) treated electrode surface by poly-dopamine modification：It will gather
Dopamine spends ion water making into poly-dopamine dispersion liquid, and poly-dopamine dispersant liquid drop is added to step (2) treated electrode
Surface, it is dry, it cleans 3-5 times, is dried with nitrogen.
It is furthermore preferred that the concentration of the poly-dopamine dispersion liquid is 0.5-5mg/mL.
Preferably, in step (4), it is in the method for step (3) treated electrode surface by mercaptophenyl boronic acid modification：It will
Mercaptophenyl boronic acid solution is added drop-wise to step (3) treated electrode surface, reacts 1.5-4h under room temperature and wet condition；Then clear
It washes 3-5 times, is dried with nitrogen.
It is furthermore preferred that the concentration of the mercaptophenyl boronic acid solution is 2.3-6.8mM.
Application of the above-mentioned Photoelectrochemistrbiosensor biosensor in detection 5hmC is also protection scope of the present invention.
The third aspect of the present invention provides a kind of method using above-mentioned Photoelectrochemistrbiosensor biosensor detection 5hmC, packet
Include following steps：
Using above-mentioned Photoelectrochemistrbiosensor biosensor as working electrode, saturated calomel electrode is reference electrode, supplemented by Pt
It helps electrode composition three-electrode system to carry out electrochemical signals detection, it is slow for the Tris-HCl containing 0.05-0.28M KCl to detect liquid
Solution (pH 5.4-8.5) is rushed, the relationship between electric current and 5-hydroxymethyl cytosine nucleotide concentration is established, to 5- methylol born of the same parents
Pyrimidine nucleoside acid content is detected.
Preferably, detection method used is current-vs-time method, is -0.5-0.2V using current potential.
Preferably, the concentration of the Tris-HCl buffer solution is 5-40mM.
It should be noted that above-mentioned detection method is non-disease diagnostic method.In terms of non-disease diagnosis, inspection can be passed through
The content for surveying 5hmC, finds relevant targeted drug, provides new method for the exploitation of novel drugs.
Beneficial effects of the present invention：
(1) present invention electron donor property excellent using the good photoelectric activity of tungsten sulfide and poly-dopamine, realizes light
The amplification of electric signal improves the detection sensitivity of 5hmC.
(2) using the covalent reaction of methylol and sulfydryl on M.HhaI catalysis 5hmC, the specificity of 5hmC detection is improved.
(3) detection method of the invention is simple, realizes instrument miniaturization, and easily operated, only to ITO electrode surface into
The simple processing of row, can be realized the detection to 5hmC.
(4) present invention is acted on based on aptamer and 5hmC specific binding, has very high detecting and selecting property.
Detailed description of the invention
Fig. 1：The schematic diagram of 5hmC detection of the invention.
Fig. 2：The optical electro-chemistry response curve of the 5hmC of various concentration；The concentration that curve a-i is represented is respectively 100,50,
10, the 5hmC of 5,1,0.5,0.1,0.05,0.01nM.
Fig. 3：The linear fit curve of photoelectric current logarithm and 5hmC concentration.
Fig. 4：The histogram of optical electro-chemistry response variation under the conditions of different nucleotide.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
The range of " room temperature " in the present invention is 20-30 DEG C.
" wet condition " in the present invention is that humidity is greater than 90%；It is preferred that humidity is 95-99%.
The ingredient of cleaning solution used in the present invention is：3-15mM Tris-HCl and 20-60mM KCl, pH 7.4.
The ingredient of M.HhaI buffer used in the present invention is：20-60mM Tris-HCl, 5-30mM EDTA,
pH6.0-8.5。
The ingredient of the solid buffer liquid of phos-tag-biotin used in the present invention is：5-30mM Tris-HCl,
0.05-0.35M NaCl, 0.1-0.5%Tween-20,0.1-0.6mM Zn (NO3)2, pH 6.0-8.5.
The ingredient of the fixation buffer of Streptavidin used in the present invention is：4-18mM Tris-HCl and 8-
44mM NaCl, pH 6.0-8.5.
Detection liquid used in the present invention is：5-40mM Tris-HCl, 0.05-0.28M KCl, pH 5.4-8.5.
As background technology part is introduced, there are certain deficiencies for the detection method of 5hmC in the prior art, for example,
5hmC in method detection DNA chain based on DNA hybridization, but this method is limited by DNA detection probe sequence and length, Bu Nengshi
Now to the detection of the 5hmC on long chain DNA, moreover, this method can not achieve the detection to 5hmC content in genomic DNA；Using
PCR method detects 5hmC, and there are expensive equipment, design of primers is complicated, needs the problems such as fluorescent marker.Based on this, structure of the present invention
A kind of Photoelectrochemistrbiosensor biosensor for detecting 5hmC has been built, it can be to single using Photoelectrochemistrbiosensor biosensor of the invention
5hmC detected, it is applied widely, can also be achieved the detection to the 5hmC in genomic DNA.
The schematic diagram of Photoelectrochemistrbiosensor biosensor building and detection of the invention is shown in Fig. 1.Using ITO electrode as matrix electricity
Pole, using the electrostatic adsorption force between the oxygen-containing group and tungsten sulfide of ITO electrode surface, by tungsten sulfide modification to electrode surface.
The physisorption between tungsten sulfide and poly-dopamine is recycled, by poly-dopamine modification to electrode surface.Then, using poly-
The idiosyncrasy of phenyl boric acid in vicinal diamines structure and mercaptophenyl boronic acid on dopamine surface, by mercaptophenyl boronic acid modification to electrode
Surface.Under the catalytic action of M.HhaI, covalent reaction occurs for sulfydryl on the upper methylol of 5hmC and mercaptophenyl boronic acid, and 5hmC is caught
Receive electrode surface.At this point, the phosphate radical on 5hmC is far from electrode surface.Recycle phos-tag-biotin and phosphate radical
Combination, by phos-tag-biotin modification to electrode surface.Finally, utilizing the knot between biotin and Streptavidin
Cooperation is used, and Streptavidin is modified to the sensor prepared to electrode surface.Poly-dopamine is a kind of excellent electronics confession
Body material, can be obviously promoted the photoelectric respone of tungsten sulfide, to improve detection sensitivity.And when Streptavidin modification to electrode
Behind surface, photoelectric current can be substantially reduced, and this is mainly due to the big molecular structures of Streptavidin, hinder light induced electron
Migration.And what the modification amount of Streptavidin was determined by the concentration of 5hmC, therefore, using 5hmC concentration and photoelectric current it is linear
Relationship is, it can be achieved that detection to 5hmC.
In one embodiment of the invention, the building process of the Photoelectrochemistrbiosensor biosensor provided is：
(1) preparation of poly-dopamine：The dopamine for weighing 0.5-1g is dissolved in 300-600mL deionized water.Magnetic force stirs
It mixes down, is heated to 45-80 DEG C.Then, the NaOH solution that 2-4mL concentration is 0.5-2M is added.Stand reaction 3-8 hours.2000-
5000rpm centrifugation, collects suspension.Suspension 10000-15000rpm is centrifuged again, collects precipitating.Precipitating is washed with deionized water
After washing 3-5 times, it is dried in vacuo in 60 DEG C.
(2) preparation of tungsten sulfide dispersion liquid：5-250mg tungsten sulfide is weighed, is added in 50mL deionized water, ultrasonic disperse
1-3 hours.
(3) preparation of poly-dopamine dispersion liquid：10-100mg poly-dopamine is weighed, is added in 20mL deionized water, is surpassed
Sound disperses 1-3 hours.
(4) ITO electrode pre-processes：ITO electro-conductive glass is divided into 5 × 1cm2, then (compared with ethyl alcohol/NaOH mixed liquor
Example is 1:1-1:6) it is cleaned by ultrasonic 30-60 minutes, is finally respectively washed again 30-60 minutes with acetone and secondary water again, and in room
It is dried under temperature, for use.
(5) fixation of tungsten sulfide：25-76 μ L tungsten sulfide dispersant liquid drop is added to pretreated ITO electrode surface, infrared lamp
Irradiate drying.Then, electrode is cleaned 3-5 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are WS2/ITO。
(6) fixation of poly-dopamine：16-55 μ L poly-dopamine dispersant liquid drop is added to WS2/ ITO electrode surface, infrared lamp
Irradiate drying.Then, electrode is cleaned 3-5 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are PDA/WS2/ITO。
(7) mercaptophenyl boronic acid is fixed：By 23-54 μ L concentration be 2.3-6.8mM to mercaptophenyl boronic acid solution (to sulfydryl benzene
Boric acid solution is formulated by 10mM tris-HCl solution (pH8.0)) it is added drop-wise to PDA/WS2/ ITO electrode surface, room temperature and tide
It is reacted 1.5-4 hours under the conditions of wet.Then, electrode is cleaned 3-5 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are
MPBA/PDA/WS2/ITO。
(8) modification of 5hmC：12-47 μ L is contained the M.HhaI's of the 50-200unit/mL and 5hmC of various concentration
M.HhaI buffer is added drop-wise to MPBA/PDA/WS2/ ITO electrode surface is reacted 1.5-4 hours under 25-45 DEG C and wet condition.
Then, electrode is cleaned 3-5 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are 5hmC/MPBA/PDA/WS2/ITO。
(9) phos-tag-biotin is fixed：20-60 μ L is contained to the phos- of 10-40 μM of phos-tag-biotin
The fixed buffer of tag-biotin is added drop-wise to 5hmC/MPBA/PDA/WS2It is reacted under/ITO electrode surface, room temperature and wet condition
0.5-3 hours.Then, electrode is cleaned 3-5 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are Phos-tag/
5hmC/MPBA/PDA/WS2/ITO。
(10) fixation of Streptavidin：15-62 μ L is contained to the strepto- parent of the Streptavidin of 0.14-0.85mg/mL
Phos-tag/5hmC/MPBA/PDA/WS is added drop-wise to the fixed buffer of element2/ ITO electrode surface, 25-55 DEG C and wet condition
Lower reaction 0.5-3.5 hours.Then, electrode is cleaned 3-5 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are SA/
Phos-tag/5hmC/MPBA/PDA/WS2/ITO。
In the building process of above-mentioned optical electro-chemistry sensor, each step complements each other, and is sequentially considered critical, Mei Yibu
All it is the fixed modification service of next step, lacks previous step, may result in subsequent modification failure.Fixed modification is in ITO electricity
The material of pole surface can be commercial product, can also voluntarily be prepared, as long as meeting requirement in performance, the present invention
It is not particularly limited.
In another embodiment of the present invention, it gives using above-mentioned Photoelectrochemistrbiosensor biosensor detection 5hmC's
Process is：
(1) SA/Phos-tag/5hmC/MPBA/PDA/WS prepared with various concentration 5hmC2/ ITO electrode is as work
Electrode, saturated calomel electrode be reference electrode, Pt be auxiliary electrode group at three-electrode system carry out optical electro-chemistry signal detection,
Light source is visible light, is-0.8-0.2V using current potential, and in detection liquid, (5-40mM Tris-HCl, 0.05-0.28M KCl, pH are
Photoelectric current is recorded in 5.4-8.5).
(2) relationship between electric current and 5-hydroxymethyl cytosine nucleotide concentration is established, treats test sample using the relational expression
The content of 5hmC in product is detected.
With the increase of 5hmC concentration, the quantity of the Streptavidin of electrode surface also increases therewith, leads to photosignal
It gradually decreases.According to 5hmC concentration and the linear relationship of photoelectric current, it can be achieved that the detection to 5hmC.
Use Photoelectrochemistrbiosensor biosensor of the invention to the detection range of 5hmC for 0.01-100nM, detection is limited to
4.12pM。
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
The test material that test material is this field routine is not specifically described used in the embodiment of the present invention,
It can be commercially available by commercial channel.
Embodiment 1：The preparation of poly-dopamine
The dopamine for weighing 0.54g is dissolved in 300mL deionized water.Under magnetic agitation, it is heated to 50 DEG C.Then, add
Enter the NaOH solution that 2mL concentration is 1M.Stand reaction 5 hours.4000rpm centrifugation, collects suspension.Again by suspension
12000rpm centrifugation, collects precipitating.After precipitating is washed with deionized 3 times, it is dried in vacuo in 60 DEG C.
Embodiment 2：The preparation of tungsten sulfide dispersion liquid
12mg tungsten sulfide is weighed, is added in 3mL deionized water, ultrasonic disperse 1-3 hours.
Embodiment 3：The preparation of poly-dopamine dispersion liquid
12mg poly-dopamine is weighed, is added in 6mL deionized water, ultrasonic disperse 1-3 hours.
Embodiment 4：ITO electrode pretreatment
ITO electro-conductive glass is divided into 5 × 1cm2, then with ethyl alcohol/NaOH mixed liquor (ratio 1:1) it is cleaned by ultrasonic 45
Minute, it is finally respectively washed again 45 minutes with acetone and secondary water again, and dry at room temperature, for use.
Embodiment 5：The fixation of tungsten sulfide
40 μ L tungsten sulfide dispersant liquid drops are added to pretreated ITO electrode surface, infrared light irradiation is dry.It then, will be electric
Pole is cleaned 3 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are WS2/ITO。
Embodiment 6：The fixation of poly-dopamine
40 μ L poly-dopamine dispersant liquid drops are added to WS2/ ITO electrode surface, infrared light irradiation are dry.Then, by electrode
It is cleaned 3 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are PDA/WS2/ITO。
Embodiment 7：Mercaptophenyl boronic acid is fixed
It is that 5mM is added drop-wise to PDA/WS to mercaptophenyl boronic acid solution by 20 μ L concentration2/ ITO electrode surface, room temperature and moist item
It is reacted 2 hours under part.Then, electrode is cleaned 3 times with cleaning solution.It is dried with nitrogen.The electrode designations of preparation are MPBA/PDA/
WS2/ITO。
Embodiment 8：The modification of 5hmC
The M.HhaI buffer of the 5hmC of the 20 μ L M.HhaI and various concentration for containing 100unit/mL is added drop-wise to MPBA/
PDA/WS2/ ITO electrode surface is reacted 2 hours under 37 DEG C and wet condition.Then, electrode is cleaned 3 times with cleaning solution.Nitrogen
Drying.The electrode designations of preparation are 5hmC/MPBA/PDA/WS2/ITO。
Embodiment 9：Phos-tag-biotin is fixed
The fixed buffer of phos-tag-biotin that 20 μ L contain 20 μM of phos-tag-biotin is added drop-wise to 5hmC/
MPBA/PDA/WS2It is reacted 2 hours under/ITO electrode surface, room temperature and wet condition.Then, electrode is cleaned 3 with cleaning solution
It is secondary.It is dried with nitrogen.The electrode designations of preparation are Phos-tag/5hmC/MPBA/PDA/WS2/ITO。
Embodiment 10：The fixation of Streptavidin
The fixed buffer of the Streptavidin of the 20 μ L Streptavidin for containing 0.2mg/mL is added drop-wise to Phos-tag/
5hmC/MPBA/PDA/WS2/ ITO electrode surface is reacted 2 hours under 37 DEG C and wet condition.Then, electrode cleaning solution is clear
It washes 3 times.It is dried with nitrogen.The electrode designations of preparation are SA/Phos-tag/5hmC/MPBA/PDA/WS2/ITO。
Embodiment 11：Optical electro-chemistry detection
Using electrochemical workstation as signal acquisition instrument, 500W xenon lamp is that visible light source (installs the mirror of filtering ultraviolet additional
Piece), SA/Phos-tag/5hmC/MPBA/PDA/WS2/ ITO electrode is working electrode, and saturated calomel electrode is reference electrode, platinum
Column electrode is to electrode, and 10mM Tris-HCl (pH 7.4) buffer containing 0.1M KCl is detection liquid, with -0.3V voltage
For operating voltage, is studied using the detection that i-t technology carries out determinand, establish the relationship between photoelectric current and 5hmC concentration, line
Property range be 0.01-100nM, calibration curve be I (nA)=- 138.22logc (nM)+535.44 (R=0.9953), detection limit
For 4.12pM (Fig. 2 and Fig. 3).
Embodiment 12：The experiment of detecting and selecting property
For the specificity of the sensor of research and establishment, 6 kinds of deoxyribonucleotides are selected as contrast agent, and such as three
Deoxyadenosine triphosphate (dATP), dTTP (dTTP), deoxyguanosine triphosphate (dGTP), dCTP
(dCTP), 5-methylcytosine deoxyribose triphosphoric acid (5m-dCTP), N-6 methyl adenine deoxyribose triphosphoric acid (m6-
dATP).And different contrast agents are participated in photocurrent variations value (the Δ I=I of the sensor of building1-I2, I1It is MPBA/PDA/
WS2The current value of/ITO, I2It is MPBA/PDA/WS2Treated that electrode continues on through phos-tag- by different nucleotide by/ITO
The concentration of the photoelectric current flow valuve of biotin and Streptavidin treated electrode, contrast agent and 5hmC are 1nM) it carries out
Comparison.The result shows that contrast agent, which participates in building sensor current value, changes obvious low 5hmC, show that the sensor of building has
Specificity (Fig. 4) well.
Embodiment 13：Stability experiment
10 SA/Phos-tag/5hmC/MPBA/PDA/WS are prepared using identical method2/ ITO electrode (5hmC concentration
For 1nM), photoelectric current is detected in detection liquid.The relative standard deviation for obtaining photoelectric current is 4.22%, illustrates that this method has very well
Reproducibility.By SA/Phos-tag/5hmC/MPBA/PDA/WS2/ ITO sensor is stored 2 weeks at 4 DEG C, is examined in detection liquid
Optical electro-chemistry signal is surveyed, 92.17% that photocurrent response is original response is obtained, illustrates that this method has good stability.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (10)
1. a kind of Photoelectrochemistrbiosensor biosensor for detecting 5-hydroxymethyl cytosine deoxyribonucleotide, which is characterized in that packet
It includes：Electrode successively modifies the tungsten sulfide in electrode surface, poly-dopamine, mercaptophenyl boronic acid, 5hmC, biotin functionalized
Phos-tag and Streptavidin.
2. Photoelectrochemistrbiosensor biosensor according to claim 1, which is characterized in that the electrode is ITO electrode.
3. the preparation method of Photoelectrochemistrbiosensor biosensor of any of claims 1 or 2, which is characterized in that include the following steps：
(1) electrode is pre-processed；
(2) tungsten sulfide is modified in step (1) treated electrode surface；
(3) poly-dopamine is modified in step (2) treated electrode surface；
(4) mercaptophenyl boronic acid is modified in step (3) treated electrode surface；
(5) under the catalytic action of M.HhaI, by 5hmC modification to step (4) treated electrode surface；
(6) it is acted on using specific bond of the phos-tag-biotin to phosphate radical on 5hmC, phos-tag-biotin is modified
To step (5) treated electrode surface；
(7) it is acted on using the specific bond between biotin and Streptavidin, Streptavidin modification is handled to step (6)
Electrode surface afterwards, that is, be prepared Photoelectrochemistrbiosensor biosensor.
4. preparation method according to claim 3, which is characterized in that in step (1), the pretreated method of electrode is：It will
Electrode first uses the mixed liquor of ethyl alcohol and sodium hydroxide to be cleaned by ultrasonic, then is cleaned with acetone and secondary water, dries.
5. preparation method according to claim 3, which is characterized in that in step (2), tungsten sulfide is modified in step (1)
The method of treated electrode surface is：Tungsten sulfide is spent into ion water making at tungsten sulfide dispersion liquid, by tungsten sulfide dispersant liquid drop
It is added to pretreated electrode surface, it is dry, it then cleans 3-5 times, is dried with nitrogen；
Preferably, the concentration of the tungsten sulfide dispersion liquid is 0.1-5mg/mL.
6. preparation method according to claim 3, which is characterized in that in step (3), poly-dopamine is modified in step
(2) method of treated electrode surface is：Poly-dopamine is spent into ion water making at poly-dopamine dispersion liquid, by poly-dopamine
Dispersant liquid drop is added to step (2) treated electrode surface, dry, cleans 3-5 times, is dried with nitrogen；
Preferably, the concentration of the poly-dopamine dispersion liquid is 0.5-5mg/mL.
7. preparation method according to claim 3, which is characterized in that in step (4), mercaptophenyl boronic acid is modified in step
(3) method of treated electrode surface is：Mercaptophenyl boronic acid solution is added drop-wise to step (3) treated electrode surface, room
1.5-4h is reacted under mild wet condition；Then it cleans 3-5 times, is dried with nitrogen；
Preferably, the concentration of the mercaptophenyl boronic acid solution is 2.3-6.8mM.
8. application of the Photoelectrochemistrbiosensor biosensor of any of claims 1 or 2 in detection 5hmC.
9. a kind of method using the detection of Photoelectrochemistrbiosensor biosensor described in as claimed in claim 1 or 22 5hmC, which is characterized in that
Include the following steps：
Using Photoelectrochemistrbiosensor biosensor of any of claims 1 or 2 as working electrode, saturated calomel electrode is reference electricity
Pole, Pt carry out electrochemical signals detection at three-electrode system for auxiliary electrode group, and detection liquid is to contain 0.05-0.28MKCl's
Tris-HCl buffer solution (pH 5.4-8.5), establishes the relationship between electric current and 5-hydroxymethyl cytosine nucleotide concentration, right
5-hydroxymethyl cytosine nucleotide content is detected.
10. according to the method described in claim 9, it is characterized in that：Detection method used is current-vs-time method, using current potential
For -0.5-0.2V.
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