CN109709170B - A method of the photoelectrochemical assay based on black titanium dioxide detects 5-hydroxymethyl cytosine - Google Patents
A method of the photoelectrochemical assay based on black titanium dioxide detects 5-hydroxymethyl cytosine Download PDFInfo
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Abstract
The method for the photoelectrochemical assay detection 5-hydroxymethyl cytosine that the invention discloses a kind of based on black titanium dioxide, the present invention construct the Photoelectrochemistrbiosensor biosensor of detection 5hmC first, comprising: electrode is sequentially fixed at the black TiO of electrode surface2, DNA probe, 5hmC, p-aminophenyl boric acid, PAMAM and amido modified zinc oxide.The present invention utilizes black TiO2Photoelectric properties, glycosyl transferase the signal of the catalysis of the specificity of 5-hydroxymethyl cytosine, the effect of PAMAM amplification of signal and ZnO is quenched, realize the Sensitive Detection to 5-hydroxymethyl cytosine.Detection method of the invention is easy to operate, is easy to implement miniaturization, only modifies ITO electrode surface the detection that can be realized to 5-hydroxymethyl cytosine.
Description
Technical field
The present invention relates to photoelectrochemical assay technical fields, and in particular to a kind of optical electro-chemistry based on black titanium dioxide
The method of analysis detection 5-hydroxymethyl cytosine.
Background technique
5-hydroxymethyl cytosine (5hmC) is an important epigenetic modification object, is 5-methylcytosine (5mC) quilt
What the enzymatic oxidation of TET family generated, the referred to as hexabasic base of DNA.5hmC is found in bacteriophage early in nineteen fifty-two, but
It is not cause enough attention at that time.Until 2009, researcher had found that 5hmC is dry thin in mouse, National People's Congress's brain and embryo
There is abundant expression just to attract people's attention in born of the same parents.Epigenetic marker 5hmC important as one is living in many life
Play a role in dynamic, such as adjust gene activity, genetic recombination, cell differentiation etc..And research discovery 5hmC is in cancer cell
Content be substantially reduced, therefore realize be of great significance to the detection of 5hmC.It is existing but since 5hmC with 5mC structure is similar
Some biological detecting methods are difficult to separate the two.
The method of currently used detection 5hmC has the real-time PCR sequencing PCR of unimolecule, thin-layered chromatography, efficient liquid phase-mass spectrum connection
With, capillary electrophoresis-laser-induced fluorescence technology etc., but the generally existing expensive equipment of these methods, it is complicated for operation, need professional
The disadvantages of operation.Therefore, it realizes particularly significant to the quick, simple of 5hmC, Sensitive Detection.
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.Black titanium dioxide is a kind of novel visible light catalytic material, due to its uniqueness
The nuclei of crystallization/lattice disorder shell structurre, its electronic structure is improved, and surface-active enhancing not only has excellent
Absorbing properties, also have excellent electrical properties, this makes it have a wide range of applications in the energy and environmental area.But mesh
It is preceding that there has been no the reports of the photoelectrochemical assay method detection 5hmC based on black titanium dioxide.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of photoelectrochemical assays based on black titanium dioxide
The method for detecting 5-hydroxymethyl cytosine realizes quick, simple and Sensitive Detection to 5hmC.
To achieve the above object, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of Photoelectrochemistrbiosensor biosensor for detecting 5hmC, comprising: electrode, successively
It is fixed on the black TiO of electrode surface2, DNA probe, the complementary dna chain containing 5hmC, amino phenyl boric acid, polyamidoamine tree
Dendritic macromolecule (PAMAM) and amido modified zinc oxide.
The base complementrity of the DNA probe and the complementary dna chain.
Preferably, the electrode is ITO electrode.
Preferably, the nucleotide sequence of the DNA probe are as follows:
Probe DNA sequence dna: 5 '-CGC GCG TAC ATC GGC CAC ATC T-P3O4-3'(SEQ ID NO.1)；
The nucleotide sequence of the complementary dna chain are as follows:
5’-AGA TGT GGC CGA TGT ACG ChmGC G-3’(SEQ ID NO.2)。
The second aspect of the present invention provides the preparation method of above-mentioned Photoelectrochemistrbiosensor biosensor, comprising the following steps:
(1) electrode is pre-processed；
(2) by black TiO2It is fixed on step (1) treated electrode surface；
(3) DNA probe is fixed on step (2) treated electrode surface；
(4) 5hmC is fixed on step (3) treated electrode surface；
(5) under the catalytic action of glycosyl transferase, glycosyl is introduced into step (4) treated electrode surface；
(6) reacting using o-dihydroxy and the amino phenyl boric acid on glycosyl, by p-aminophenyl boric acid modified to step (5)
Treated electrode surface；
(7) reacting using amino and carboxyl, by PAMAM modification to step (6) treated electrode surface；
(8) ZnO for modifying amino is fixed to step (7) treated electrode surface by reacting using amino and carboxyl,
The biosensor prepared.
Preferably, in step (1), the pretreated method of electrode are as follows: by electrode ethyl alcohol-sodium hydroxide mixed liquor, acetone
It is cleaned by ultrasonic 30-60min respectively with secondary water, dries.It is furthermore preferred that in the ethyl alcohol-sodium hydroxide mixed liquor, ethyl alcohol and hydrogen
The mass ratio of sodium oxide molybdena is 1:1-1:4.
Biggish overpotential is generally had on not pretreated electrode, is increased so as to cause slow in reacting, energy consumption.In order to
The advantage for playing electrode, improves the activity of electrode, needs to pre-process electrode surface.It is pre-processed using electrode of the invention
The electrode overpotential that method can reduce, to effectively improve the activity of electrode.
Preferably, in step (2), the black TiO2It is prepared by the following method:
By white TiO2Be by weight with sodium borohydride (0.5-2): (0.5-2) is ground, 200-400 DEG C of nitrogen atmosphere
Lower calcining 0.5-2h is enclosed, obtains black solid, washing, drying are to get black TiO2。
Preferably, in step (2), by black TiO2Method fixed to pretreated electrode surface are as follows:
By black TiO2It is add to deionized water, ultrasonic disperse, black TiO is made2Dispersion liquid, by black TiO2Dispersion
Drop is added to pretreated electrode surface, drying under infrared light irradiation.
It is furthermore preferred that the black TiO2And the ratio of deionized water additional amount is (10-20) mg:(3-10) mL.
It is furthermore preferred that the time of ultrasonic disperse is 1-3h.
Preferably, in step (3), method that DNA probe is fixed on step (2) treated electrode surface are as follows:
Probe fixer comprising DNA probe is added drop-wise to step (2) treated electrode surface, in 37 DEG C of wet conditions
Lower hatching 1-4h.
It is furthermore preferred that containing in the probe fixer: 3-15mM Tris-HCl, 1-5mM EDTA, 1-5M NaCl, 1-
5mM TCEP；pH 6.0-8.5.
Preferably, in step (4), 5hmC is fixed on step (3) treated the method for electrode surface are as follows:
The 10-40 μ L DNA hybridization drop for including 5hmC is added to step (4) treated ITO electrode surface, is put into hatching
Hatch 1-4h under 37 DEG C of wet conditions in case.
Preferably, contain in the DNA hybridization liquid: 3-15mM Tris-HCl, 1-5mM EDTA, 1-5M NaCl；pH
6.0-8.5。
Preferably, in step (5), method that glycosyl is introduced into step (4) treated electrode surface are as follows:
Glycosyl transferase reaction solution comprising glycosyl transferase is added drop-wise to step (4) treated electrode surface, in 37
Hatch 1-4h under DEG C wet condition.
It is furthermore preferred that containing in the glycosyl transferase reaction solution: 3-15mM Tris-HCl, 10-100mM NaCl, 5-
20mM MgCl2,0.5-4mM DTT；pH 6.0-8.5.
Preferably, in step (6), by the method for p-aminophenyl boric acid modified to step (5) treated electrode surface are as follows:
P-aminophenyl boric acid solution is added drop-wise to step (5) treated electrode surface, is hatched under 37 DEG C of wet conditions
1-4h。
Preferably, in step (7), by the method for PAMAM modification to step (6) treated electrode surface are as follows:
It will be added drop-wise to step (6) to PAMAM solution treated electrode surface, hatch 1-4h under 37 DEG C of wet conditions.
Preferably, in step (8), by the method for the ZnO for the modifying amino electrode surface that is fixed to that step (7) treated
Are as follows:
The ZnO dispersant liquid drop for modifying amino is added to step (7) treated electrode surface, is incubated under 37 DEG C of wet conditions
Change 1-4h.
It is furthermore preferred that the ZnO of the modification amino is prepared by the following method:
Zinc acetate is added in dehydrated alcohol, is stirred evenly, NaOH solution is added under agitation, in 120-180
4-8h is reacted under the conditions of DEG C, obtains the first product；
First product is washed, is then added in NaOH solution, 60-90 DEG C is stirred at reflux reaction 15-25h, obtains the
Two products；
Second product is washed, is then added in ethanol-water mixture, APTES is added under agitation, is flowed back
10-25h, by reaction product washing, it is dry to get.
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
Electrode composition three-electrode system is helped to carry out optical electro-chemistry signal detection, the Tris-HCl containing 0.01-1M ascorbic acid (AA) is slow
Solution (pH 6.0-8.5) is rushed, the relationship between electric current and 5hmC concentration is established, 5hmC content is detected.
Preferably, detection method used is current-vs-time method, is -0.5-0.5V using current potential.
Preferably, the concentration of the Tris-HCl buffer solution is 10-100mM.
It should be noted that above-mentioned detection method can be used in non-disease diagnosis aspect, it can containing by detection 5hmC
Amount, finds relevant targeted drug, provides new method for the exploitation of novel drugs.
Beneficial effects of the present invention:
(1) present invention utilizes black TiO2Good photoelectric activity and ZnO are to black TiO2The quenching effect of photosignal,
Realize the Sensitive Detection to 5hmC.
(2) expand the quantity for introducing signaling molecule using PAMAM dendrimer, improve the detection sensitivity of 5hmC.
(3) it is acted on using specific catalytic of the glycosyl transferase to methylol on 5hmC, improves the specificity of 5hmC detection.
(4) 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.
Detailed description of the invention
Fig. 1: the schematic diagram of 5hmC detection of the invention.
Fig. 2: the 5hmC of various concentration optical electro-chemistry response curve；The concentration that curve a-i is represented is respectively 0.05,0.1,
0.5, the 5hmC of 1,5,10,50,100,200nM.
Fig. 3: the linear fit curve of photoelectric current logarithm and 5hmC concentration.
Fig. 4: the histogram of the 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 are as follows: 3-15mM Tris-HCl and 20-60mM KCl, pH 6.0-
8.5。
The ingredient of probe fixer used in the present invention are as follows: 3-15mM Tris-HCl, 1-5mM EDTA, 1-5M
NaCl, 1-5mM TCEP, pH 6.0-8.5.
The ingredient of DNA hybridization liquid used in the present invention are as follows: 3-15mM Tris-HCl, 1-5mM EDTA, 1-5M
NaCl, pH 6.0-8.5.
The ingredient of glycosyl transferase reaction solution used in the present invention are as follows: 3-15mM Tris-HCl, 10-100mM
NaCl、5-20mM MgCl2, 0.5-4mM DTT, pH 6.0-8.5.
Detection liquid used in the present invention are as follows: 10-100mM Tris-HCl, 0.01-1M AA, pH 6.0-8.5.
As background technology part is introduced, there are expensive equipments for existing 5hmC detection method, complicated for operation, need special
The disadvantages of industry personnel operate, it is difficult to realize quick, the Sensitive Detection to 5hmC.
Based on this, the object of the present invention is to provide it is a kind of new can quickly, the method for Sensitive Detection 5hmC.
Black TiO2The energy and environmental area are applied to properties such as its unique optics, electrochemistry.But it utilizes black
Color TiO2Photoelectric activity detect 5hmC, yet there are no and have been reported that.
The present invention constructs a kind of based on black TiO for the first time2The optical electro-chemistry sensor of photoelectric activity, utilizes black TiO2
Photoelectric properties, glycosyl transferase is to the catalysis of the specificity of 5-hydroxymethyl cytosine, the effect of PAMAM amplification of signal and ZnO
Signal quenching, realizes quick, the Sensitive Detection to 5-hydroxymethyl cytosine.
The building process and testing principle of optical electro-chemistry sensor of the invention are as shown in Figure 1.First by black TiO2
It modifies in electrode surface, utilizes black TiO2Excellent photoelectric activity generates a stable photoelectric current.Followed by phosphate
Group and TiO2Reaction the DNA probe of one terminal modified phosphate group is fixed on electrode surface.Then, base pair complementarity is utilized
5hmC is fixed on electrode surface.Then, glycosyl is introduced using specific catalytic of the glycosyl transferase to 5hmC, using on glycosyl
O-dihydroxy and aminobenzene acid reaction, introduce electrode surface for amino.Finally, successively will using amino with reacting for carboxyl
PAMAM and amido modified ZnO introduce electrode surface.Due to ZnO and black TiO2Competition, which absorbs light and free electron, to be caused
Photosignal reduces.Since the concentration of 5hmC determines the quantity of introducing ZnO.Therefore, the line of the concentration based on 5hmC and photoelectric current
Sexual intercourse is, it can be achieved that Sensitive Detection to 5hmC.
In one embodiment of the invention, the building process of the Photoelectrochemistrbiosensor biosensor provided are as follows:
(1) ITO electrode pre-processes: ITO electro-conductive glass is divided into 1 × 5cm2, then (compared with ethyl alcohol/NaOH mixed liquor
Example is 1:1-1:4), acetone and secondary water are cleaned by ultrasonic 30-60 minutes respectively, and dry at room temperature, for use.
(2) black TiO2Preparation: weigh 0.5-2g white TiO2, 0.5-2g sodium borohydride is ground in mortar.It will
Mixture is put into porcelain boat, calcines 0.5-2h under 200-400 DEG C of nitrogen atmosphere.It obtains black solid and uses deionized water and nothing respectively
Water-ethanol is washed three times, and 60 DEG C of vacuum drying are spare.
(3) it the preparation of amido modified zinc oxide: weighs 0.1-1g zinc acetate and is added in 50mL dehydrated alcohol, stir evenly.
Then, 10-30mL 0.5M NaOH solution is added under stirring state.Then above-mentioned solution is transferred to 120-180 in reaction kettle
DEG C reaction 4-8h.Product deionized water and dehydrated alcohol are washed three times, then weigh the above-mentioned product of 0.5-1.5g and 100mL is added
60-90 DEG C of reflux 15-25h is stirred in 0.5M NaOH solution.Product is added in the mixed solution of second alcohol and water after washing three times
(1:1-1:6).Under stirring state, 3-5mL APTES is added and then flows back 10-25h.Finally, product washes three times and 60 DEG C very
Empty drying for standby.
The amido modified zinc oxide of preparation is add to deionized water, ultrasonic disperse, is prepared amido modified ZnO points
Dispersion liquid, in the amido modified ZnO dispersion liquid, the concentration of amido modified ZnO is 0.5-6mg/mL.
(4) black TiO2The preparation of dispersion liquid: 10-20mg black TiO is weighed2, it is added in 3-10mL deionized water, surpasses
Sound disperses 1-3 hours.
(5) black TiO2Fixation: by 10-40 μ L black TiO2Dispersant liquid drop is added to pretreated electrode surface, red
It is dried under outer lamp.The electrode designations of preparation are TiO2/ITO。
(6) the 10-40 μ L probe fixer for including 1 μM of DNA probe the fixation of DNA probe: is added drop-wise to TiO2/ ITO electricity
Pole surface is put into 37 DEG C of wet environment hatching 1-4h in incubator.Then, by electrode washing 3-5 times.The electrode designations of preparation are
ssDNA/TiO2/ITO。
(7) the 10-40 μ L DNA hybridization drop for including the DNA complementary strand containing 5hmC the fixation of 5hmC: is added to ssDNA/
TiO2/ ITO electrode surface is put into 37 DEG C of wet environment hatching 1-4h in incubator.Then, by electrode washing 3-5 times.Preparation
Electrode designations are 5hmC-dsDNA/TiO2/ITO。
(8) glycosylation of 5hmC: including the glycosyl transferase reaction solution (60unit/mL) of glycosyl transferase by 10-40 μ L
It is added drop-wise to 5hmC-dsDNA/TiO2/ ITO electrode surface is put into 37 DEG C of wet environment hatching 1-4h in incubator.It then, will be electric
Pole is rinsed 3-5 times.The electrode designations of preparation are Glu/5hmC-dsDNA/TiO2/ITO。
(9) 10-40 μ L p-aminophenyl boric acid solution (1 μM) fixation of p-aminophenyl boric acid: is added drop-wise to Glu/5hmC-
dsDNA/TiO2/ ITO electrode surface is put into 37 DEG C of wet environment hatching 1-4h in incubator.Then, by electrode washing 3-5 times.
The electrode designations of preparation are M-APBA/Glu/5hmC-dsDNA/TiO2/ITO。
(10) 10-40 μ L PAMAM solution (10wt.%in methanol) fixation of PAMAM: is added drop-wise to M-APBA/
Glu/5hmC-dsDNA/TiO2/ ITO electrode surface is put into 37 DEG C of wet environment hatching 1-4h in incubator.Then, by electrode
It rinses 3-5 times.The electrode designations of preparation are PAMAM/M-APBA/Glu/5hmC-dsDNA/TiO2/ITO。
(11) the amido modified ZnO dispersion liquid (0.5-6mg/mL) of 10-40 μ L the fixation of ZnO: is added drop-wise to PAMAM/M-
APBA/Glu/5hmC-dsDNA/TiO2/ ITO electrode surface is put into 37 DEG C of wet environment hatching 1-4h in incubator.Then, will
Electrode washing 3-5 times.The electrode designations of preparation are ZnO/PAMAM/M-APBA/Glu/5hmC-dsDNA/TiO2/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 are as follows:
(1) ZnO/PAMAM/M-APBA/Glu/5hmC-dsDNA/TiO is prepared with the 5hmC of various concentration2/ ITO electrode,
And using it as working electrode, saturated calomel electrode, platinum filament form three-electrode system respectively as reference electrode and auxiliary electrode.
It is -0.5-0.5V using current potential using 500W xenon lamp as light source, detection liquid is 10-100mM Tris-HCl, 0.01-1M AA
(pH6.0-8.5) photoelectric current detection is carried out；
(2) relationship between electric current and 5hmC concentration is established, using the relational expression to the content of the 5hmC in sample to be tested
It is detected.
With the increase of 5hmC concentration, the ZnO quantity of electrode surface increases, and photo-signal is caused to reduce.According to 5hmC
Concentration and the linear relationship of electric current are, it can be achieved that detection to 5hmC.
It should be noted that Photoelectrochemistrbiosensor biosensor of the invention is mainly used for detecting in nucleotide sample
The content of 5hmC.On the basis of obtaining the sequence of the nucleotide sample containing 5hmC, according to base pair complementarity principle, design
DNA probe constructs Photoelectrochemistrbiosensor biosensor of the invention, to realize the detection to 5hmC content in nucleotide sample.
Photoelectrochemistrbiosensor biosensor of the invention is 0.05-200nM to the detection range of 5hmC, and detection is limited to
2.12pM。
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: black TiO2Preparation
Weigh 1g white TiO2, 1g sodium borohydride is ground in mortar.Mixture is put into porcelain boat, 250 DEG C of nitrogen
Atmosphere encloses lower calcining 1h.It obtains black solid to be washed three times with deionized water and dehydrated alcohol respectively, 60 DEG C of vacuum drying are spare.
Embodiment 2: the preparation of amido modified zinc oxide
It weighs 0.4g zinc acetate to be added in 50mL dehydrated alcohol, stir evenly.Then, 25mL is added under stirring state
0.5M NaOH solution.Then above-mentioned solution is transferred to 150 DEG C of reaction 6h in reaction kettle.Product deionized water and anhydrous second
Alcohol is washed three times, then weighs the above-mentioned product of 1g and 70 DEG C of reflux 20h of stirring in 100mL 0.5M NaOH solution are added.Product washing
It is added after three times in the mixed solution of second alcohol and water (1:1).Under stirring state, 3mL APTES is added and then flows back 20h.Most
Afterwards, product is washed three times and 60 DEG C of vacuum drying are spare.
The pretreatment of embodiment 3:ITO electrode
ITO electro-conductive glass is divided into 1 × 5cm2, then use ethyl alcohol/NaOH mixed liquor (1:1), acetone and secondary moisture
Qing Xi not be 45 minutes, and dry at room temperature, for use.
Embodiment 4: black TiO2Fixation
Black TiO2The preparation of dispersion liquid: 12mg black TiO is weighed2Solid is added in 3mL deionized water, ultrasonic disperse
2 hours, obtain black TiO2Dispersion liquid.
By 40 μ L black TiO2Dispersant liquid drop is added to pretreated ITO electrode (1 × 5cm2) surface, infrared light irradiation is dry
It is dry.The electrode designations of preparation are TiO2/ITO。
The fixation of embodiment 5:5hmC
The 20 μ L probe fixer for including 1 μM of DNA probe (shown in SEQ ID NO.1) is added drop-wise to TiO2/ ITO electrode table
Face is put into 37 DEG C of wet environment hatching 2h in incubator.Then, by electrode washing 3 times.It then include to contain 5hmC's by 20 μ L
The DNA hybridization drop of DNA complementary strand (shown in SEQ ID NO.2) is added to electrode surface, is put into 37 DEG C of wet environments in incubator
Hatch 2h.Then, by electrode washing 3 times.The electrode designations of preparation are 5hmC-dsDNA/TiO2/ITO。
The glycosylation of embodiment 6:5hmC
The 40 μ L glycosyl transferase reaction solution (60unit/mL) for including glycosyl transferase is added drop-wise to 5hmC-dsDNA/
TiO2/ ITO electrode surface is put into 37 DEG C of wet environment hatching 2h in incubator.Then, by electrode washing 3 times.The electrode of preparation
Labeled as Glu/5hmC-dsDNA/TiO2/ITO。
Embodiment 7: the fixation of amido modified ZnO
40 μ L p-aminophenyl boric acid solutions (1 μM) are added drop-wise to Glu/5hmC-dsDNA/TiO2/ ITO electrode surface, is put into
37 DEG C of wet environments hatch 2h in incubator.Then, by electrode washing 3 times.Then by 40 μ L PAMAM solution (10wt.%in
Methanol it) is added drop-wise to electrode surface, is put into 37 DEG C of wet environment hatching 2h in incubator.Then, by electrode washing 3 times.Most
Afterwards, the amido modified ZnO dispersant liquid drop of 40 μ L is added to electrode surface, is put into 37 DEG C of wet environment hatching 2h in incubator.Then,
By electrode washing 3 times.The electrode designations of preparation are ZnO/PAMAM/M-APBA/Glu/5hmC-dsDNA/TiO2/ITO。
Embodiment 8: optical electro-chemistry detection
With ZnO/PAMAM/M-APBA/Glu/5hmC-dsDNA/TiO2/ ITO electrode, saturated calomel electrode, platinum electrode
Respectively working electrode, reference electrode, auxiliary electrode, 10mM Tris-HCl (pH 7.4) buffer containing 0.1M AA are inspection
Liquid is surveyed, using -0.3V voltage as operating voltage, 500W xenon lamp is visible light source (eyeglass for installing filtering ultraviolet additional) in electrochemistry work
It stands upper progress photo-signal acquisition.Establish the relationship between photoelectric current and 5hmC concentration, range of linearity 0.05-200nM,
Calibration curve is I (nA)=- 21.81logc (nM)+173.89 (R=0.9944), and detection is limited to 2.12pM (Fig. 2 and Fig. 3).
Embodiment 9: selective enumeration method
Selectivity is an important indicator of optical electro-chemistry sensor performance, therefore we select 5-methylcytosine
(5mC) and N-6 methyl adenine (m6A) are studied as selectivity of the chaff interferent to sensor.And to disturbance reagent
Participate in photocurrent variations value (the Δ I=I of the sensor of building1-I2, I1It is ssDNA/TiO2The current value of/ITO, I2It is ssDNA/
TiO2/ ITO is by treated electrode continues on through glycosyl transferase, p-aminophenyl boric acid, PAMAM, the ZnO processing of disturbance object
The concentration of the photoelectric current flow valuve of electrode afterwards, chaff interferent and 5hmC are 1nM) it is compared.The result shows that chaff interferent participates in
It constructs sensor current value and changes obvious low 5hmC, show that the sensor of building has specific well (Fig. 4).
Embodiment 10: stability experiment
10 ZnO/PAMAM/M-APBA/Glu/5hmC-dsDNA/TiO are prepared using identical method2/ ITO electrode, so
Photo-signal detection is carried out in the 10mM Tris-HCl buffer solution (pH7.4) containing 0.1M AA afterwards, obtains electric current
Relative standard deviation is 4.32%, illustrates that this method has good reproducibility.By ZnO/PAMAM/M-APBA/Glu/5hmC-
dsDNA/TiO2/ ITO sensor is stored 2 weeks at 4 DEG C, then carries out photoelectric current detection, and obtaining current-responsive is original response
92.56%, illustrate 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.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>a kind of method of the photoelectrochemical assay detection 5-hydroxymethyl cytosine based on black titanium dioxide
<130> 2019
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
cgcgcgtaca tcggccacat ct 22
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
agatgtggcc gatgtacgcg cg 22
Claims (15)
1. a kind of Photoelectrochemistrbiosensor biosensor for detecting 5hmC characterized by comprising electrode is sequentially fixed at electrode table
The black TiO in face2, DNA probe, the complementary dna chain containing 5hmC, amino phenyl boric acid, PAMAM and amido modified zinc oxide；
The base complementrity of the DNA probe and the complementary dna chain；
For the nucleotide sequence of the DNA probe as shown in SEQ ID NO.1, the 3 ' of DNA probe are terminal modified phosphate group；
The nucleotide sequence of the complementary dna chain containing 5hmC is as shown in SEQ ID NO.2.
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 comprises the following steps:
(1) electrode is pre-processed；
(2) by black TiO2It is fixed on step (1) treated electrode surface；
(3) DNA probe is fixed on step (2) treated electrode surface；
(4) 5hmC is fixed on step (3) treated electrode surface；
(5) under the catalytic action of glycosyl transferase, glycosyl is introduced into step (4) treated electrode surface；
(6) reacting using o-dihydroxy and amino phenyl boric acid, by p-aminophenyl boric acid modified to step (5) treated electrode
Surface；
(7) reacting using amino and carboxyl, by PAMAM modification to step (6) treated electrode surface；
(8) reacting using amino and carboxyl, by the ZnO for modifying amino be fixed to step (7) treated electrode surface to get
To the biosensor of preparation.
4. preparation method according to claim 3, which is characterized in that in step (1), the pretreated method of electrode are as follows: will
Electrode ethyl alcohol-sodium hydroxide mixed liquor, acetone and secondary water is cleaned by ultrasonic 30-60min respectively, dries.
5. the preparation method according to claim 4, which is characterized in that in the ethyl alcohol-sodium hydroxide mixed liquor, ethyl alcohol and
The mass ratio of sodium hydroxide is 1:1-1:4.
6. preparation method according to claim 3, which is characterized in that in step (2), by black TiO2Fixed to pretreatment
The method of electrode surface afterwards are as follows:
By black TiO2It is add to deionized water, ultrasonic disperse, black TiO is made2Dispersion liquid, by black TiO2Dispersant liquid drop
It is added to pretreated electrode surface, drying under infrared light irradiation；
The black TiO2And the ratio of deionized water additional amount is (10-20) mg:(3-10) mL；
The black TiO2It is prepared by the following method:
By white TiO2Be by weight with sodium borohydride (0.5-2): (0.5-2) is ground, under 200-400 DEG C of nitrogen atmosphere
0.5-2h is calcined, obtains black solid, washing, drying are to get black TiO2。
7. preparation method according to claim 6, which is characterized in that the time of ultrasonic disperse is 1-3h.
8. preparation method according to claim 3, which is characterized in that in step (3), DNA probe is fixed on step (2)
The method of treated electrode surface are as follows:
Probe fixer comprising DNA probe is added drop-wise to step (2) treated electrode surface, is incubated under 37 DEG C of wet conditions
Change 1-4h；
Contain in the probe fixer: 3-15mM Tris-HCl, 1-5mM EDTA, 1-5M NaCl, 1-5mM TCEP；pH
6.0-8.5。
9. preparation method according to claim 3, which is characterized in that in step (4), 5hmC is fixed at step (3)
The method of electrode surface after reason are as follows:
The 10-40 μ L DNA hybridization drop for including 5hmC is added to step (4) treated ITO electrode surface, is put into incubator
Hatch 1-4h under 37 DEG C of wet conditions；
Contain in the DNA hybridization liquid: 3-15mM Tris-HCl, 1-5mM EDTA, 1-5M NaCl；pH 6.0-8.5.
10. preparation method according to claim 3, which is characterized in that in step (5), glycosyl is introduced at step (4)
The method of electrode surface after reason are as follows:
Glycosyl transferase reaction solution comprising glycosyl transferase is added drop-wise to step (4) treated electrode surface, in 37 DEG C of tides
Hatch 1-4h under the conditions of wet；
Contain in the glycosyl transferase reaction solution: 3-15mM Tris-HCl, 10-100mM NaCl, 5-20mM Mg Cl2、
0.5-4mM DTT；pH 6.0-8.5.
11. preparation method according to claim 3, which is characterized in that in step (8), the ZnO for modifying amino is fixed to
The method of step (7) treated electrode surface are as follows:
The ZnO dispersant liquid drop for modifying amino is added to step (7) treated electrode surface, hatches 1- under 37 DEG C of wet conditions
4h；
The ZnO of the modification amino is prepared by the following method:
Zinc acetate is added in dehydrated alcohol, is stirred evenly, NaOH solution is added under agitation, in 120-180 DEG C of item
4-8h is reacted under part, obtains the first product；
First product is washed, is then added in NaOH solution, 60-90 DEG C is stirred at reflux reaction 15-25h, obtains the second production
Object；
Second product is washed, is then added in ethanol-water mixture, APTES is added under agitation, flow back 10-
25h, by reaction product washing, it is dry to get.
12. application of the Photoelectrochemistrbiosensor biosensor of any of claims 1 or 2 in detection 5hmC.
13. 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
The following steps are included:
Using Photoelectrochemistrbiosensor biosensor of any of claims 1 or 2 as working electrode, saturated calomel electrode is reference electricity
Pole, Pt carry out optical electro-chemistry signal detection, the Tris-HCl containing 0.01-1M AA at three-electrode system for auxiliary electrode group
Buffer solution, pH 6.0-8.5 establish the relationship between electric current and 5hmC concentration, detect to 5hmC content.
14. according to the method for claim 13, which is characterized in that detection method used is current-vs-time method, using current potential
For -0.5-0.5V.
15. according to the method for claim 13, which is characterized in that the concentration of the Tris-HCl buffer solution is 10-
100mM。
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