CN111690722B - ATP detection nucleic acid sensor based on entropy driving and hybrid chain reaction and preparation method thereof - Google Patents
ATP detection nucleic acid sensor based on entropy driving and hybrid chain reaction and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an ATP detection nucleic acid sensor based on entropy driving and hybrid chain reaction and a preparation method thereof. The nucleic acid sensor comprises 5 probes, namely H, F, A/B/S, H1 and H2; the invention is based on ATP aptamer conformation transition, releases initiation chain, drives entropy to drive catalytic amplification primary circulation and hybridization chain reaction secondary circulation, and finally detects ATP molecules through fluorescence signal enhancement. The nucleic acid sensor has the advantages of rapid reaction, high sensitivity, strong anti-interference capability, mild reaction conditions and the like, can make up for the defects of the existing ATP detection method, and realizes the rapid and accurate quantitative detection of ATP.
Description
Technical Field
The invention belongs to the field of nucleic acid sensors, and particularly relates to an ATP detection nucleic acid sensor based on entropy driving and hybridization chain reaction and a preparation method thereof, in particular to an entropy driving circuit coupling hybridization chain reaction double amplification circuit based on nucleic acid aptamer regulation and a fluorescence biosensor for fluorescence detection of ATP.
Background
ATP has important reference significance in clinical medical diagnosis, the content of ATP is often related to the health degree of organisms, and the content of ATP in human serum is about 1 mu mol.L -1 When the content of ATP is abnormal, various diseases such as cardiovascular and cerebrovascular diseases, Parkinson's syndrome, ischemia, hypoglycemia, malignant tumors and the like can be caused, and meanwhile, ATP has wide clinical application and can provide important auxiliary treatment for diseases such as cerebral hemorrhage sequelae, myocardial diseases, chronic hepatitis and the like. Therefore, the rapid, accurate, sensitive and specific ATP detection method is designed, which is not only beneficial to promoting the exploration of the mysteries of life on the molecular level, but also has important significance for the analysis of the latest drugs, clinical diagnosis, overcoming of a plurality of difficult and complicated diseases and the like, and has potential application value in the fields of environmental science, biology, medicine and the like.
Disclosure of Invention
The invention aims to provide an ATP detection nucleic acid sensor based on entropy driving and hybrid chain reaction and a preparation method thereof.
The following technical scheme is adopted for achieving the purpose:
the invention provides a nucleic acid sensor for detecting ATP cascade method, an upstream soil moisture driving circuit comprises: reconstructing a probe H, pre-assembling a template probe A/B/S and a stimulating probe F; the downstream hybridization chain reaction circuit comprises: fluorescent dye-modified hairpin probes H1 and hairpin probe H2. The probe sequences are respectively as follows:
and (3) reconstructing a probe H:
CAGGTTACCCTACGTCTCCATAGGGTAACCTGGGGGAGTATTGCGGAGGAAGGT；
pre-assembling a template probe A: CCACATACATCATATTCCCTCAGGTTACCCTACG, respectively;
pre-assembling a template probe B: GTCACTCGATCCAATCTACAGCTCTCTTACGG;
pre-assembling a template probe S:
TGGAGACGTAGGGTAACCTGAGGGCCGTAAGAGAGCTGTAGATTGGATCG；
stimulation probe F: CGATCCAATCTACAGCTCTCTTACGGCCCTCATTC AATACCCTACG, respectively;
hairpin probe H1:
CGATCCAA(FAM)TCTACAGCAGATGTGTAGCTGTAGA(Dabcy 1)TTGGATCGAGTGAC；
hairpin probe H2: TACACATCTGCTGTAGATTGGATCGGTCACTCGATCCAATCTACAGC, respectively;
the preparation method of the nucleic acid sensor comprises the following steps:
(1) dissolving the probe A, B, S in Tris-HCl buffer solution, annealing for 8 minutes at 95 ℃ to ensure that the probe is completely hybridized to obtain a preassembled template probe A/B/S mixed solution;
(2) pre-dissolving a hairpin probe H1 in a Tris-HCl buffer solution, and annealing at 95 ℃ for 8 minutes to obtain a stable H1 hairpin solution;
(3) pre-dissolving a hairpin probe H2 in a Tris-HCl buffer solution, and annealing at 95 ℃ for 8 minutes to obtain a stable H2 hairpin solution;
(4) pre-dissolving the reconstructed hairpin probe H in Tris-HCl buffer solution, and annealing for 8 minutes at 95 ℃ to obtain stable H hairpin solution;
(5) and (3) mixing the solutions prepared in the steps (1), (2), (3) and (4), and adding a stimulation probe F to obtain a sensing solution.
The Tris-HCl buffer contained the following final concentration components: 5 mM Tris-HCl, 40 mM sodium chloride, pH 7.4; in the sensing solution, the final concentration of each probe was 200 nM.
The working principle of the sensor is shown in figure 1:
in the presence of ATP, the recognition probe H undergoes conformational transition, a hidden HI region of an H chain is exposed, and the 3-terminal base of HI is not hybridized with the 5-terminal base of S; and then HI continues to be complementarily matched with the subsequent structure of S, the base at the position is the original hybridization part of B and A, and A is replaced to form an HI/B/S hybrid. In this case, the S-middle part of the hybrid T/B/S has a 4-base unhybridized portion. F in the solution can be hybridized with the 4 bases, and is gradually hybridized with S towards two ends by taking the F as a fulcrum, HI and B originally hybridized with S are gradually replaced, so that HI and B are released, and the first cycle process is completed. The HI released into the solution can be continuously repeated in the first cycle process to continuously release B, so that signal amplification is realized.
The 5-terminus of B released into solution can hybridize with the 3' -end of hairpin H1, followed by gradual hybridization with the neck of H1, opening hairpin H1, forming a hybrid of B/H1. The 5 'end of H1 of the formed B/H1 hybrid can be continuously hybridized with the 3' end base of H2, so that the hairpin structure of H2 is opened, the B/H1/H2 hybrid is formed, the 5 'end of H2 can be continuously opened to the 3' end of H1, and the cycle is repeated to realize signal amplification. The stem position of H1 of the hairpin structure is modified with a fluorescent group FAM, and a complementary site thereof is modified with a quenching group Dabcy 1. While H1 maintains the hairpin structure, the fluorescence of FAM is quenched, with no fluorescent signal. When B opens H1 and further opens H2 to form a B/H1/H2/H1/H2/H1/H2/H1/H2 … … hybrid, FAM is far away from Dabcy 1, FAM fluorescence is recovered, and fluorescence signals are enhanced. The concentration of ATP in the solution can be measured by measuring the increase in the fluorescent signal.
Further, the application of the nucleic acid sensor to ATP ions comprises the following steps:
(1) respectively adding ATP standard solutions with different concentrations and solutions to be detected into a sensing solution, uniformly mixing, and incubating for 1 hour at 37 ℃ in a constant-temperature metal bath;
(2) taking out each group of mixed solution from the constant-temperature metal bath, and measuring the fluorescence value of each mixed solution;
(3) and (3) making a standard curve of the ATP concentration to the fluorescence value according to the fluorescence values of the ATP standard solutions with different concentrations, calculating a regression equation, and finally calculating the ATP concentration according to the fluorescence value of the sample to be detected.
And (3) the fluorescence value is measured, the excitation wavelength is 488 nm, and the emission wavelength is 520 nm.
ATP detection concentration is 0.1. mu.M-2. mu.M.
The invention has the beneficial effects that: the invention provides a nucleic acid sensor for detecting ATP cascade signal amplification and a preparation method thereof, which realizes high specificity identification of ATP by utilizing ATP aptamer conformation transformation; the entropy is utilized to drive the catalytic amplification primary circulation and the hybridization chain reaction secondary circulation, so that the fluorescence signal is amplified, and the ultra-sensitive detection of the target ATP is realized; the reaction condition is mild, the reaction speed is high, the anti-interference capability is strong, and the detection sensitivity is improved.
Drawings
FIG. 1 is a diagram showing the operation of a nucleic acid biosensor constructed according to the present invention.
FIG. 2 is a standard curve of ATP concentration-fluorescence intensity in the examples.
FIG. 3 is a bar graph of the selectivity of the sensor for different interfering analytes.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments and the accompanying drawings, but the present invention is not limited by the following embodiments.
Seven DNA probes, namely, a pre-assembled template probe (hereinafter referred to as A, B and S), a reconstituted probe (hereinafter referred to as H), a stimulation probe (hereinafter referred to as F), a fluorescent dye Dabcy 1, a FAM-modified hairpin probe 1 (hereinafter referred to as H1) and a hairpin probe 2 (hereinafter referred to as H2), were designed in the examples of the present invention.
Example 1
The sequence design of the preassembly template probe, the reconstruction probe (ATP recognition DNA probe), the stimulation probe, the fluorescent dye Dabcy 1 and the FAM modified hairpin probe and the hairpin probe is shown in the table 1;
TABLE 1 complete sequence of sensors
The preparation method of the nucleic acid sensor comprises the following steps:
(1) dissolving the probe A, B, S in Tris-HCl buffer solution, annealing for 8 minutes at 95 ℃ to ensure that the probe is completely hybridized to obtain a preassembled template probe A/B/S mixed solution;
(2) pre-dissolving a hairpin probe H1 in a Tris-HCl buffer solution, and annealing at 95 ℃ for 8 minutes to obtain a stable H1 hairpin solution;
(3) pre-dissolving a hairpin probe H2 in a Tris-HCl buffer solution, and annealing at 95 ℃ for 8 minutes to obtain a stable H2 hairpin solution;
(4) pre-dissolving the reconstructed hairpin probe H in Tris-HCl buffer solution, and annealing at 95 ℃ for 8 minutes to obtain stable H hairpin solution;
(5) and (3) mixing the solutions prepared in the steps (1), (2), (3) and (4), and adding a stimulation probe F to obtain a sensing solution. The final concentration of each component was 200 nM.
EXAMPLE 2 annealing of hairpin probes
The hairpin probe solution was prepared at a concentration of 2. mu.M using Tris-HCl buffer, and 1. mu.L of hairpin probe (100. mu.M) and 49. mu.L of Tris-HCl buffer (5 mM Tris-HCl, 40 mM sodium chloride, pH 7.4) were added to one 200. mu.L centrifuge tube. The centrifuge tubes were kept at 95 ℃ for 8 minutes in a thermostatted metal bath and then rapidly placed in ice water for 30 minutes to allow the formation of stable secondary structures.
EXAMPLE 3 detection Limit of sensor
The ATP standard solutions of different concentrations were detected by the sensor of example 3, and the detection range was determined. The method comprises the following specific steps:
(1) standard solutions of ATP were prepared at different concentrations.
(2) mu.L of the sensing solution was mixed with 5. mu.L of ATP standards of different concentrations, and then 35. mu.L of Tris-HCl buffer was added to 6 centrifuge tubes, respectively, to a final concentration of 0.1. mu.M, 0.2. mu.M, 0.5. mu.M, 1. mu.M and 2. mu.M ATP. The solution was shaken for 10 seconds to homogenize the solution, and the mixture was placed in a constant temperature metal bath and reacted at 37 ℃ in the dark for 1 hour.
(3) The mixed solutions of each group were taken out from the constant temperature metal bath, and the fluorescence intensity value at 520 nm of the mixed solutions of each group was measured.
(4) And (3) according to the fluorescence intensity values of the ATP standard solutions with different concentrations, making a standard curve of the ATP concentration-fluorescence intensity values, and calculating a regression equation.
(5) The standard curve is shown in fig. 2: the linear range of the standard curve is 0.1-2 mu M, and the detection limit is 38 nM.
Example 4 sensor interference rejection verification
(1) Control solutions (GTP, CTP, UTP) containing different analogues were prepared at a concentration of 20. mu.M.
(2) According to the procedure of example 3, 10. mu.L of the sensing solution was mixed with 5. mu.L of different analog solutions, and then 35. mu.L of Tris-HCl buffer was added to each of the 3 centrifugal tubes to a solution volume of 50. mu.L and an analog concentration of 20. mu.M. The solution was shaken for 10 seconds to homogenize the solution, and the mixture was placed in a constant temperature metal bath and reacted at 37 ℃ in the dark for 1 hour.
(3) The mixed solutions of each group were taken out from the constant temperature metal bath, and the fluorescence intensity value at 520 nm of the mixed solutions of each group was measured.
(4) Histogram of fluorescence value versus interfering analyte species is made
(5) The sensor interference rejection capability is shown in fig. 3: the sensor has no response ability to GTP, CTP, UTP and the like, and has selectivity to ATP.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Minjiang academy
<120> ATP detection nucleic acid sensor based on entropy driving and hybrid chain reaction and preparation method thereof
<130> 7
<160> 7
<170> PatentIn version 3.3
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caggttaccc tacgtctcca tagggtaacc tgggggagta ttgcggagga aggt 54
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tggagacgta gggtaacctg agggccgtaa gagagctgta gattggatcg 50
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ccacatacat catattccct caggttaccc tacg 34
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gtcactcgat ccaatctaca gctctcttac gg 32
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<213> Artificial sequence
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cgatccaatc tacagctctc ttacggccct cattcaatac cctacg 46
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cgatccaatc tacagcagat gtgtagctgt agattggatc gagtgac 47
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<212> DNA
<213> Artificial sequence
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tacacatctg ctgtagattg gatcggtcac tcgatccaat ctacagc 47
Claims (4)
1. An ATP-detecting nucleic acid sensor based on entropy-driven and hybrid strand reactions, characterized in that an upstream moisture-driving circuit comprises: reconstructing a probe H, pre-assembling a template probe A/B/S and a stimulating probe F; the downstream hybridization strand reaction circuit comprises: fluorescent dye-modified hairpin probes H1 and H2; the probe sequences are respectively as follows:
and (3) reconstructing a probe H:
CAGGTTACCCTACGTCTCCATAGGGTAACCTGGGGGAGTATTGCGGAGGAAGGT；
pre-assembling a template probe A: CCACATACATCATATTCCCTCAGGTTACCCTACG;
pre-assembling a template probe B: GTCACTCGATCCAATCTACAGCTCTCTTACGG;
pre-assembling a template probe S:
TGGAGACGTAGGGTAACCTGAGGGCCGTAAGAGAGCTGTAGATTGGATCG；
stimulation probe F:
CGATCCAATCTACAGCTCTCTTACGGCCCTCATTC AATACCCTACG；
hairpin probe H1:
CGATCCAA(FAM)TCTACAGCAGATGTGTAGCTGTAGA(Dabcy1)TTGGATCGAGTGAC；
hairpin probe H2:
TACACATCTGCTGTAGATTGGATCGGTCACTCGATCCAATCTACAGC；
the preparation method of the ATP detection nucleic acid sensor based on entropy driving and hybridization chain reaction comprises the following steps:
(1) dissolving the pre-assembled template probe A, B, S in Tris-HCl buffer solution, annealing for 8 minutes at 95 ℃ to ensure that the pre-assembled template probe A/B/S mixed solution is obtained after complete hybridization;
(2) pre-dissolving a hairpin probe H1 in a Tris-HCl buffer solution, and annealing at 95 ℃ for 8 minutes to obtain a stable H1 hairpin solution;
(3) pre-dissolving the hairpin probe H2 in Tris-HCl buffer solution, and annealing for 8 minutes at 95 ℃ to obtain stable H2 hairpin solution;
(4) pre-dissolving the reconstructed hairpin probe H in Tris-HCl buffer solution, and annealing at 95 ℃ for 8 minutes to obtain stable H hairpin solution;
(5) and (3) mixing the solutions prepared in the steps (1), (2), (3) and (4), and adding a stimulation probe F to obtain a sensing solution.
2. An ATP-detecting nucleic acid sensor based on entropy-driven and hybridized strand reactions according to claim 1, wherein Tris-HCl buffer is composed of: 5 mM Tris-HCl, 40 mM sodium chloride, pH 7.4; in the sensing solution, the final concentration of each probe was 200 nM.
3. Use of the ATP detection nucleic acid sensor based on entropy-driven and hybrid strand reactions of claim 1 in the preparation of a reagent for detecting ATP, comprising the steps of:
(1) respectively adding ATP standard solutions with different concentrations and a sample to be detected into the sensing solution, and incubating for 1h at 37 ℃ in a constant-temperature metal bath;
(2) taking out each group of standard sample solution, and measuring the fluorescence value of each standard solution by using a fluorescence spectrometer; and obtaining an ATP concentration-fluorescence intensity standard curve;
(3) and calculating the concentration of ATP to be detected according to the fluorescence value of the sample to be detected.
4. An ATP detection nucleic acid sensor based on entropy-driven and hybridized chain reaction of claim 3, wherein the fluorescence value in step (2) is measured with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.
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