CN106520913B - Preparation method of graphene oxide-DNA sensor based on enzymatic cleavage and cyclic amplification and its application in detecting thrombin - Google Patents

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

Preparation method and method of graphene oxide-DNA sensor based on enzymatic cleavage cyclic amplification Application in the detection of thrombin

technical field

The invention belongs to the field of protein detection in the field of biomedicine, and relates to a method for detecting thrombin with high sensitivity of a graphene oxide-DNA sensor based on enzymatic cleavage cycle amplification, in particular to a method based on exonuclease Graphene oxide (GO)-aptamer sensor for amplifying detection signal and method for detecting thrombin.

Background technique

Thrombin is a serine protease in the blood, involved in some physiological and pathological reactions of the human body, such as inflammation, wound repair, blood coagulation, platelet activation and other processes. Differences in the amount of thrombin in the blood can cause abnormal coagulation. In addition, thrombin has a close relationship with the development of many diseases and is used as a disease marker. Therefore, the detection of thrombin with high sensitivity in the early stage of clinical diagnosis is particularly important

Aptamers are single-stranded oligonucleotides that can specifically recognize various target molecules such as antibodies, bacteria, proteins, and cells. Nucleic acid aptamers have the characteristics of stable activity, low cost, easy modification, and easy long-term storage. Nucleic acid aptamer-based sensors have good applications in many aspects, such as food safety, drug analysis, environmental monitoring, and biochemical analysis. aspect. Due to the importance of thrombin, the interaction between thrombin and nucleic acid aptamers has also been widely studied. The nucleic acid aptamer (TBA) that can specifically bind to thrombin has high affinity and high selectivity for thrombin. It combines with the surface epitope of human α-thrombin to form a stable G-quadruplex structure. Many sensors for detecting thrombin are developed based on this principle.

At present, signal amplification technology has been increasingly used in the research to improve the detection sensitivity of thrombin, such as gold nano-assisted signal amplification technology, DNase-assisted signal amplification technology, nucleic acid aptamer-GO signal amplification technology, rolling circle amplification technology amplification technology, hybrid chain reaction amplification technology, enzyme labeling amplification technology, exonuclease catalyzed targeted cycle electrochemical technology, etc. Among them, the detection technology based on enzyme cycle amplification signal is one of them. Exonuclease is an enzyme that acts on a single nucleotide. It can sequentially hydrolyze phosphodiester bonds from one end of the sequence to become multiple single nucleotide fragments, so exonuclease can degrade and bind to the target protein. The nucleic acid aptamer released from the target protein is recombined with other nucleic acid aptamers. This cycle of use provides a good new method to achieve high-sensitivity detection.

In this study, a detection technique combining exonuclease-catalyzed targeted cycling technology with a GO sensor was developed to improve the sensitive detection of thrombin.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for detecting thrombin by a GO sensor based on the cyclic amplification signal of enzymatic cleavage. The capture-DNA is immobilized on the surface of GO by covalent connection, and then hybridized with a FAM-modified thrombin aptamer (TBAaptamer). , and blocked non-specific sites on the GO surface with a small amount of PEG, the thrombin nucleic acid aptamer immobilized on the GO surface was quenched fluorescence and effectively avoided the digestion of exonuclease. When thrombin is specifically bound to the aptamer, the thrombin-aptamer complex leaves the surface of GO, and the exonuclease recognizes the aptamer in the hydrolysis complex, releases thrombin, and realizes the recycling of thrombin. The signal gradually increases. On the basis of covalent binding, the sensor avoids false positive signals, introduces enzyme-catalyzed targeted cycling technology, and develops a new high-efficiency, convenient, and highly sensitive thrombin detection technology. Thrombin for detection.

The preparation method of the graphene oxide-DNA sensor based on enzymatic cleavage cycle amplification includes the following steps:

(1) Preparation of GO: GO was prepared by the modified Hummers method, GO was vacuum dried for use, and before use, ultrasonically dispersed in an aqueous solution to obtain a GO dispersion;

(2) Activation of GO: Mix the aqueous solution A containing 50 mM NHS and 200 mM EDC with the 2 mg/ml GO dispersion prepared in step (1), then add ultrapure water, react at room temperature for 0.5 hours, then elute, and store for later use ;

(3) Preparation of GO-aptamer sensor: first connect the aminated capture DNA to the GO surface obtained in step (2), then dropwise add TBA, due to the specificity of TBA, combine with capture DNA, and then dropwise add PEG, namely The GO-DNA sensor was prepared. GO was able to quench the fluorescence of TBA end-labeled FAM.

In step (2), the volume ratio of the aqueous solution A, the GO dispersion liquid and the ultrapure water is 1:2:1.

In step (3), the sequence of the capture DNA is: 5'-NH2-AGTCACCCCAACCTG CCCTACCACGGACT--3', and the capture DNA itself will form a stem-loop structure.

In step (3), the sequence of the TBA is: 5'AAAA GTCCG TG GTAGGGCA GGTTGGGGTGA CT-FAM-3', and the single-chain TBA makes it easier for the exonuclease to recognize the single-chain in the thrombin-TBA complex. TBA.

In step (3), the concentration ratio of the capture DNA:TBA is 1:1, and the concentrations are both 10 nM; the concentration of PEG is 50 nM, and the concentration of GO is 0-25 μg/mL.

The graphene oxide-DNA sensor prepared by the invention based on enzymatic cleavage cycle amplification detects thrombin.

The following steps are involved in the detection of thrombin:

S1: Detect the fluorescence value of the GO-DNA sensor;

S2: Thrombin is added to the GO-DNA sensor, then exonuclease is added, or Thrombin-Exonuclease is directly added to the thrombin-exonuclease mixture, and after 30 minutes of reaction, the fluorescence value is detected again; the fluorescence change is analyzed.

After TBA binds to thrombin, the TBA-thrombin complex leaves the surface of GO, and the exonuclease recognizes the aptamer in the hydrolysis complex and releases thrombin, so that the thrombin can be recycled, and the FAM fluorescence signal is gradually enhanced. Detect it.

In step S2, the concentration of thrombin Thrombin is 1 nM; the concentration of exonuclease is 0.03 U/mL.

The present invention has the following advantages:

(1) In the present invention, GO is easy to obtain, the method is simple, and the cost is low. It makes full use of the fact that GO can quench the fluorescence labeled at the end of single-stranded DNA. When the protein binds to the single-stranded DNA, the fluorescence recovers. Sensitivity to detect thrombin.

(2) The present invention uses exonuclease to hydrolyze and cut single-stranded nucleic acid, release the target protein in the complex, and realize the recycling of the target protein, thereby driving the fluorescence signal to gradually increase.

(3) Aminated capture DNA was immobilized to the surface of GO, and a small amount of PEG was used to solve the problem of non-specific adsorption on the surface of GO, thereby increasing the specificity of detection and further improving the sensitivity of detection.

Description of drawings

Fig. 1: the schematic flow chart of the present invention;

Figure 2: Sensitivity detection of thrombin based on GO sensor; the concentration of fluorescently labeled nucleic acid aptamer (aptamer) in the figure is 10 nM, and the concentration of graphene oxide is 20 μg/mL.

Figure 3: Selective detection of thrombin based on GO sensor;

Figure 4: The detection diagram of 1pM thrombin by the GO sensor. The curves in the figure from top to bottom are the detection result curves of aptamer(c), aptamer-GO-1pM Thrombin-Exonuclease(b), and aptamer-GO(a).

Figure 5: The detection diagram of 1nM thrombin by the GO sensor, the curves in the figure from top to bottom are aptamer(c), aptamer-GO-1nM Thrombin-Exonuclease(b), and aptamer-GO(a) detection result curve.

Detailed ways

The present invention will be further described below with reference to the examples, which are used to illustrate the present invention rather than to limit the scope of the present invention.

Example 1:

(1) Preparation of GO: GO was prepared in large quantities by the modified Hummers method. In a three-necked flask, 3 g of flake graphite powder, 1.5 g of NaNO ₃ and 69 mL of concentrated sulfuric acid were added and stirred in a constant temperature water bath. After 1 hour of reaction, 1 g of KMnO ₄ was added, and 150 mL of deionized water was added after 5 hours of reaction at 35°C. After reacting for 30 min at a temperature of 98 °C, 50 mL of deionized water, 5 mL of H ₂ O ₂ and 250 mL of 10% dilute hydrochloric acid were added, and the solution was poured into a 1000 mL large beaker and washed to a pH of 5-6. The oxidized product was vacuum-dried for use, and was sonicated at 1000 W in an aqueous solution for 30 min before use. GO activation: 0.5ml containing 50mM NHS, 200mMEDC and 1ml 2mg/ml GO, then add 0.5ml ultrapure water, react at room temperature for 0.5 hours and then elute, store for later use

(2) Synthesize a specific aptamer sequence: thrombin nucleic acid aptamer sequence: capture DNA: 5'-NH2-AGTCACCCCAACCTGCCC TACCACGGACT--3'. The underlined portion is what makes the DNA form a stem-loop structure. TBA: 5'AAAAGTCCG TG GTAGGGCA GGTTGGGGTGA CT-FAM-3', (purchased from Shanghai Bioengineering Co., Ltd.) The underlined part is to make it easier for exonuclease to recognize the single-stranded TBA in the thrombin-TBA complex.

(3) Fluorescence quenching: The aminated capture DNA and TBA-aptamer were immobilized in the activated GO aqueous solution, that is, a GO-aptamer sensor was prepared; GO could quench the fluorescence of TBA-aptamer end-labeled FAM; capture DNA: TBA-aptamer is 1:1, the concentration is 10 nM; the concentration of GO is 20 μg/mL;

(4) Detection of thrombin based on GO sensor: 1ml reaction system contains 10nM capture-DNA, 20μg/ml activated GO, (adding 50nmol PEG to prevent the non-specific adsorption of Thrombin/TBA to GO), react for 3-5h After that, Thrombin/Thrombin-Exonuclease (1nM thrombin/0.03UmL ^-1 exonuclease) was added, and the reaction was carried out at room temperature for 30min, and the change of fluorescence value was analyzed.

The fluorescence intensity was detected by Synergy H4 produced in the United States. The excitation light was 488 nm, the emission light was 518 nm, the emission wavelength was 510-650 nm, and the fluorescence intensity value at 520 nm was observed. As shown in Figure 2, the concentration of FAM-labeled aptamer is 10 nM, and the concentration of GO is 20 μg/mL. Detection found that the sensitivity can reach 1pM.

Figure 1 is a schematic flow chart of the present invention. In the figure, TBA-aptamer is combined with thrombin to form a TBA-thrombin complex, the structure of the aptamer itself changes, and it is separated from the surface of GO, and the exonuclease in the solution recognizes the TBA-thrombin complex. The single-chain TBA in the body is hydrolyzed to release thrombin. The free thrombin continues to bind to the TBA on the surface of GO, thereby increasing the fluorescence signal.

(5) Select other proteins lysozyme, BSA, and IgG that interact non-specifically with TBA-aptamer for selective detection. Under the same experimental conditions, it is found that GO-aptamer can specifically bind to thrombin, and can clearly distinguish it from other proteins. The difference in protein is shown in Figure 3. Experiments show that the detection method has good selectivity for thrombin. The concentration of FAM-labeled aptamer in the figure is 10 nM, and the concentration of GO is 20 μg/mL.

Example 2:

Steps (1), (2) and (3) are the same as in Example 1.

(4) Detection of thrombin by GO sensor based on enzyme digestion and amplification: Thrombin/Thrombin-Exonuclease (1nM thrombin/0.03UmL-1exonuclease) was added to step (3), and the reaction was carried out at room temperature for 30min. Synergy H4 produced in the United States was used to measure the fluorescence intensity. Detection, the excitation light is 485nm, the emission light is 518nm, the emission wavelength is 510-650nm, the fluorescence intensity value at 520nm is observed, and the intensity of the labeled FAM on the aptamer is analyzed by Origin 8.0, and it is found that the detection sensitivity reaches 1pM.

Example 3:

Steps (1), (2) and (3) are the same as in Example 1.

(4) Detection of thrombin by the GO sensor based on enzyme digestion and amplification: Thrombin/Thrombin-Exonuclease (1nM thrombin/0.03UmL ^-1 exonuclease) was added to step (3), and the reaction was carried out at room temperature for 30min. Synergy H4 produced in the United States changed the fluorescence intensity For detection, the excitation light is 485nm, the emission light is 518nm, the emission wavelength is 510-650nm, the fluorescence intensity value at 520nm is observed, and the intensity of FAM labeled on the aptamer is analyzed by Origin 8.0, and it is found that the detection sensitivity reaches 0.5nM.

SEQUENCE LISTING

<110> Jiangsu University

<120> Preparation method of graphene oxide-DNA sensor based on enzymatic cleavage cyclic amplification and detection of thrombin

Applications

<130> Preparation method of graphene oxide-DNA sensor based on enzymatic cleavage and cyclic amplification and detection of thrombin

Applications

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> DNA

<213> Artificial sequences

<400> 1

agtcacccca acctgcccta ccacggact 29

<210> 2

<211> 32

<212> DNA

<213> Artificial sequences

<400> 2

aaaagtccgt ggtagggcag gttggggtga ct 32

Claims (9)

1. the preparation method of the graphene oxide-DNA sensor based on enzymatic cleavage cycle amplification, is characterized in that, comprises the steps: (1) preparation of graphene oxide: graphene oxide is prepared by the improved Hummers method, the graphene oxide is vacuum-dried for subsequent use, and before use, ultrasonically dispersed in an aqueous solution to obtain a graphene oxide dispersion; (2) Activation of graphene oxide: Mix the aqueous solution A containing 50 mM NHS and 200 mM EDC with the 2 mg/ml graphene oxide dispersion obtained in step (1), add ultrapure water, and react at room temperature for 0.5 Elute after hours and store for later use; (3) First connect the aminated capture DNA to the graphene oxide surface obtained in step (2), then dropwise add TBA, due to the specificity of TBA, combine with capture DNA, and then dropwise add PEG to obtain graphene Oxide-DNA sensor. 2. the preparation method of the graphene oxide-DNA sensor based on enzymatic cleavage cycle amplification according to claim 1, is characterized in that, in step (2), described aqueous solution A, graphene oxide dispersion liquid and ultrapure The volume ratio of water is 1:2:1. 3. the preparation method of the graphene oxide-DNA sensor based on enzyme digestion cycle amplification according to claim 1, is characterized in that, in step (3), the sequence of described capture DNA is: 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, is characterized in that, in step (3), the sequence of described TBA is: 5' AAAA GTCCG TG GTAGGGCA GGTTGGGGTGA CT-FAM-3'. 5. the preparation method of the graphene oxide-DNA sensor based on enzyme digestion cycle amplification according to claim 1, is characterized in that, in step (3), the concentration ratio of described capture DNA:TBA is 1:1, The concentrations were all 10 nM; the concentration of PEG was 50 nM, and the concentration of graphene oxide was 20-25 μg/mL. 6. The graphene oxide-DNA sensor based on enzymatic cleavage cycle amplification, wherein the graphene oxide-DNA sensor is amplified based on enzymatic cleavage cycle as described in any one of claims 1-5 The preparation method of graphene oxide-DNA sensor is obtained. 7. the application of the graphene oxide-DNA sensor based on enzyme digestion cycle amplification as claimed in claim 6, is characterized in that, described graphene oxide-DNA sensor is used for the detection coagulation of non-diagnostic purpose enzymes. 8. the application of the graphene oxide-DNA sensor based on enzymatic cleavage cycle amplification according to claim 7, is characterized in that, comprises the following steps when detecting thrombin for non-diagnosis and treatment purpose: S1: Detect the fluorescence value of the graphene oxide-DNA sensor; S2: Add thrombin to the graphene oxide-DNA sensor, then add exonuclease, or directly add a mixture of thrombin-exonuclease, and after 30 minutes of reaction, detect the fluorescence value; analyze the fluorescence change. 9. The application of the graphene oxide-DNA sensor based on enzymatic digestion cycle amplification according to claim 8, wherein in step S2, the concentration of thrombin is 1 nM; the concentration of exonuclease is 0.03U/mL .

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