CN111705113A - Functional nucleic acid fluorescence sensor and application thereof in lead ion detection - Google Patents
Functional nucleic acid fluorescence sensor and application thereof in lead ion detection Download PDFInfo
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Abstract
The invention relates to a functional nucleic acid fluorescence sensor and application thereof in lead ion detection, belonging to the technical field of biochemistry. The deoxyribozyme and the molecular beacon designed by the invention can be used as a recognition element, a signal amplification mediation element and a signal emission element of lead ions. The method comprises the following specific steps: hybridizing and mixing deoxyribozyme and ribozyme substrate in a buffer solution, and adding a molecular beacon, DNA polymerase, restriction endonuclease, polynucleotide kinase, dTNPs and a solution to be detected; incubating the mixed system at constant temperature under specific conditions, cooling to room temperature, and detecting the fluorescence intensity of the system to realize high-sensitivity rapid detection of lead ions. According to the invention, an automatic isothermal amplification strategy is constructed through the designed deoxyribozymes and molecular beacons, separation operation is not required, the processes of sample adding, incubation and detection are completed in a single tube, and high-sensitivity and rapid detection of lead ions in a sample is realized.
Description
Technical Field
The invention relates to a functional nucleic acid fluorescence sensor and application thereof in lead ion detection, belonging to the technical field of biochemistry.
Background
The GR-5 deoxyribozyme is a single-stranded DNA obtained by an in vitro screening technique, and is a recognition element with high specificity to lead ions. The molecular beacon is a stem-loop oligonucleotide, the basic principle is that a fluorescent group and a quenching group are respectively marked at two ends of the oligonucleotide, and when target DNA and the molecular beacon are hybridized with each other, the energy resonance transfer process of the fluorescent group and the quenching group of the molecular beacon is cut off, so that a fluorescent signal is recovered.
The isothermal nucleic acid signal amplification strategy is a high-efficiency nucleic acid sequence amplification strategy, and does not need precise temperature control equipment and complex operation processes in the signal amplification process. Compared with the PCR technology, isothermal nucleic acid signal amplification has the advantages of simplicity, rapidness, low price and the like, and has huge potential application prospect and value in high-sensitivity detection of DNA, RNA, cells, proteins, small molecules and metal ions.
Lead ions are one of the dangerous heavy metal pollution sources, seriously harms human health, and particularly has serious influence on the health and intelligence development of young children. And, lead ion (Pb)2+) Pollution is also widely distributed in natural environments, living goods, and foods, and thus highly sensitive detection thereof is required. The traditional detection method needs complex and expensive instruments, a fussy detection process and professional technicians, and is difficult to meet the urgent requirement of people on detecting the content of lead ions with high sensitivity, low price, simplicity, real time and rapidness.
Disclosure of Invention
In view of the above-mentioned defects of the prior art, the present invention provides a functional nucleic acid fluorescence sensor and the application of the fluorescence sensor in lead ion detection.
In order to realize the purpose, the invention designs the deoxyribozyme, the ribozyme substrate and the molecular beacon, constructs a fluorescence analysis method of an isothermal cascade amplification strategy, and realizes high-sensitivity and rapid detection on lead ions.
The functional nucleic acid fluorescence sensor provided by the invention comprises deoxyribozyme, ribozyme substrate, molecular beacon, DNA polymerase, restriction enzyme, polynucleotide kinase and dTNPs.
The deoxyribozyme sequence is shown in SEQ No. 1; it is designated GR-5E1 in the present invention for convenience of description.
The ribozyme substrate sequence is shown as SEQ No.2, wherein rA is adenine ribonucleotide; it is designated GR-5S1 in the present invention for convenience of description.
The molecular beacon sequence is shown as SEQ No.3, wherein the 5 'end is marked by a fluorescent group, the 3' end is marked by a fluorescence quenching group, the fluorescent group used in the invention is FAM, and the fluorescence quenching group is Dabcyl; the molecular beacon is named MB in the present invention for convenience of description.
Further, the DNA polymerase is a large fragment Bsm DNA polymerase.
Further, the restriction enzyme is nb.
Further, the polynucleotide kinase is T4 PNK.
Further, the dTNPs are equimolar mixtures of dATP, dCTP, dGTP and dTTP, and 10mM each of them is used in the present invention.
The invention also provides an application of the functional nucleic acid fluorescence sensor in lead ion detection.
The steps of applying the functional nucleic acid fluorescence sensor to lead ion detection are as follows:
(1) preparing deoxyribozyme buffer solution, ribozyme substrate buffer solution and molecular beacon buffer solution
Dissolving the deoxyribozyme, the ribozyme substrate and the molecular beacon in a buffer solution to obtain a deoxyribozyme buffer solution, a ribozyme substrate buffer solution and a molecular beacon buffer solution;
(2) drawing a standard curve
Mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, adding molecular beacon buffer solution, DNA polymerase, restriction endonuclease, polynucleotide kinase, dTNPs solution and lead ion standard solution with known concentration, and uniformly mixing;
adding buffer solution to fix the volume of the mixed solution obtained in the step one;
placing the mixed reaction system obtained in the step two in a constant-temperature incubator for incubation;
fourthly, performing fluorescence detection on the mixed reaction system obtained in the third step, and recording the fluorescence intensity peak value corresponding to the lead ion standard solution;
replacing the lead ion standard solution in the step I with a series of lead ion standard solutions with known concentrations, repeating the operation of the step I to obtain a series of lead ion standard solution fluorescence intensity peak values, and drawing a standard curve by taking the lead ion concentration logarithm value of the lead ion standard solution as a horizontal coordinate and the lead ion standard solution fluorescence intensity peak value as a vertical coordinate;
(3) detection of sample to be tested
Replacing the lead ion standard solution in the step (2) with a sample to be detected, repeating the operation of the step (2) and the step (c) to obtain the fluorescence intensity peak value of the sample to be detected, substituting the fluorescence intensity peak value of the sample to be detected into a regression equation of a standard curve, and calculating the concentration of lead ions in the sample to be detected.
Further, the buffer used in step (1) and step (2) ② was Bsm R buffer consisting of 10mM Tris-HCl (pH 8.5), 10mM MgCl2、100mM KCl、0.1mg/ml BSA。
Further, the third step (2) is to place the mixed reaction system obtained in the second step in a constant temperature incubator to incubate for 40min at 37 ℃, and then incubate for 15min at 80 ℃.
Further, when the fluorescence intensity is measured in the step (2), the adopted excitation wavelength and emission wavelength are determined according to the excitation wavelength and emission wavelength of the fluorescent group. The fluorescent group based on the molecular beacon used in the invention is FAM, and the fluorescence quenching group is Dabcyl; the conditions for fluorescence detection are therefore: the excitation wavelength was 492nm and the emission wavelength was 520 nm.
The principle of applying the functional nucleic acid fluorescence sensor to lead ion detection is as follows:
when Pb is present in the system, as shown in FIG. 12+GR-5E1 cleaves its corresponding substrate strand into two parts. Then, BsmDNA polymerase was replicated with the 5' end portion of the fragment as a primer and GR-5E1 as a template to form a double-stranded DNA. Next, the restriction enzyme nb. bpu10i nicks a specific recognition site of the double-stranded DNA, and then Bsm DNA polymerase performs strand displacement replication again with the nick as an origin to form the double-stranded DNA. At the same time, oligo-chain T capable of binding to MB is generatedarget DNA. Hybridization of TargetDNA to MB opens the hairpin structure to release a fluorescent signal, and the substrate fragment (right 3' -end part P) is liberated by GR-5E1 cleavage2) Hybridizing with the left end of the MB-Target DNA complex to form MB-Target-P2. Then, Bsm DNA polymerase was expressed as P2As a primer, the MB is used as a template to perform isothermal strand substitution to form double-stranded DNA and simultaneously substitute to generate new Target DNA, and the new Target DNA is hybridized with the next MB again. Thus, isothermal amplification cascade was carried out, and trace amount of Pb was obtained2+A large number of fluorescence-quenched MB stem-loop structures can be opened, releasing a strong fluorescent signal. Therefore, the system amplified by isothermal feedback series can detect Pb in the system with ultrahigh sensitivity2+The content of (a).
The invention has the beneficial effects that:
(1) the invention constructs an automatic isothermal cascade amplification strategy through the designed deoxyribozymes and molecular beacons, does not need separation operation, completes the processes of sample adding, incubation and detection in a single tube, and realizes high-sensitivity rapid detection of lead ions in a sample.
(2) The deoxyribozyme and the molecular beacon designed by the invention are stable, easy to synthesize and modify, low in cost and good in biocompatibility.
(3) Compared with other traditional detection methods, the detection method provided by the invention is simple to operate and short in detection time.
(4) The detection method has high sensitivity and low detection limit.
Drawings
FIG. 1 is a schematic diagram of the principle of lead ion detection by a functional nucleic acid fluorescence sensor.
FIG. 2 is a diagram of gel electrophoresis feasibility for detecting lead ions.
FIG. 3 is a graph showing the effect of various substances of the system on the fluorescence release of molecular beacons.
FIG. 4 is a standard curve of functional nucleic acid fluorescence sensor for detecting lead ions.
FIG. 5 shows different Pb2+Fluorescence intensity of lead ion standard solution of concentration.
FIG. 6 is a graph of fluorescence intensity detected for different interfering ions.
Detailed Description
In order to make the purpose and technical solution of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings.
Example 1: electrophoretic feasibility verification
(1) Preparation of 17% urea modified polyacrylamide gel
6.8mL of 30% acrylamide gel stock solution, 10 XTBE 1.2mL and 5.04g of urea were added to a 50mL centrifuge tube, then ultrapure water was added to a constant volume of 12mL, the mixture was mixed well, and air bubbles were removed by sonication for 5 min. Adding 6 mu L of tetramethyl ethylenediamine and 80 mu L of 10% ammonium persulfate solution, quickly mixing uniformly, immediately injecting the glue solution into a rubber plate, and standing for about 1h until the gel is solidified.
The composition of the 10 × TBE buffer was: 890mM Tris-boronic acid, 20mM EDTA, pH 8.2-8.4(25 ℃ C.)
(2) Preparation of the sample to be tested
Preparing a deoxyribozyme buffer solution, a ribozyme substrate buffer solution and a molecular beacon buffer solution for later use by using Bsm R buffer solution;
mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, then adding lead ion solution to be detected, molecular beacon buffer solution, 2 units of DNA polymerase, 3 units of restriction endonuclease, 4 units of T4 polynucleotide kinase and dTNPs, uniformly mixing, then adding Bsm R buffer solution to fix the volume of the reaction solution to 50 muL, so that the concentrations of deoxyribozyme, ribozyme substrate, molecular beacon, dTNPs and lead ions are respectively 1 muM, 250 muM and 1 muM. And placing the mixed reaction system in a constant-temperature incubator for incubation at 37 ℃ for 40min, then adjusting the temperature of the constant-temperature incubator to 80 ℃ for incubation for 15min, and cooling to room temperature for later use.
(3) Electrophoretic analysis process
Firstly, the gel prepared in the step (1) is pre-electrophoresed for 1h at 90V in an electrophoresis tank. Then, 10 mu L of the sample to be detected prepared in the step (2) and 2 mu L of the sample loading buffer solution are uniformly mixed to be used as a sample, and 6 mu L of the sample is loaded by using a micro sample injection needle. Then, electrophoresis was carried out at 90V for 30 min. Electrophoresis was then stopped at 160V until the lowest blue indicator reached the bottom of the gel. The buffer used during electrophoresis was 1 × TBE.
The composition of the loading buffer was: 1% SDS, 100nM EDTA, 60% glycerol, 0.03% bromophenol blue, 0.03% xylyl blue FF, pH 7.6.
(4) Silver staining process
The indicator is washed off from the gel subjected to the step (3) in 10% acetic acid solution until the gel is transparent and colorless, and then the gel is rinsed 3 times with ultrapure water for about 3min each time. Then adding silver dye solution for silver dyeing for 25min, quickly rinsing once, and washing off redundant silver. Finally adding a developing solution to develop for about 10min on a shaking table. Gel electrophoresis results were collected using ChemiDoc XRS gel imaging.
The silver dye solution comprises the following components: every 100mL of silver staining solution contains 0.1g of silver nitrate, 150 muL of formaldehyde solution and 20 muL of 10% sodium thiosulfate solution;
the composition of the developer solution is as follows: each 100mL of the developer contained 3g of sodium bicarbonate and 150. mu.L of formaldehyde solution.
FIG. 2 is a gel electrophoresis image showing feasibility analysis of urea denaturing gel electrophoresis for detecting lead ions. Lanes 1,2,3,4 correspond to GR-5E1, GR-5S1, Target DNA, MB, respectively; lane 5 corresponds to GR-5E1 (1. mu.M) + GR-5S1 (1. mu.M) + MB (1. mu.M) without Pb addition2+The system of (1); lane 6 corresponds to GR-5E1 (1. mu.M) + GR-5S1 (1. mu.M) + MB (1. mu.M) with the addition of Pb2+The system of (1). The electrophoresis result shows that the lead ions can trigger the isothermal amplification process, thereby realizing the synthetic amplification of the target DNA.
Example 2: effect of the substances on the fluorescence Release of molecular beacons
(1) The deoxyribozyme, the ribozyme substrate and the molecular beacon are prepared into a deoxyribozyme buffer solution, a ribozyme substrate buffer solution and a molecular beacon buffer solution for standby by using a Bsm R buffer solution.
(2) Mixing molecular beacon buffer solution with P2Mixing uniformly; adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L so as to ensure that the final concentration of the molecular beacon is 200 nM; a mixed reaction system 1 was prepared.
(3) Molecular beacon buffer, P2And 2.5. mu.L of 10mMUniformly mixing the dTNPs; adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L so as to ensure that the final concentration of the molecular beacon is 200 nM; to prepare a mixed reaction system 2.
(4) Molecular beacon buffer, P22.5 mu L of 10mM dTNPs and 3 units of restriction enzyme are mixed evenly; adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L so as to ensure that the final concentration of the molecular beacon is 200 nM; to prepare a mixed reaction system 3.
(5) Molecular beacon buffer, P22.5 μ L10 mM dTNPs, 3 units restriction endonuclease, 2 units DNA polymerase mixing well; adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L so as to ensure that the final concentration of the molecular beacon is 200 nM; to prepare a mixed reaction system 4.
(6) Molecular beacon buffer, P22.5 μ L10 mM dTNPs, 3 units restriction endonuclease, 2 units DNA polymerase and ribozyme substrate buffer solution; adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L, so that the final concentration of the molecular beacon and the ribozyme substrate is 200nM and 9 nM; to prepare a mixed reaction system 5.
(7) Mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, and adding molecular beacon buffer solution and P22.5. mu.L of 10mM dTNPs, 3 units of restriction enzyme, 2 units of DNA polymerase; adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L, so that the final concentration of the molecular beacon, the ribozyme substrate and the deoxyribozyme is 200nM, 9nM and 10 nM; to prepare a mixed reaction system 6.
(8) Mixing the molecular beacon buffer solution and the Target DNA, adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L, and enabling the final concentration of the molecular beacon and the Target DNA to be 200nM and 50 nM; to prepare a mixed reaction system 7.
(9) Mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, and adding molecular beacon buffer solution and P22.5. mu.L of 10mM dTNPs, 3 units of restriction enzyme, 2 units of DNA polymerase, Target DNA; adding Bsm R bufferThe volume of the reaction solution is fixed to 100 mu L by the solution, so that the final concentrations of the molecular beacon, the ribozyme substrate, the deoxyribozyme and the Target DNA are 200nM, 9nM, 10nM and 50 nM; to prepare a mixed reaction system 8.
(10) And placing the mixed reaction systems 1-8 in a constant-temperature incubator for incubation at 37 ℃ for 40min, and then adjusting the temperature of the constant-temperature incubator to 80 ℃ for incubation for 15 min.
(11) The mixed reaction system 1-8 was cooled to room temperature, and the fluorescence intensity of the mixed reaction system was measured with an Edinburgh FS5 type fluorescence spectrometer under the conditions of an excitation wavelength of 492nm and an emission wavelength of 520 nm.
FIG. 3 is a graph showing the effect of various substances of the system on the fluorescence release of molecular beacons. It can be observed that only systems where Target DNA is present can detect a strong fluorescence signal at 520 nm.
Example 3: drawing a standard solution curve
(1) Preparing lead ion standard solution
Preparation of Pb2+Lead ion standard solutions at concentrations of 1pM, 10pM, 100pM, 500pM, 1nM, 5nM, 10nM, 50nM, 100nM, respectively.
(2) Drawing a standard curve
Preparing deoxyribozyme buffer solution, ribozyme substrate buffer solution and molecular beacon buffer solution for later use by using Bsm R buffer solution;
mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, then adding molecular beacon buffer solution, 2 units of DNA polymerase, 3 units of restriction endonuclease, 4 units of polynucleotide kinase, 2.5 mu L of 10mM dTNPs and 10 mu L of 1pM lead ion solution to be detected, and uniformly mixing;
adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L, so that the final concentrations of the deoxyribozyme, the ribozyme substrate and the molecular beacon are 10nM, 9nM and 200 nM;
and fourthly, placing the mixed reaction system in a constant-temperature incubator for incubation for 40min at 37 ℃, and then adjusting the temperature of the constant-temperature incubator to 80 ℃ for incubation for 15 min.
Cooling the mixed reaction system to room temperature, measuring the fluorescence intensity of the mixed reaction system by using an Edinburgh FS5 type fluorescence spectrometer under the conditions that the excitation wavelength is 492nm and the emission wavelength is 520nm, and recording the fluorescence intensity peak value of the lead ion standard solution.
⑥ sequential use of Pb2+Replacing the lead ion standard solution in the step ② with lead ion standard solutions with the concentrations of 10pM, 100pM, 500pM, 1nM, 5nM, 10nM, 50nM and 100nM, repeating the operation in the step ②③④⑤ to obtain a series of fluorescence intensity peaks of the lead ion standard solutions, drawing a standard curve by taking the lead ion concentration logarithm value of the lead ion standard solution as the abscissa and the fluorescence intensity peak value of the lead ion standard solution as the ordinate, and fitting a regression equation of the standard curve as F (81.10 +22.87 × lgC), R (R under the condition that the regression equation of the standard curve is F (81.10 +22.87 lgC), wherein R is R under the condition that the regression equation of the standard20.986, where F represents fluorescence intensity and C represents Pb2+The concentration, standard curve is shown in FIG. 4.
FIG. 5 shows different Pb2+Fluorescence release profile of lead ion standard solution at concentration. A rapid increase in the fluorescence intensity of the system with increasing lead ion concentration can be observed.
Example 4: selectivity for other metal ions
① was provided with 10nM Zn2+、Co2+、Mn2+、Cu2+、Ni2+、Fe2+、Ca2+、Pb2+The solution is ready for use; configuration of Zn2+、Co2+、Mn2+、Cu2+、Ni2+、Fe2+、Ca2+The mixed solution of (1), wherein each ion concentration is 10 nM; configuration of Zn2+、Co2 +、Mn2+、Cu2+、Ni2+、Fe2+、Ca2+、Pb2+The mixed solution of (4) was prepared, wherein each ion concentration was 10 nM.
Preparing the deoxyribozyme buffer solution, the ribozyme substrate buffer solution and the molecular beacon buffer solution for later use by using Bsm R buffer solution;
mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, then adding molecular beacon buffer solution, 2 units of DNA polymerase, 3 units of restriction enzyme, 4 units of polynucleotide kinase, 2.5 mu L of 10mM dTNPs and 10 mu L of ultrapure water, and uniformly mixing;
adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L, so that the final concentrations of the deoxyribozyme, the ribozyme substrate and the molecular beacon are 10nM, 9nM and 200 nM;
fifthly, placing the mixed reaction system in a constant temperature incubator for incubation for 40min at 37 ℃, and then adjusting the temperature of the constant temperature incubator to 80 ℃ for incubation for 15 min.
Sixthly, cooling the mixed reaction system to room temperature, measuring the fluorescence intensity of the mixed reaction system by using an Edinburgh FS5 type fluorescence spectrometer under the conditions that the excitation wavelength is 492nm and the emission wavelength is 520nm, and recording the fluorescence intensity peak value of the sample.
⑦ sequential use of single Zn2+、Co2+、Mn2+、Cu2+、Ni2+、Fe2+、Ca2+、Pb2+Solution and Zn2+、Co2+、Mn2+、Cu2 +、Ni2+、Fe2+、Ca2+Mixed solution of (2) and Zn2+、Co2+、Mn2+、Cu2+、Ni2+、Fe2+、Ca2+、Pb2+The procedure of step ③④⑤⑥ was repeated, replacing the ultrapure water of step ③ with the mixed solution of (a), and the peak value of the fluorescence intensity of each sample was recorded.
FIG. 6 is a graph of fluorescence intensity detected for different interfering ions. It can be observed that the detection system exhibits a significant increase in fluorescence intensity only in the presence of lead ions, while ultrapure water, other metal ions alone or in a mixture thereof cause little increase in fluorescence intensity of the system. This indicates that other metal ions do not interfere with Pb2+Detection of (3).
Example 5:
① collecting tap water from public laboratory building of Shanghai oceanic university and lake water from Ming lake of Shanghai oceanic university campus, filtering the above water samples with sterile microporous ultrafiltration membrane (0.22 μm) to remove particulate impurities, mixing 0.9mL tap water or lake water sample with 0.1mL Pb2+Storage solutionAdding a standard to lead Pb in the sample2+The concentrations were 5nM, 10nM and 20nM, and the samples were sequentially recorded as sample solutions # 1- # 6.
Preparing the deoxyribozyme buffer solution, the ribozyme substrate buffer solution and the molecular beacon buffer solution for later use by using Bsm R buffer solution;
mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, then adding molecular beacon buffer solution, 2 units of DNA polymerase, 3 units of restriction endonuclease, 4 units of polynucleotide kinase, 2.5 mu L of 10mM dTNPs and 10 mu L of 1# sample solution, and uniformly mixing;
adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L, so that the final concentrations of the deoxyribozyme, the ribozyme substrate and the molecular beacon are 10nM, 9nM and 200 nM;
fifthly, placing the mixed reaction system in a constant temperature incubator for incubation for 40min at 37 ℃, and then adjusting the temperature of the constant temperature incubator to 80 ℃ for incubation for 15 min.
Sixthly, cooling the mixed reaction system to room temperature, measuring the fluorescence intensity of the mixed reaction system by using an Edinburgh FS5 type fluorescence spectrometer under the conditions that the excitation wavelength is 492nm and the emission wavelength is 520nm, and recording the fluorescence intensity peak value of the sample.
And seventhly, replacing the 1# sample solution in the step III with the 2# to 6# sample solution in sequence, repeating the operation of the step III, recording the fluorescence intensity peak value of each sample.
⑧ the fluorescence intensity peaks of the sample solutions 1# to 6# were respectively substituted into the regression equation of the standard curve obtained in example 3 to calculate Pb in each sample solution2+The results of concentration are shown in Table 1.
TABLE 1
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and all equivalent modifications, equivalents and improvements made within the spirit and principle of the present invention are included in the scope of the present invention/utility model.
Sequence listing
<110> Shanghai ocean university
<120> functional nucleic acid fluorescence sensor and application thereof in lead ion detection
<130>2020.6.24
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<170>SIPOSequenceListing 1.0
<210>1
<211>73
<212>DNA
<213>DNAzyme
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tggcgtcgca gaatatgcgc ccatcctcag ccgacgccat ctgaagtagc gccgccgtat 60
agtgactcgt gac 73
<210>2
<211>32
<212>DNA
<213>ribozyme substrates
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gtcacgagtc actatragga agatggcgtc gc 32
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<211>41
<212>DNA
<213>molecular beacons
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tggcgtcgca gaatatgcgc ccatcctcag ctgcgacgcc a 41
Claims (10)
1. A functional nucleic acid fluorescence sensor, which is characterized by comprising deoxyribozyme, ribozyme substrate, molecular beacon, DNA polymerase, restriction enzyme, polynucleotide kinase and dTNPs;
the deoxyribozyme sequence is shown in SEQ No. 1;
the ribozyme substrate sequence is shown in SEQ No. 2;
the molecular beacon sequence is shown as SEQ No.3, wherein the 5 'end is marked by a fluorescent group, and the 3' end is marked by a fluorescence quenching group.
2. The functional nucleic acid fluorescence sensor of claim 1, wherein the DNA polymerase is Bsm DNA polymerase.
3. The functional nucleic acid fluorescence sensor according to claim 1, wherein the restriction enzyme is nb.
4. The functional nucleic acid fluorescence sensor of claim 1, wherein the polynucleotide kinase is T4 PNK.
5. The functional nucleic acid fluorescence sensor of claim 1, wherein dTNPs is an equimolar mixture of dATP, dCTP, dGTP, dTTP.
6. Use of the functional nucleic acid fluorescence sensor according to claims 1-5 in lead ion detection.
7. Use according to claim 6, characterized in that it comprises the following steps:
(1) preparing deoxyribozyme buffer solution, ribozyme substrate buffer solution and molecular beacon buffer solution
Dissolving the deoxyribozyme, the ribozyme substrate and the molecular beacon in a buffer solution to obtain a deoxyribozyme buffer solution, a ribozyme substrate buffer solution and a molecular beacon buffer solution;
(2) drawing a standard curve
Mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, adding molecular beacon buffer solution, DNA polymerase, restriction endonuclease, polynucleotide kinase, dTNPs solution and lead ion standard solution with known concentration, and uniformly mixing;
adding buffer solution to fix the volume of the mixed solution obtained in the step one;
placing the mixed reaction system obtained in the step two in a constant-temperature incubator for incubation;
fourthly, performing fluorescence detection on the mixed reaction system obtained in the third step, and recording the fluorescence intensity peak value corresponding to the lead ion standard solution;
replacing the lead ion standard solution in the step I with a series of lead ion standard solutions with known concentrations, repeating the operation of the step I to obtain a series of lead ion standard solution fluorescence intensity peak values, and drawing a standard curve by taking the lead ion concentration logarithm value of the lead ion standard solution as a horizontal coordinate and the lead ion standard solution fluorescence intensity peak value as a vertical coordinate;
(3) detection of sample to be tested
Replacing the lead ion standard solution in the step (2) with a sample to be detected, repeating the operation of the step (2) and the step (c) to obtain the fluorescence intensity peak value of the sample to be detected, substituting the fluorescence intensity peak value of the sample to be detected into a regression equation of a standard curve, and calculating the concentration of lead ions in the sample to be detected.
8. The use of claim 7, wherein the buffer used in step (1) and step (2) is BsmR buffer.
9. The use of claim 7, wherein step (2) is performed by placing the mixed reaction system obtained in step (2) in a constant temperature incubator to incubate at 37 ℃ for 40min, and then incubate at 80 ℃ for 15 min.
10. The use according to claim 7, wherein the excitation wavelength and the emission wavelength used in the measurement of the fluorescence intensity in step (2) are determined based on the excitation wavelength and the emission wavelength of the fluorophore.
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