CN106520913B - 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 field of protein detection in the field of biomedicine, and relates to a method for detecting thrombin (thrombin) with high sensitivity by using a graphene oxide-DNA sensor based on enzyme digestion cycle amplification, in particular to a Graphene Oxide (GO) -aptamer (aptamer) sensor based on an exonuclease amplified detection signal and a method for detecting thrombin by using the same.
Background
Thrombin is a serine protease in blood, and is involved in physiological and pathological reactions of human body, such as: inflammation, wound repair, blood clotting, platelet activation, and the like. Differences in thrombin levels in blood can cause abnormal coagulation functions. In addition, thrombin has a close relationship with the development of many diseases and is used as a disease marker, and therefore, high sensitivity in the early stage of clinical diagnosis is particularly important for thrombin detection
The aptamer is a single-chain oligonucleotide and can specifically recognize various target molecules such as antibodies, bacteria, proteins and cells, and the aptamer has the characteristics of stable activity, low cost, easy modification, easy long-term storage and the like, and a sensor based on the aptamer has good application in many aspects such as food safety, drug analysis, environmental monitoring, biochemical analysis and the like.
At present, signal amplification technologies are increasingly used in research for improving the detection sensitivity of thrombin, such as gold nanoparticle-assisted signal amplification technology, dnase-assisted signal amplification technology, aptamer-GO signal amplification technology, rolling circle amplification technology, hybrid chain reaction amplification technology, enzyme labeling amplification technology, exonuclease-catalyzed targeted circular electrochemical technology, and the like. Among them, a detection technique based on an enzyme cycle amplification signal is one of them. Exonuclease (exoclease) is an enzyme acting on a single nucleotide, and can hydrolyze phosphodiester bonds from one end of a sequence in sequence to form a plurality of single nucleotide fragments, so that the Exonuclease can degrade an aptamer combined with a target protein, release the target protein, and be combined with other aptamers, and the Exonuclease is recycled, thereby providing a novel method for realizing high-sensitivity detection.
The present study develops a detection technique that combines the targeted cycling technique catalyzed by exonuclease with the GO sensor to improve the sensitivity detection of thrombin.
Disclosure of Invention
The invention aims to provide a method for detecting thrombin by a GO sensor based on enzyme digestion catalytic cycle amplification signals. After thrombin is specifically combined with the aptamer, the thrombin-aptamer complex leaves the GO surface, exonuclease recognizes and hydrolyzes the aptamer in the complex, thrombin is released, cyclic utilization of thrombin is achieved, and fluorescence signals are gradually enhanced. On the basis of covalent bonding, the sensor avoids false positive signals, introduces an enzyme catalysis targeting circulation technology, develops a new thrombin detection technology with high efficiency, convenience and high sensitivity, and thus detects thrombin with high sensitivity, rapidness and low cost.
The preparation method of the graphene oxide-DNA sensor based on enzyme digestion cycle amplification comprises the following steps:
(1) preparing GO: GO is prepared by an improved Hummers method, vacuum drying is carried out on GO for later use, and before use, the GO is subjected to ultrasonic dispersion in an aqueous solution to obtain a GO dispersion liquid;
(2) activation of GO: mixing an aqueous solution A containing 50mM NHS and 200mM EDC with the 2mg/ml GO dispersion prepared in the step (1), adding ultrapure water, reacting for 0.5 hour at room temperature, eluting, and storing for later use;
(3) preparing a GO-aptamer sensor: and (3) firstly connecting the aminated capture DNA to the surface of the GO prepared in the step (2), then dropwise adding TBA (tert-butyl-N-terminal), combining with the capture DNA due to the specificity of the TBA, and then dropwise adding PEG (polyethylene glycol), thus obtaining the GO-DNA sensor. GO is able to quench fluorescence of TBA end-labeled FAM.
In the step (2), the volume ratio of the aqueous solution A, GO dispersion to the ultrapure water is 1: 2: 1.
in the step (3), the sequence of the capture DNA is as follows: 5 '-NH 2-AGTCACCCCAACCTG CCCTACCACGGACT-3', the capture DNA itself will form a stem-loop structure.
In the step (3), the sequence of the TBA is as follows: 5 'AAAA GTCCG TG GTAGGGCA GGTTGGGGTGA CT-FAM-3', which makes it easier for exonuclease to recognize single-stranded TBA in thrombin-TBA complex.
In the step (3), the capture DNA: concentration ratio of TBA is 1:1, the concentration is 10 nM; the concentration of PEG is 50nM, and the concentration of GO is 0-25 mug/mL.
The prepared graphene oxide-DNA sensor based on enzyme digestion cyclic amplification detects thrombin.
The method for detecting thrombin comprises the following steps:
s1: detecting a fluorescence value of the GO-DNA sensor;
s2: adding Thrombin Thrombin and Exonuclease into the GO-DNA sensor, or directly adding Thrombin-Exonuclease mixture Thrombin-Exonuclease, reacting for 30min, and detecting a fluorescence value; the change in fluorescence was analyzed.
After TBA is combined with thrombin, the TBA-thrombin compound leaves the surface of GO, exonuclease recognizes aptamers in the hydrolyzed compound, thrombin is released, the thrombin is recycled, FAM fluorescence signals are gradually enhanced, and the thrombin is detected according to the change of fluorescence intensity.
In step S2, the concentration of Thrombin Thrombin is 1 nM; the concentration of the exonuclease exouchase is 0.03U/mL.
The invention has the following advantages:
(1) the method has the advantages that GO is easy to obtain, the method is simple, the cost is low, the characteristic that GO can quench fluorescence labeled at the tail end of single-stranded DNA is fully utilized, and when protein is combined with the single-stranded DNA, the fluorescence is recovered, so that the method can be used for detecting the thrombin quickly, specifically and highly sensitively.
(2) The invention adopts exonuclease to carry out hydrolytic cleavage on single-stranded nucleic acid, releases target protein in the compound, realizes the recycling of the target protein and drives the fluorescent signal to be gradually enhanced.
(3) The amination capture DNA is fixedly connected to the GO surface, and a small amount of PEG is used for solving the problem of nonspecific adsorption of the GO surface, so that the detection specificity is increased, and the detection sensitivity is further improved.
Drawings
FIG. 1: the flow chart of the invention is schematic;
FIG. 2: (ii) thrombin sensitivity detection assay based on GO sensor; the concentration of the fluorescently labeled aptamer (aptamer) in the figure was 10nM, and the concentration of graphene oxide was 20. mu.g/mL.
FIG. 3: thrombin selective detection assay based on GO sensor;
FIG. 4: the GO sensor detects 1pM Thrombin, and curves in the graph are aptamer (c), aptamer-GO-1pM Thrombin-Exonase (b) and aptamer-GO (a) detection result curves from top to bottom in sequence.
FIG. 5: the GO sensor detects a graph with 1nM Thrombin, and curves in the graph are an aptamer (c), an aptamer-GO-1nM Thrombin-Exonaclease (b) and an aptamer-GO (a) detection result curve from top to bottom in sequence.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1:
(1) preparing GO: the GO is prepared in large scale by an improved Hummers method, 3g of flake graphite powder and 1.5g of NaNO are added into a three-neck flask3Then the mixture is put into a constant temperature water bath kettle to be stirred with 69mL of concentrated sulfuric acid. After 1h of reaction 1g of KMnO was added4After reaction at 35 ℃ for 5 hours, 150mL of deionized water was added. After reacting at 98 ℃ for 30min, 50mL of deionized water and 5mLH were added2O2And 250mL of 10% diluted hydrochloric acid, the solution was poured into a 1000mL beaker and washed to pH 5-6. The oxidized product was dried in vacuum for use and sonicated in an aqueous solution at 1000W for 30min before use. GO activation: 0.5ml NHS containing 50mM and 200mM EDTA and 1ml 2mg/ml GO, adding 0.5ml ultrapure water, reacting at room temperature for 0.5 hr, eluting, and storing for use
(2) Synthesis of specific aptamer sequence: thrombin aptamer sequence: capture DNA 5 '-NH 2-AGTCACCCCAACCTGCCC TACCACGGACT-3'. The underlined part is to make the DNA form a stem-loop structure. TBA 5 'AAAAGTCCG TG GTAGGGCA GGTTGGGGTGA CT-FAM-3', streaking (available from Shanghai bioengineering, Inc.) is intended to facilitate exonuclease recognition of single-stranded TBA in thrombin-TBA complexes.
(3) Fluorescence quenching: fixing the aminated capture DNA and the TBA-aptamer into an activated GO aqueous solution to prepare a GO-aptamer sensor; GO can quench the fluorescence of FAM labeled by the end of TBA-aptamer; wherein the capture DNA used: TBA-aptamer is 1:1, and the concentration is 10 nM; the concentration of GO is 20 mug/mL;
(4) detecting the thrombin based on the GO sensor: 1ml reaction system contains 10nM capture-DNA and 20. mu.g/ml activated GO (50 nmol PEG is added to prevent non-specific adsorption of GO by Thrombin/TBA), and after 3-5h of reaction, Thrombin/Thrombin-Exonase (1nM Thrombin/0.03 UmL) is added-1exonuclease), reacting for 30min at room temperature, and analyzing the change of the fluorescence value.
The fluorescence intensity is detected by Synergy H4 of Mei-Shi province, the excitation light is 488nm, the emission light is 518nm, the emission wavelength is 510-650nm, and the fluorescence intensity value at 520nm is observed. As shown in FIG. 2, the concentration of FAM labeled aptamer was 10nM and GO was 20. mu.g/mL. The detection finds that the sensitivity can reach 1 pM.
Wherein, FIG. 1 is a schematic flow chart of the invention, in the diagram, TBA-aptamer is combined with thrombin to form a TBA-thrombin complex, the structure of the aptamer changes and is separated from the surface of GO, exonuclease in the solution recognizes single-chain TBA in the TBA-thrombin complex, and the single-chain TBA is hydrolyzed to release thrombin free from thrombin and is continuously combined with TBA on the surface of GO, so that the fluorescence signal is continuously enhanced.
(5) Selecting several other proteins lysozyme, BSA and IgG which have non-specific effects with the TBA-aptamer to carry out selective detection, and finding that the GO-aptamer can specifically bind to the thrombin and can obviously distinguish differences with other proteins under the same experimental condition, as shown in figure 3, experiments prove that the detection method has good selectivity on the thrombin, the concentration of the FAM-labeled aptamer in the figure is 10nM, and the concentration of GO is 20 mug/mL.
Example 2:
steps (1), (2) and (3) were the same as in example 1.
(4) Detecting the thrombin by using a GO sensor based on enzyme digestion amplification: adding Thrombin/Thrombin-Exonase (1nM Thrombin/0.03UmL-1 Exonase) into the step (3), reacting at room temperature for 30min, detecting the fluorescence intensity by American Synergy H4, wherein the excitation light is 485nM, the emission light is 518nM, the emission wavelength is 510-650nM, observing the fluorescence intensity value at 520nM, analyzing the change of the intensity of FAM marked on the aptamer through Origin 8.0, and finding that the detection sensitivity reaches 1 pM.
Example 3:
steps (1), (2) and (3) were the same as in example 1.
(4) Detecting the thrombin by using a GO sensor based on enzyme digestion amplification: adding Thrombin/Thrombin-Exonase (1nM Thrombin/0.03 UmL) to step (3)-1Exonuclease) reacting for 30min at room temperature, detecting the fluorescence intensity by American Synergy H4, observing the fluorescence intensity value at 520nM, analyzing the change of the intensity of FAM marked on the aptamer through Origin 8.0, and finding that the detection sensitivity reaches 0.5 nM.
SEQUENCE LISTING
<110> university of Jiangsu
<120> preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection
Application of
<130> preparation method of graphene oxide-DNA sensor based on enzyme digestion cycle amplification and application of graphene oxide-DNA sensor in thrombin detection
Application of
<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 based on enzyme digestion cycle amplification is characterized by comprising the following steps:
(1) preparing graphene oxide: preparing graphene oxide by an improved Hummers method, drying the graphene oxide in vacuum for later use, and performing ultrasonic dispersion in an aqueous solution before use to obtain a graphene oxide dispersion liquid;
(2) activation of graphene oxide: mixing an aqueous solution A containing 50mM NHS and 200mM EDC with the graphene oxide dispersion liquid prepared in the step (1) at a concentration of 2mg/ml, adding ultrapure water, reacting at room temperature for 0.5 hour, eluting, and storing for later use;
(3) connecting aminated capture DNA to the surface of the graphene oxide prepared in the step (2), then dropwise adding TBA (tert-butyl-ammonium-methacrylate) and combining the capture DNA due to the specificity of the TBA, and then dropwise adding PEG (polyethylene glycol) to obtain the graphene oxide-DNA sensor.
2. The method for preparing a graphene oxide-DNA sensor based on enzyme digestion cycle amplification according to claim 1, wherein in the step (2), the volume ratio of the aqueous solution A to the graphene oxide dispersion liquid to the ultrapure water is 1: 2: 1.
3. the method for preparing the graphene oxide-DNA sensor based on enzyme digestion cycle amplification according to claim 1, wherein in the step (3), the sequence of the capture DNA is as follows: 5 '-NH 2-AGTCACCCCAACCTG CCCTACCACGGACT-3', the capture DNA itself will form a stem-loop structure.
4. The preparation method of the graphene oxide-DNA sensor based on enzyme digestion cycle amplification according to claim 1, wherein in the step (3), the sequence of the TBA is as follows: 5 'AAAA GTCCG TG GTAGGGCA GGTTGGGGTGA CT-FAM-3'.
5. The method for preparing the graphene oxide-DNA sensor based on enzyme digestion cycle amplification according to claim 1, wherein in the step (3), the capture DNA: concentration ratio of TBA is 1:1, the concentration is 10 nM; the concentration of PEG is 50nM, and the concentration of graphene oxide is 20-25 mug/mL.
6. The graphene oxide-DNA sensor based on enzyme digestion cycle amplification is characterized by being prepared by the preparation method of the graphene oxide-DNA sensor based on enzyme digestion cycle amplification according to any one of claims 1 to 5.
7. The use of the graphene oxide-DNA sensor based on amplification of the digestion cycle according to claim 6, wherein the graphene oxide-DNA sensor is used for detecting thrombin for non-diagnostic purposes.
8. The application of the graphene oxide-DNA sensor based on enzyme digestion cycle amplification according to claim 7, wherein the step of detecting thrombin for non-diagnosis and treatment purposes comprises the following steps:
s1: detecting the fluorescence value of the graphene oxide-DNA sensor;
s2: adding thrombin into the graphene oxide-DNA sensor, adding exonuclease or directly adding a thrombin-exonuclease mixture, reacting for 30min, and detecting a fluorescence value; the change in fluorescence was analyzed.
9. The use of the graphene oxide-DNA sensor based on amplification by enzymatic digestion cycle of claim 8, wherein in step S2, the concentration of thrombin is 1 nM; the concentration of exonuclease was 0.03U/mL.
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| 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 |
| CN107843631B (en) * | 2017-12-25 | 2019-08-23 | 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 |
| CN109001167B (en) * | 2018-05-21 | 2021-02-19 | 南京医科大学 | Method and kit for detecting Adenosine Triphosphate (ATP) by using strand displacement signal amplification fluorescent sensor based on aptamer and carbon dot |
| 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 |
| CN115792231B (en) * | 2022-11-04 | 2023-07-25 | 中拓生物有限公司 | DNase I biosensor based on thrombin aptamer-regulated enzyme cascade reaction |
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