CN106520913A - Preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection - Google Patents
Preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection Download PDFInfo
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Abstract
The invention belongs to the field of protein detection in the field of biomedicine, and particularly relates to a graphene oxide-DNA sensor based on enzyme digestion cycle amplification and a thrombin detection method thereof. The method mainly comprises the following steps: 1) preparing graphene oxide; 2) adding FAM-labeled TBA aptamer to the amino-activated graphene oxide; 3) thrombin with different concentrations is added, and detection is carried out through the change of fluorescence intensity of aptamer marks adsorbed on the surface of the graphene oxide. The invention utilizes the characteristic of quenching fluorescence of graphene oxide, adopts exonuclease III to identify thrombin single-chain aptamer (TBA), hydrolyzes the thrombin single-chain aptamer (TBA) to release thrombin, thereby realizing the cyclic utilization of the thrombin, gradually enhancing the fluorescence signal, realizing the trace detection of the thrombin with high sensitivity, high speed and low cost, avoiding non-specific adsorption by covalent bonding and adding polyethylene glycol, and improving the detection limit of the graphene oxide-aptamer sensor to the thrombin to 0.024 pM.
Description
Technical field
The invention belongs to the protein detection field in biomedical sector, is related to a kind of stone for circulating based on digestion and amplifying
The method that black olefinic oxide-DNA sensor is detected to fibrin ferment (thrombin) high sensitivity, and in particular to a kind of to be based on nucleic acid
The graphene oxide (GO) of excision enzyme amplification detection signal-aptamer (aptamer) sensor and its detection fibrin ferment
Method.
Background technology
Fibrin ferment is a kind of serine protease in blood, participates in some reactions of Human Physiology and pathology, such as：Inflammation,
The processes such as wound repair, blood clotting, platelet activation.In blood, the difference of thrombin amount can cause dysfunction of blood coagulation.
Additionally, the development of fibrin ferment and numerous disease has a close relationship, and by as disease marker, therefore, at the beginning of clinical diagnosis
Phase high sensitivity to fibrin ferment detect it is particularly important
Aptamer is single strain oligonucleotide, and the various target molecules of energy specific recognition are such as：Antibody, bacterium, protein,
And cell.The features such as active stable, low cost of aptamer, easy modification, easy long term storage, based on aptamer
Sensor has application well at many aspects, such as in terms of food security, Pharmaceutical Analysis, environmental monitoring and biochemical analysis etc..
Due to the importance of fibrin ferment, fibrin ferment is equally widely studied with the interaction of aptamer, can be specifically bound solidifying
The aptamer (TBA) of hemase has high-affinity and high selectivity to fibrin ferment, can be with the surface antigen of people α-fibrin ferment
Determinant combines, and forms stable G- tetrad structures, and the sensor of many detection fibrin ferments is exactly to send out according to this principle
Exhibition.
At present, signal amplification technique has increasingly been used in the detection sensitivity research for improve fibrin ferment, such as Jenner
Rice auxiliary signal amplifying technique, DNA enzymatic auxiliary signal amplifying technique, aptamer-GO signal amplification techniques, rolling circle amplification skill
Art, cross chain reaction amplifying technique, enzyme mark amplifying technique, circumscribed enzymatic targeting circulation electrochemical techniques etc..Wherein, it is based on
It is exactly one kind therein that the detection technique of signal is amplified in enzyme circulation.Exonuclease (Exonuclease) is to single nucleotides
The enzyme of effect, can hydrolyze phosphodiester bond successively from one end of sequence, become multiple mononucleotide fragments, therefore exonuclease
The aptamer combined with target protein by energy degraded, discharges target protein, with other aptamers in conjunction with so circulation makes
With, there is provided a good new method for realizing high sensitivity detection.
A kind of detection that the targeting circulating technology of Exonucleolytic enzymatic is combined with GO sensors of this research and development
Technology, to improve the sensitivity technique to fibrin ferment.
The content of the invention
It is an object of the present invention to provide a kind of amplify inspection of the GO sensors of signal to thrombin based on digestion catalytic cycle
Capture-DNA is fixed to GO surfaces, then the thrombin aptamer (TBA with FAM modifications by being covalently attached by survey method
Aptamer) hybridize, and with a small amount of PEG closing GO surfaces non-specific sites, be fixed on the fibrin ferment aptamer on GO surfaces
Fluorescent quenching and effectively prevent the digestion of exonuclease.After fibrin ferment is specifically bound with aptamer, fibrin ferment-
Aptamer compound leaves GO surfaces, and the aptamers in excision enzyme identification hydrolyzing composition discharge fibrin ferment, realize blood coagulation
Enzyme is recycled, and fluorescence signal gradually strengthens.The sensor is on the basis of covalent bond, it is to avoid false positive signal, introduces enzyme
Catalysis targeting circulating technology, has developed a kind of high new blood coagulation enzymatic detection techniques of efficient, convenient, sensitivity, so as to highly sensitive, fast
Speed, low cost are detected to thrombin.
The preparation method of the graphene oxide-DNA sensor amplified is circulated based on digestion, is comprised the steps：
(1) prepare GO：By improve Hummers methods prepare GO, GO is vacuum dried it is standby, using front, in the aqueous solution
Middle ultrasonic disperse, obtains GO dispersion liquids；
(2) activation of GO：By containing 50mM NHS with and 200mM EDC water solution A and 2mg/ml obtained in step (1)
GO dispersion liquids mix, and add ultra-pure water, elute, store for future use under room temperature after reacting 0.5 hour；
(3) preparation of GO-aptamer sensors：First connect GO obtained in amidized capture DNA to step (2)
Surface, then TBA is added dropwise, due to the specificity of TBA, combined with capture DNA, then PEG is added dropwise, that is, GO-DNA sensings are obtained
Device.GO can be quenched the fluorescence of the end-labelled FAM of TBA.
In step (2), the volume ratio of the water solution A, GO dispersion liquids and ultra-pure water is 1：2：1.
In step (3), the sequence of the capture DNA is：5’-NH2-AGTCACCCCAACCTG CCC
TACCACGGACT--3 ', the capture DNA itself can form loop-stem structure.
In step (3), the sequence of the TBA is：5’AAAA GTCCG TG GTAGGGCA GGTTGGGGTGA CT-
FAM-3 ', the single-stranded TBA make the single-stranded TBA that exonuclease is easier in identification fibrin ferment-TBA compounds.
In step (3), the capture DNA：The concentration ratio of TBA is 1：1, concentration is 10nM；The concentration of PEG is
The concentration of 50nM, GO is 0-25 μ g/mL.
Prepared by the present invention circulates the graphene oxide-DNA sensor detection fibrin ferment for amplifying based on digestion.
Comprise the steps during detection fibrin ferment：
S1：The fluorescent value of detection GO-DNA sensors；
S2：Fibrin ferment Thrombin is added in GO-DNA sensors, adds excision enzyme exonuclease, Huo Zhezhi
The mixture Thrombin Exonuclease of addition fibrin ferment-excision enzyme are met, after reaction 30min, then fluorescent value is detected；Analysis
Its change in fluorescence.
With reference to after thrombin, TBA-thrombin compounds leave GO surfaces to TBA, in excision enzyme identification hydrolyzing composition
Aptamers, discharge fibrin ferment so that fibrin ferment is recycled, and FAM fluorescence signals gradually strengthen, according to the change of fluorescence intensity
Change, so as to detect to which.
In step S2, the concentration of fibrin ferment Thrombin is 1nM；The concentration of excision enzyme exonuclease is 0.03U/mL.
The present invention has advantages below：
(1) in the present invention, GO is easily obtained, and method is simple, low cost, makes full use of GO that single stranded DNA end mark can be quenched
Fluorescence, after protein combines single stranded DNA, the characteristics of fluorescence recovers, can quick, specificity, high sensitivity to thrombin
Detected.
(2) present invention is hydrolyzed to single-chain nucleic acid cutting using exonuclease, the target protein in release compound, reality
Existing target protein is recycled, and thus drives fluorescence signal gradually to strengthen.
(3) GO surfaces are fixedly attached to amination capture DNA, and the non-specific of GO surfaces is solved with a small amount of PEG
Property absorption problem, so as to increase the specificity of detection, further improves the sensitivity of detection.
Description of the drawings
Fig. 1：The schematic flow sheet of the present invention；
Fig. 2：The detection of fibrin ferment thrombin sensitivitys is schemed based on GO sensors；Fluorescently-labeled aptamer in figure
(aptamer) concentration is 10nM, and the concentration of graphene oxide is 20 μ g/mL.
Fig. 3：Based on GO sensors to fibrin ferment thrombin selective enumeration method figures；
Fig. 4：GO sensors to 1pM thrombin detection scheme, in figure curve be from top to bottom followed successively by aptamer (c),
Aptamer-GO-1pM Thrombin Exonuclease (b), aptamer-GO (a) testing result curves.
Fig. 5：GO sensors to 1nM thrombin detection scheme, in figure curve be from top to bottom followed successively by aptamer (c),
Aptamer-GO-1nM Thrombin Exonuclease (b), aptamer-GO (a) testing result curves.
Specific embodiment
The present invention will be further described with reference to embodiments, and embodiment is for illustrating rather than for limiting
The scope of the present invention processed.
Embodiment 1：
(1) prepare GO：GO is prepared on a large scale by the Hummers methods for improveing, in there-necked flask, 3g crystalline flake graphites is added
Powder, 1.5g NaNO3Stir with being put into after the 69mL concentrated sulfuric acids in thermostat water bath.1g KMnO are added after reaction 1h4, it is anti-at 35 DEG C
150mL deionized waters are added after answering 5 hours.After 30min being reacted at 98 DEG C of temperature, add deionized water, the 5mL of 50mL
H2O2And 10% watery hydrochloric acid of 250mL, solution is poured in the large beaker of 1000mL, it is 5-6 to wash to pH value.Oxidation is produced
Thing vacuum drying is standby, using front, 1000W ultrasounds 30min in aqueous.GO is activated：0.5ml NHS containing 50mM and 200mM
EDC and 1ml 2mg/ml GO, add 0.5ml ultra-pure waters, elute, store for future use under room temperature after reacting 0.5 hour
(2) special aptamer sequences are synthesized：Fibrin ferment nucleic acid aptamer sequence：capture DNA:5’-NH2-
AGTCACCCCAACCTGCCC TACCACGGACT--3’.Dashed part is to make DNA form loop-stem structure.TBA:5’AAAA
GTCCG TG GTAGGGCA GGTTGGGGTGA CT-FAM-3 ', (being purchased from Shanghai bioengineering Co., Ltd) dashed part is
Single-stranded TBA in making exonuclease easily recognize fibrin ferment-TBA compounds.
(3) fluorescent quenching：Amidized capture DNA and TBA-aptamer is fixed to the GO aqueous solution for having activated
In, that is, preparing becomes GO-aptamer sensors；GO can be quenched the fluorescence of the end-labelled FAM of TBA-aptamer；Wherein institute
Use capture DNA：TBA-aptamer is 1:1, concentration is 10nM；The concentration of GO is 20 μ g/mL；
(4) thrombin is detected based on GO sensors：Contain middle 10nM capture-DNA in 1ml reaction systems,
The GO of 20 μ g/ml activation, (adding the PEG of 50nmol to prevent non-specific adsorptions of the Thrombin/TBA to GO), after reaction 3-5h,
Add Thrombin/Thrombin-Exonuclease (1nM thrombin/0.03UmL-1Exonuclease), room temperature reaction
30min, analysis of fluorescence value changes.
The beautiful Synergy H4 that produce are detected to fluorescence intensity, and exciting light is 488nm, and launching light is 518nm, launch wavelength
510-650nm, fluorescence intensity level at observation 520nm.Such as Fig. 2, in figure, the concentration of the aptamer of FAM marks is 10nM, GO concentration
For 20 μ g/mL.Detection finds that sensitivity can reach 1pM.
Wherein Fig. 1 is the schematic flow sheet of the present invention, is combined TBA-aptamer with thrombin and form TBA- in figure
Thrombin complexs, aptamers self structure change, and depart from GO surfaces, and the exonuclease in solution recognizes TBA-
Single-stranded TBA in thrombin complexs, and hydrolyzed the TBA knots of the free fibrin ferment continuation of release fibrin ferment and GO surfaces
Close, so that fluorescence signal constantly strengthens.
(5) several protein lysozyme, BSA, the IgG for choosing other with the non-specific effects of TBA-aptamer are selected
Selecting property is detected, under same experiment condition, is found GO-aptamer energy specific bonds thrombin, and substantially can be distinguished and which
The difference of his several albumen, such as Fig. 3, it is selective well that experiment proves that the detection method has to thrombin, and in figure, FAM is marked
The concentration of the aptamer of note is 10nM, and GO concentration is 20 μ g/mL.
Embodiment 2：
Step (1), (2) and (3) is with embodiment 1.
(4) the GO sensors expanded based on digestion are detected to thrombin：Thrombin/ is added in step (3)
Thrombin-Exonuclease (1nM thrombin/0.03UmL-1exonuclease), room temperature reaction 30min are beautiful to produce
Synergy H4 are detected to fluorescence intensity, and exciting light is 485nm, and launching light is 518nm, and launch wavelength 510-650nm is seen
Fluorescence intensity level at 520nm is surveyed, the FAM intensity for marking is analyzed by Origin 8.0 and is changed, find detection on aptamer
Sensitivity reach 1pM.
Embodiment 3：
Step (1), (2) and (3) is with embodiment 1.
(4) the GO sensors expanded based on digestion are detected to thrombin：Thrombin/ is added in step (3)
Thrombin-Exonuclease(1nM thrombin/0.03UmL-1Exonuclease), room temperature reaction 30min is beautiful to produce
Synergy H4 are detected to fluorescence intensity, and exciting light is 485nm, and launching light is 518nm, and launch wavelength 510-650nm is seen
Fluorescence intensity level at 520nm is surveyed, the FAM intensity for marking is analyzed by Origin 8.0 and is changed, find detection on aptamer
Sensitivity reach 0.5nM.
SEQUENCE LISTING
<110>Jiangsu University
<120>The preparation method of the graphene oxide-DNA sensor amplified is circulated and on detection fibrin ferment based on digestion
Application
<130>The preparation method of the graphene oxide-DNA sensor amplified is circulated and on detection fibrin ferment based on digestion
Application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213>Artificial sequence
<400> 1
agtcacccca acctgcccta ccacggact 29
<210> 2
<211> 32
<212> DNA
<213>Artificial sequence
<400> 2
aaaagtccgt ggtagggcag gttggggtga ct 32
Claims (9)
1. the preparation method of the graphene oxide-DNA sensor amplified is circulated based on digestion, it is characterised in that including as follows
Step：
(1) prepare GO：GO is prepared by the Hummers methods for improveing, GO is vacuum dried standby, using front, is surpassed in aqueous
Sound disperses, and obtains GO dispersion liquids；
(2) activation of GO：By containing 50mM NHS with and 200mM EDC water solution A and 2mg/ml GO obtained in step (1)
Dispersion liquid mixes, and adds ultra-pure water, elutes, store for future use under room temperature after reacting 0.5 hour；
(3) preparation of GO-aptamer sensors：First connect GO surfaces obtained in amidized capture DNA to step (2),
TBA is added dropwise again, due to the specificity of TBA, is combined with capture DNA, then PEG is added dropwise, that is, GO-DNA sensors are obtained.
2. it is according to claim 1 based on digestion circulate amplify graphene oxide-DNA sensor preparation method,
Characterized in that, in step (2), the volume ratio of the water solution A, GO dispersion liquids and ultra-pure water is 1：2：1.
3. it is according to claim 1 based on digestion circulate amplify graphene oxide-DNA sensor preparation method,
Characterized in that, in step (3), the sequence of the capture DNA is：5’-NH2-AGTCACCCCAACCTG
CCCTACCACGGACT--3 ', the capture DNA itself can form loop-stem structure.
4. it is according to claim 1 based on digestion circulate amplify graphene oxide-DNA sensor preparation method,
Characterized in that, in step (3), the sequence of the TBA is：5’AAAA GTCCG TG GTAGGGCA GGTTGGGGTGACT-
FAM-3 ', the single-stranded TBA make the single-stranded TBA that exonuclease is easier in identification fibrin ferment-TBA compounds.
5. it is according to claim 1 based on digestion circulate amplify graphene oxide-DNA sensor preparation method,
Characterized in that, in step (3), the capture DNA：The concentration ratio of TBA is 1：1, concentration is 10nM；The concentration of PEG
For 50nM, the concentration of GO is 0-25 μ g/mL.
6. graphene oxide-the DNA sensor amplified is circulated based on digestion, it is characterised in that described graphene oxide-
DNA sensor is obtained by the preparation method of the imprinted polymer as any one of claim 1-5.
7. a kind of application for circulating the graphene oxide-DNA sensor amplified based on digestion as claimed in claim 6, its
It is characterised by, described graphene oxide-DNA sensor is used for detecting fibrin ferment.
8. the application for circulating the graphene oxide-DNA sensor amplified based on digestion according to claim 7, which is special
Levy and be, comprise the steps during detection fibrin ferment：
S1：The fluorescent value of detection GO-DNA sensors；
S2：Fibrin ferment Thrombin is added in GO-DNA sensors, is added excision enzyme exonuclease, or is directly added
Enter the mixture Thrombin Exonuclease of fibrin ferment-excision enzyme, after reaction 30min, then detect fluorescent value；Analyze which glimmering
Light changes.
9. the application for circulating the graphene oxide-DNA sensor amplified based on digestion according to claim 8, which is special
Levy and be, in step S2, the concentration of fibrin ferment Thrombin is 1nM；The concentration of excision enzyme exonuclease is 0.03U/mL.
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| CN107764790A (en) * | 2017-10-10 | 2018-03-06 | 广西师范学院 | Method based on enzyme and graphene oxide aptamer sensor detection fibrin ferment |
| CN107843631A (en) * | 2017-12-25 | 2018-03-27 | 安阳师范学院 | Protease detection electrochemical sensor and preparation method and detection method |
| CN108866063A (en) * | 2018-06-28 | 2018-11-23 | 中国水产科学研究院珠江水产研究所 | A kind of aptamer and its preparation method and application of PEG modification |
| CN109001167A (en) * | 2018-05-21 | 2018-12-14 | 南京医科大学 | A kind of method and kit based on the strand displacement signal of aptamers and carbon dots amplification fluorescent optical sensor detection atriphos |
| CN109975542A (en) * | 2019-02-22 | 2019-07-05 | 中山大学 | A kind of Biomolecule detection kit and biomolecule detecting method |
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| CN113866146A (en) * | 2021-09-29 | 2021-12-31 | 上海交通大学 | Construction of graphene oxide-based aptamer sensor, method for detecting fumonisin B1 and application |
| CN115792231A (en) * | 2022-11-04 | 2023-03-14 | 山东大学 | DNase I biosensor based on enzyme cascade reaction regulated by thrombin aptamer |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107764790A (en) * | 2017-10-10 | 2018-03-06 | 广西师范学院 | Method based on enzyme and graphene oxide aptamer sensor detection fibrin ferment |
| CN107764790B (en) * | 2017-10-10 | 2020-01-10 | 广西师范学院 | Method for detecting thrombin based on enzyme and graphene oxide aptamer sensor |
| CN107843631A (en) * | 2017-12-25 | 2018-03-27 | 安阳师范学院 | Protease detection electrochemical sensor and preparation method and detection method |
| WO2019168467A1 (en) * | 2018-02-27 | 2019-09-06 | Agency For Science, Technology And Research | A method and system for determining membrane protein recycling rates |
| CN109001167A (en) * | 2018-05-21 | 2018-12-14 | 南京医科大学 | A kind of method and kit based on the strand displacement signal of aptamers and carbon dots amplification fluorescent optical sensor detection atriphos |
| CN108866063A (en) * | 2018-06-28 | 2018-11-23 | 中国水产科学研究院珠江水产研究所 | A kind of aptamer and its preparation method and application of PEG modification |
| CN109975542A (en) * | 2019-02-22 | 2019-07-05 | 中山大学 | A kind of Biomolecule detection kit and biomolecule detecting method |
| CN113866146A (en) * | 2021-09-29 | 2021-12-31 | 上海交通大学 | Construction of graphene oxide-based aptamer sensor, method for detecting fumonisin B1 and application |
| CN115792231A (en) * | 2022-11-04 | 2023-03-14 | 山东大学 | DNase I biosensor based on enzyme cascade reaction regulated by thrombin aptamer |
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