CN104568878B - It is a kind of based on detection method of the graphene oxide chip to copper ion - Google Patents
It is a kind of based on detection method of the graphene oxide chip to copper ion Download PDFInfo
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- CN104568878B CN104568878B CN201410832706.4A CN201410832706A CN104568878B CN 104568878 B CN104568878 B CN 104568878B CN 201410832706 A CN201410832706 A CN 201410832706A CN 104568878 B CN104568878 B CN 104568878B
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Abstract
The invention belongs to the heavy metal detection method in biomedical sector, a kind of detection method of the copper ion based on graphene oxide chip.The invention mainly comprises following steps：（1）Prepare the slide with graphene oxide dot matrix；（2）Connect Cu2+Correspondence cutting DNAzyme sequences to have graphene oxide dot matrix slide；（3）Cu2+On above-mentioned slide dissection occurs for detection.The present invention can be quenched with the fluorescence of graphene oxide by AuNP, Cu2+After effect, contain Cu2+Cleavage site single stranded DNA zyme is cut off, and depart from the single stranded DNA zyme for being fixed on the amido modified other end AuNP marks in one end on graphene oxide, AuNP and the distance on graphene oxide surface is set to change, so that the principle that the fluorescence of graphene oxide changes, with reference to high-throughout microarray chip technology, this ﹑ of energy Di Cheng are in high sensitivity to Cu2+Detected.
Description
Technical field
It is especially a kind of based on graphene oxide life the invention belongs to the heavy metal detection method in biomedical sector
Thing chip is to Cu2+Detection method.
Background technology
Cu2+Can occur strong interaction with protein and various enzymes in human body, them is lost activity, it is also possible to
Be enriched with some organs of human body, if it exceeds the limit that human body is resistant to, human body acute poisoning can be caused, it is subacute in
Poison, slow poisoning etc., know from experience to people and cause very big harm.Therefore, Cu2+Pollution has been drawn in terms of medicine, food, environment
Play the attention of people's height.Research at present to copper ion detection method is more and more, is mainly lied prostrate including immunoassay, dissolution
An Fa, test paper method etc., existing certain methods lack higher sensitivity, relatively low to the detection flux of sample, therefore set up
A kind of highly sensitive, high-flux detection method system is the current research contents for being badly in need of development.
Two scientist An Deliehaimu of Univ Manchester UK in 2004（Andre Geim）And Constant
Ding Nuowoxiaoluofu（Konstantin Novoselov）Graphene is prepared first, and obtains Nobel's physics of 2010
Prize, receives the concern of whole world researcher.The oxide of graphene prepares simple, and cost is than relatively low, and surface has more
Functional group, such as aldehyde radical, carboxyl, golden nanometer particle（AuNP）The fluorescence of graphene oxide itself can be quenched in the DNA of mark,
DNA enzymatic (DNAzyme) is a kind of DNA molecular with catalysis, with higher stability, when being reacted with object,
The change of structure can be caused（Work as Cu2+During the DNAzyme of correspondence cutting, its DNAzyme structure is caused to change, wherein one
DNA is cut off, and departs from former matched sequence）, so as to be used for the detection of heavy metal.The DNAzyme of AuNP marks is being cut
Before and after cutting, changed with the distance on graphene oxide surface, the fluorescence intensity of graphene oxide changes, and passes through stone
The change of the fluorescence intensity of black olefinic oxide is to Cu2+Concentration is detected.Biochip is a kind of high-throughout instrument, utilizes stone
The characteristic of black olefinic oxide, is combined biochip with graphene oxide, develops a kind of Cu in higher sensitivity2+Concentration is examined
Survey method.
The content of the invention
For Shortcomings in the prior art, the invention provides a kind of Cu in graphene oxide chip2+Detection
Method, energy low cost, high flux, high sensitivity are to Cu2+Detected..
The present invention is to realize above-mentioned technical purpose by following technological means.
It is a kind of based on detection method of the graphene oxide chip to copper ion, comprise the following steps：
（1）Prepare the slide with graphene oxide dot matrix：
1ml graphene oxides solution (0.3 mg/ml) point is in amido modified slide（Long 76mm, wide 25mm are thick
1mm）On, after drying, with ultrapure water, on the slide with graphene oxide dot matrix, add 1 ml and contain 0.6 nM
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides（EDCI）With 6.0 nM N- hydroxy thiosuccinimides
（sulfo-NHS）Mixture after 12h at room temperature,.
（2）Connect Cu2+Correspondence cutting DNAzyme sequences to have graphene oxide dot matrix slide：
The single stranded DNA zyme sequences of the amido modified other end AuNP marks in one end contain cleavage site with complementary one
DNAzyme sequences are according to 1:After 1 ratio mixing, hybridizing box neutralization procedure is placed on（1）The slide of preparation is chemically reacted
12h, unnecessary probe is washed away with ultra-pure water；The fluorescence intensity F of graphene oxide on scanning record slide2。
（3）Cu2+Detection：
The copper ion solution of various concentrations is added to by step（2）On treated slide, below 15min is reacted,
With ultrapure water, scanning record graphene oxide is in Cu2+Fluorescence intensity F after dissection3。
The present invention mechanism be：Add the Cu of various concentrations2+By to step under different time（2）Middle DNAzyme's
Effect, Cu2+Cutting, which is connected to graphene oxide, to be had after the DNAzyme that AuNP is marked, and DNAzyme structure changes：
The single stranded DNA zyme for having cleavage site departs from graphene oxide surface, and graphene oxide is fixed on containing what AuNP was marked
On single stranded DNA zyme with being changed before the distance on graphene oxide surface and cutting, cause the glimmering of graphene oxide
Luminous intensity changes, and with scanning and analyzing after ultrapure water, passes through graphene oxide fluorescence intensity Cu2+Before and after effect
Change, works as Cu2+Dissection rear oxidation graphene fluorescence intensity F3/Cu2+The fluorescence intensity F of graphene oxide before dissection2
Ratio close to 0 when, illustrate there is Cu in solution2+, detection sensitivity during this method detection copper ion is up to 0.1nM.
The beneficial effects of the invention are as follows：
（1）The present invention is combined graphene oxide and microarray chip technology, and energy low cost, Gao Tong Liang ﹑ are highly sensitive
Property is to Cu2+Concentration is detected.
（2）It is of the invention to be fully quenched on biochip on graphene oxide with the AuNP DNAzyme sequences marked
Fluorescence, but when DNAzyme is in Cu2+Under effect, structure changes, and AuNP is different from the distance of graphene oxide,
The different principle of its efficiency being quenched, to Cu2+Detected, it is relatively easy and effective.
Brief description of the drawings
Fig. 1 is schematic flow sheet of the present invention.
Fig. 2 is Cu2+Cutting DNA zyme course of reaction figures.
Embodiment
Below in conjunction with the accompanying drawings and specific embodiment the present invention is further illustrated, but protection scope of the present invention is simultaneously
Not limited to this.
1. the preparation of amido modified slide：5 ml hydrogen peroxide are added in the beaker for be placed with slide（H2O2）, then with shifting
Liquid pipe adds the 15 ml concentrated sulfuric acids（H2SO4）, slowly vibrating or 30 min of standing are allowed to abundant reaction on shaking table, and this reaction can make
Surface hydroxylation, outwells the liquid of step reaction, is cleaned with deionized water 3 times.First use washes of absolute alcohol 2 times, be subsequently poured into
20 ml absolute ethyl alcohols, the hydroxylating silicon chip of reacted acquisition is transferred in beaker, with washes of absolute alcohol 3 times.Cleaning is completed
After outwell ethanol, be rapidly added APTES（APTES）With the mixed liquor of absolute ethyl alcohol（Volume ratio is 1：
15）, or first add 15ml absolute ethyl alcohols, then add 2 h of shaking reaction on 1 ml APTES, shaking table with pipette.
2. the preparation of graphene oxide：According to improved Hummer methods, in three-necked flask, 3g crystalline flake graphites are added
Powder, 1.5 g NaNO3Stirred with being put into after the 69 ml concentrated sulfuric acids in thermostat water bath.React and 1g KMnO are added after 1h4, at 35 DEG C
150 ml deionized waters are added after 5 hours of reaction.After reacting 30 min at 98 DEG C of temperature, 50 ml deionization is added
Water, 5 ml H2O2And 250 ml 10% watery hydrochloric acid, in the large beaker that solution is poured into 1000 ml, washing to pH value is 5-
6。
3. prepare the slide with graphene oxide dot matrix：1 ml graphene oxides solution (0.3 mg/ml) point
In amido modified slide（Long 76mm, wide 25mm, thick 1mm）On, after drying, use ultrapure water.Add 1 ml EDCI（0.6
nM）And sulfo-NHS（6.0 nM) 12h at room temperature after mixture.
4. linking objective DNAzyme sequences：Synthetic DNA zyme sequences, pass through 0.7ul on graphene oxide chip
The graphene oxide of activation is 5pmol Cu with the concentration modified through terminal amino group2+The DNAzyme mixtures of correspondence cutting enter
Row connection, obtains connecting DNAzyme chip.The DNA sequence dna used is as follows：a. 5¢-TTCTTT CTAA TACG GC
TTACC-3 ￠, with the b.5 ￠-NH2-GGTAAGCCTGGGCCTCTTTC TTT TTA AG marked at 3 ' ends with AuNP
AAAGAAC-3 ￠, the DNA for forming double-strand pouch-type conformation, Cu are denatured by a and b2+Being capable of the specific certain bits for cutting the DNA sequence dna
The 17-24 1-8 bit bases complementations from 5 ￠ ends with sequence b from 5 ￠ ends of point, wherein sequence a, and sequence b is from 5 ￠ ends
15-22 25-32 bit bases are complementary from 5 ￠ ends with sequence b, according to 1：1 ratio hybridizes 2h after being mixed under 37 degree,
Liquid after 0.7ul hybridization is added dropwise 1）The surface of glass slide of preparation, carries out 12 h of chemical reaction in hybridizing box, and fixed to graphite
On olefinic oxide dot matrix, the fluorescence that graphene oxide can be quenched by fluorescent energy transmission in the AuNP of DNA sequence dna mark is strong
Degree, is scanned in 532nm to chip, (F after the fluorescence intensity of graphene oxide is quenched2) be quenched before (F1) ratio be
80%-95%。
5. Cu2+Detection：Add 0.1 nM Cu2+Solution (sodium ascorbate containing 50 uM) is to being connected to graphite
DNAzyme is carried out after cutting 10-15 min, dissection on olefinic oxide, and DNAzyme structure changes, AuNP marks
DNAzyme and the distance on graphene oxide surface change, chip is scanned in 532nm, stone is able to observe that
Further change, its fluorescence intensity (F occur for the fluorescence intensity of black olefinic oxide3) and Cu2+Graphene oxide is glimmering before cutting
Luminous intensity (F2) ratio be close to 0, carry out repeated experiment under same experimental conditions, above-mentioned experimental phenomena is consistent, shows
Chip surface fluorescence intensity change comes from the DNAzyme sequences for being connected to graphene oxide surface in Cu2+The lower structure of effect
Change, the sensitivity of detection can reach 0.1 nM.The higher detection sensitivity reported is 1 nM.(Y. Hao, L.
Liu, Y. Long, J. Wang, Y. Liu, F. Zhou, Biosens. Bioelectron., 2013, 41,723–
729.）10 times are improved in sensitivity.
The present invention has carried out control experiment under identical treatment conditions, with other heavy metal ion of same concentrations such as
Pb2+, Mg2+, Al3+Deng cutting, its fluorescence intensity does not have significant change.The above results show, Cu2+Effectively DNAzyme can be entered
Row cutting, by the influence to graphene oxide fluorescence intensity, so as to Cu2+Concentration carries out specific detection.
The present invention has carried out control experiment under identical treatment conditions, with other heavy metal ion of same concentrations such as
Pb2+, Mg2+, Al3+Deng cutting, its fluorescence intensity does not have significant change.The above results show, Cu2+Effectively DNAzyme can be entered
Row cutting, by the influence to graphene oxide fluorescence intensity, so as to Cu2+Concentration carries out specific detection.
For the present invention preferred embodiment, but the present invention is not limited to above-mentioned embodiment to the embodiment, not
In the case of the substantive content of the present invention, any conspicuously improved, replacement that those skilled in the art can make
Or modification belongs to protection scope of the present invention.
Claims (4)
1. it is a kind of based on detection method of the graphene oxide chip to copper ion, it is characterised in that：
(1) slide with graphene oxide dot matrix is prepared；
(2) DNAzyme of other end AuNP marks amido modified to one end is with containing Cu2+The single stranded DNA zyme of cleavage site is pressed
According to 1:After 1 denaturation hybridization, progress chemical reaction 12h on the slide with graphene oxide dot matrix is connected to, with ultrapure washing
Remove the fluorescence intensity F of graphene oxide on unnecessary DNA sequence dna, scanning record slide2；
(3) copper ion solution of various concentrations is added on the slide treated by step (2), reacts 10-15min, used
Ultrapure water, scanning record graphene oxide is in Cu2+Fluorescence intensity F after dissection3。
2. detection method according to claim 1, it is characterised in that：The step (1) is that 1ml graphene oxides is molten
Liquid point is on amido modified slide, after drying, and with ultrapure water, then adds 1ml EDCI and sulfo-NHS mixtures
Stand 12h at room temperature afterwards.
3. detection method according to claim 2, it is characterised in that：The concentration of the graphene oxide solution is
0.3mg/ml；The concentration of EDCI solution is 0.6nM；The concentration of sulfo-NHS solution is 6.0nM.
4. detection method according to claim 1, it is characterised in that：Work as Cu2+The fluorescence of dissection rear oxidation graphene
Intensity F3/Cu2+Graphene oxide fluorescence intensity F before dissection2Ratio close to 0 when, illustrate there is Cu in solution2+, should
Detection sensitivity during method detection copper ion is up to 0.1nM.
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| CN105842209A (en) * | 2016-03-18 | 2016-08-10 | 中国科学院合肥物质科学研究院 | Paper sensor for rapid on-site detection of fluorine ions in water and preparation method thereof |
| CN109557155A (en) * | 2019-01-17 | 2019-04-02 | 杭州电子科技大学 | It is a kind of based on graphene-In Glassy Carbon Electrode Modified With Nano-gold preparation method and application |
| CN111965151A (en) * | 2020-07-31 | 2020-11-20 | 江苏大学 | Graphene oxide biochip and preparation method and application thereof |
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| CN103031375A (en) * | 2012-12-10 | 2013-04-10 | 江苏大学 | DNA (deoxyribonucleic acid) methylation detection kit and detection method |
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| WO2009012309A2 (en) * | 2007-07-16 | 2009-01-22 | The Board Of Trustees Of The University Of Illinois | Nucleic acid based fluorescent sensor for divalent copper ion detection |
| WO2009045632A2 (en) * | 2007-08-10 | 2009-04-09 | The Board Of Trustees Of The University Of Illinois | Nucleic acid based fluorescent sensor for mercury detection |
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Non-Patent Citations (5)
| Title |
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| A molecular beacon and grapheme oxide-based fluorescent biosensor for Cu2＋ detection;Jiahao Huang et al.;《Biosensors and Bioelectronics》;20130515;第43卷;Abstract，第380页第1段，3.1 Detection principle，Fig.1b * |
| Adsorption of DNA onto gold nanoparticles and graphene oxide:surface science and applications;Juewen Liu;《Physical Chemistry Chemical Physics》;20120814;第14卷(第30期);第10485-10496页 * |
| Graphene oxide-DNA based sensors;Li Gao et al.;《 Biosensors and Bioelectronics》;20141015;第60卷;第23页 2.2.1 DNA detection * |
| 基于氧化石墨烯的荧光生物传感器新方法研究;朱文平;《万方学位论文》;20131129;正文第1-80页 * |
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