CN104611419A - DNA helicase detection method based on graphene oxide chip - Google Patents
DNA helicase detection method based on graphene oxide chip Download PDFInfo
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- CN104611419A CN104611419A CN201410832817.5A CN201410832817A CN104611419A CN 104611419 A CN104611419 A CN 104611419A CN 201410832817 A CN201410832817 A CN 201410832817A CN 104611419 A CN104611419 A CN 104611419A
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Abstract
The invention belongs to protein detection in the biomedical field, and in particular provides a DNA helicase detection method based on a graphene oxide chip. The DNA helicase detection method based on the graphene oxide chip comprises the following steps: (1), preparing a slide with graphene oxide dot matrixes; (2), connecting a double-chain DNA sequence with the slide with the graphene oxide dot matrixes; (3) detecting DNA helicase, to be specific, adding different quantities of DNA helicases at different action time, performing PBS washing to scan and analyze, and detecting according to FAM fluorescence intensity change. According to the DNA helicase detection method based on the graphene oxide chip, fluorescene on FAM marked by a single-stranded DNA can be quenched by utilizing a graphene oxide; after the DNA helicase is applied to a double-stranded DNA, a single-stranded DNA separates from the complementary single-stranded DNA fixed on the graphene oxide, and the distance between the FAM and the surface of the graphene oxide is changed, so that the change of FAM fluorescence intensity is caused; by combining a high-throughput microarray chip technique, the DNA helicase can be detected with low cost and high sensitivity.
Description
Technical field
The invention belongs to the protein detection in biomedical sector, especially a kind of DNA helicase detection method based on biochip.
Background technology
DNA helicase is a kind of protein, utilizes Triphosaden (ATP) to be hydrolyzed the energy provided and opens complementary nucleic acid double chain, obtain strand, all play an important role in the metabolic processes such as copying, repair, recombinate and transcribe of DNA.They usually depend on the existence of strand, and can identify the single-stranded structure of replication fork.The general effect played catalysis double-stranded DNA and untwist in DNA replication dna process, this needs there is very important effect in the reaction of single stranded DNA at virus replication and cell proliferation, be used to treat many virus diseases, the activity therefore detecting DNA helicase has extremely important meaning.
The detection method majority of the DNA helicase activity of current existence passes through
32one in the marker DNA chain of P, detected by the method for gel electrophoresis, but this method cost is higher, consuming time, the flux of detection is low.
Two scientist An Deliehaimu (Andre Geim) and the Constantine Nuo Woxiaoluofu (Konstantin Novoselov) of Univ Manchester UK in 2004 prepare Graphene first, and obtain the Nobel Prize in physics of 2010, receive the concern of whole world researchist.The Preparation of Graphene is simple, cost compare is low, there is more functional group on surface, as aldehyde radical, carboxyl etc., at the fixing FAM(Fluoresceincarboxylic acid in graphene oxide surface) double-stranded DNA that marks time, when DNA becomes strand from double-strand, the distance on FAM and graphene oxide surface changes, graphene oxide is different by the degree of fluorescence resonance energy transmission (FRET) energy cancellation FAM fluorescence, based on this feature, by the change of FAM fluorescence intensity, DNA helicase activity is detected.Biochip is a kind of high-throughout instrument, utilizes the characteristic of graphene oxide, biochip is combined with graphene oxide, develops the DNA helicase detection method that a kind of sensitivity is higher.
Summary of the invention
For Shortcomings in prior art, the invention provides a kind of based on the detection method of graphene oxide chip to DNA helicase, energy low cost, high-throughput, high sensitivity detect DNA helicase.
The present invention realizes above-mentioned technical purpose by following technique means.
Based on a detection method for the DNA helicase of Graphene chip, comprise the steps:
(1) preparation has the slide of graphene oxide dot matrix:
1ml graphene oxide solution (0.3 mg/ml) point is at amido modified slide (long 76mm, wide 25mm, thick 1mm) on, after drying, with ultrapure water, on the slide with graphene oxide dot matrix, add 1 ml and to contain after the mixture of 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCI) of 0.6 nM and the N-hydroxy thiosuccinimide (sulfo-NHS) of 6.0 nM at room temperature 12h.
(2) double chain DNA sequence is connected to the slide with graphene oxide dot matrix:
The single-stranded DNA sequence that the amido modified the other end FAM in one end marks, after mixing according to the ratio of 1:1 with the single stranded DNA of complementation, be placed on slide prepared by hybridizing box neutralization procedure (1) and carry out chemical reaction 12h, unnecessary probe is washed away, the FAM fluorescence intensity F of DNA marker on scanning record slide with PBS
1.
(3) DNA helicase detects:
When ATP is 10 mM, joined by the DNA helicase of different quantities on slide that step (2) processed, effect below 30min, PBS rinse, the FAM fluorescence intensity F of DNA marker on scanning record slide
2.
Mechanism of the present invention is: the DNA helicase adding different quantities in different action time by the effect to step (2) double center chain DNA, change before and after DNA helicase effect, the fluorescence intensity of FAM is caused to change, by the comparison before and after the change of FAM fluorescence intensity, scanning and analysis after PBS rinses, thus to whether there is DNA helicase in solution detect, as FAM fluorescence intensity F after DNA helicase effect
2fAM fluorescence intensity F before the effect of/DNA helicase
1ratio close to 0 time, illustrate in solution to there is DNA helicase.
The invention has the beneficial effects as follows:
(1) the present invention combines graphene oxide and microarray chip technology, and energy low cost, Gao Tong Liang ﹑ high sensitivity detect DNA helicase.
(2) the present invention fully uses the FAM that complementary double-stranded DNA and single stranded DNA mark biochip, different from the distance on graphene oxide surface, DNA helicase is to after double-stranded DNA effect, the distance of FMA and graphene oxide changes, cause the fluorescence intensity change of FAM, around this principle DNA helicase is detected, relatively simple and effective.
Accompanying drawing explanation
Fig. 1 is schematic flow sheet of the present invention.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to this.
(1) preparation of amido modified slide: add 5 ml hydrogen peroxide (H in the beaker being placed with slide
2o
2), so
The 15 ml vitriol oil (H are added afterwards with transfer pipet
2sO
4), on shaking table, slowly vibration or standing 30 min make it
Abundant reaction, this reaction can make surface hydroxylation, outwells the liquid of step reaction, by washed with de-ionized water 3 times.First use washes of absolute alcohol 2 times, then pour 20 ml dehydrated alcohols into, the hydroxylation of reacted acquisition
Silicon chip is transferred in beaker, with washes of absolute alcohol 3 times.Outwell ethanol after having cleaned, add rapidly the mixed solution (volume ratio is 1:15) of APTES (APTES) and dehydrated alcohol, or first add 15ml dehydrated alcohol, then add 1 ml APTES with transfer pipet, on shaking table, 2 h are reacted in jolting.
(2) preparation of graphene oxide: according to the Hummer method improved, in there-necked flask, add 3g crystalline graphite powder, 1.5 g NaNO
3with put into thermostat water bath after the 69 ml vitriol oils and stir.1g KMnO is added after reaction 1h
4, react at 35 DEG C after 5 hours and add 150 ml deionized waters.React 30 min at temperature 98 DEG C after, then add deionized water, the 5 ml H of 50 ml
2o
2and 250 10% dilute hydrochloric acid of ml, solution is poured in the large beaker of 1000 ml, wash to pH value be 5-6.
(3) preparation has the slide of graphene oxide dot matrix: 1 ml graphene oxide solution (0.3 mg/ml) point, on amido modified slide, after drying, uses ultrapure water.Add 1 ml EDCI(0.6 nM) and sulfo-NHS(6.0 nM) after at room temperature 12h.
(4) linking objective double-stranded DNA: synthetic DNA sequence, 5 '-NH
2-GAG CGG ATT ACTATA CTA CAT TAG AAT TCC-FAM-3 ', with 5 '-GGA ATT CTA ATG TAG TAT AGT AAT CCG CTC-3 ', according to the mixed solution of 1:1 ratio under concentration is 10 pmol, after hybridizing 2 h containing 50 mM tri-(methylol) aminomethanes (Tris-HCl) and sex change in pH 8.0 damping fluid of 50 mM sodium-chlor (NaCl), get 0.6 ul fluid drips and be added to 1) surface of glass slide prepared, in hybridizing box, carry out chemical reaction 12 h, and be fixed on graphene oxide dot matrix.
(5) detection of DNA helicase: containing 0.5 mM ethylenediamine tetraacetic acid (EDTA) (EDTA), 10 mM Triphosadens (ATP), 1.3 mM magnesium chloride (MgCl
2) and 10% glycerin solution in add the SARS virus of 10 nM DNA helicase carry out reaction 30 below min, article one, single stranded DNA disengaging has the amido modified single stranded DNA be fixed on graphene oxide, the distance of FMA and graphene oxide changes, cause the fluorescence intensity change of FAM, thus make graphene oxide to the fluorescence generation cancellation being fixed on the FAM that surperficial single stranded DNA marks, after rinsing with PBS, scanning and analysis under 520 nm on the scanner, measures reacted fluorescence intensity F
2fAM fluorescence intensity F before the effect of/DNA helicase
1ratio is close to 0, the above results shows, DNA helicase can effectively untwist to double-stranded DNA, further measurement is to the different concns DNA helicase degree that FAM fluorescence intensity reduces under different action time, the activity of DNA helicase can be reflected, thus the carrying out of SARS virus DNA helicase is detected.
Described embodiment is preferred embodiment of the present invention; but the present invention is not limited to above-mentioned embodiment; when not deviating from flesh and blood of the present invention, any apparent improvement that those skilled in the art can make, replacement or modification all belong to protection scope of the present invention.
Claims (5)
1. based on a detection method for graphene oxide chip DNA helicase, it is characterized in that: comprise the steps:
(1) preparation has the slide of graphene oxide dot matrix;
(2) after the single stranded DNA that the amido modified the other end FAM in one end marks hybridize with complementary single stranded DNA sex change, be connected to there is graphene oxide dot matrix slide on, react rear washing, has scanned the FAM fluorescence intensity F recording DNA marker on slide
1;
(3) DNA helicase detects:
When ATP is 10 mM, joined by the DNA helicase of different quantities on slide that step (2) processed, act on 30 below min, PBS rinses, the FAM fluorescence intensity F of DNA marker on scanning record slide
2.
2. detection method as claimed in claim 1, it is characterized in that: described step (1) be by 1ml graphene oxide solution point on amido modified slide, after drying, with ultrapure water, then to add after 1ml EDCI and sulfo-NHS mixture at room temperature 12h.
3. detection method as claimed in claim 2, is characterized in that: the concentration of described graphene oxide solution is 0.3 mg/ml; The concentration of EDCI solution is 0.6 nM; The concentration of sulfo-NHS solution is 6.0 nM.
4. detection method as claimed in claim 1, it is characterized in that: the concrete grammar of described step (2) is: the single-stranded DNA sequence that the amido modified the other end FAM in one end marks, after mixing according to the ratio of 1:1 with the single stranded DNA of complementation, be placed on slide prepared by hybridizing box neutralization procedure (1) and carry out chemical reaction 12h, wash away unnecessary probe with PBS.
5. detection method as claimed in claim 1, is characterized in that: as FAM fluorescence intensity F after DNA helicase effect
2fAM fluorescence intensity F before the effect of/DNA helicase
1ratio close to 0 time, illustrate in solution to there is DNA helicase.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861295A (en) * | 2016-05-24 | 2016-08-17 | 长沙医学院 | Biosensor for detecting salmonella typhimurium and preparation and detection methods |
| CN106520913A (en) * | 2016-09-22 | 2017-03-22 | 江苏大学 | Preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection |
| CN107190060A (en) * | 2017-05-25 | 2017-09-22 | 太原理工大学 | MicroRNA detection probes and graphene detection method |
| CN110907421A (en) * | 2019-12-13 | 2020-03-24 | 深圳市人民医院 | Detection method and kit for copper ions based on graphdiyne and click chemistry and application |
| CN111965151A (en) * | 2020-07-31 | 2020-11-20 | 江苏大学 | Graphene oxide biochip and preparation method and application thereof |
| CN114561442A (en) * | 2022-03-21 | 2022-05-31 | 中山大学 | Helicase activity determination method based on double-stranded DNA and application thereof |
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| CN1120353A (en) * | 1993-03-27 | 1996-04-10 | 邓迪大学董事会 | Anti-microbial agents and screening method therefor |
| CN1522300A (en) * | 2001-05-01 | 2004-08-18 | 独立行政法人产业技术综合研究所 | Novel maxizyme |
| CN104237352A (en) * | 2014-10-20 | 2014-12-24 | 中国人民解放军第三军医大学第一附属医院 | Electrode modified by oxidized graphene, gold nanotube and locked nucleic acid probe as well as preparation method and application of electrode |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1120353A (en) * | 1993-03-27 | 1996-04-10 | 邓迪大学董事会 | Anti-microbial agents and screening method therefor |
| CN1522300A (en) * | 2001-05-01 | 2004-08-18 | 独立行政法人产业技术综合研究所 | Novel maxizyme |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861295A (en) * | 2016-05-24 | 2016-08-17 | 长沙医学院 | Biosensor for detecting salmonella typhimurium and preparation and detection methods |
| CN106520913A (en) * | 2016-09-22 | 2017-03-22 | 江苏大学 | Preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection |
| CN106520913B (en) * | 2016-09-22 | 2020-02-21 | 江苏大学 | Preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection |
| CN107190060A (en) * | 2017-05-25 | 2017-09-22 | 太原理工大学 | MicroRNA detection probes and graphene detection method |
| CN110907421A (en) * | 2019-12-13 | 2020-03-24 | 深圳市人民医院 | Detection method and kit for copper ions based on graphdiyne and click chemistry and application |
| CN110907421B (en) * | 2019-12-13 | 2022-06-17 | 深圳市人民医院 | Detection method and kit for copper ions based on graphdiyne and click chemistry and application |
| CN111965151A (en) * | 2020-07-31 | 2020-11-20 | 江苏大学 | Graphene oxide biochip and preparation method and application thereof |
| CN114561442A (en) * | 2022-03-21 | 2022-05-31 | 中山大学 | Helicase activity determination method based on double-stranded DNA and application thereof |
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