CN111705113B - 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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- CN111705113B CN111705113B CN202010588364.1A CN202010588364A CN111705113B CN 111705113 B CN111705113 B CN 111705113B CN 202010588364 A CN202010588364 A CN 202010588364A CN 111705113 B CN111705113 B CN 111705113B
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The utility model 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 utility model can be used as a lead ion identification element, a signal amplification mediation element and a signal emission element. The method comprises the following specific steps: hybridization of deoxyribozyme and ribozyme substrate is mixed in buffer solution, and molecular beacons, DNA polymerase, restriction endonuclease, polynucleotide kinase, dTNPs and liquid to be detected are added; incubating the mixed system at a constant temperature under a specific condition, cooling to room temperature, and detecting the fluorescence intensity of the system to realize high-sensitivity and rapid detection of lead ions. According to the utility model, an automatic isothermal progression amplification strategy is constructed through the designed deoxyribozyme and molecular beacon, separation operation is not needed, and the sample adding-incubation-detection process is completed in a single tube, so that high-sensitivity and rapid detection of lead ions in a sample is realized.
Description
Technical Field
The utility model relates to a functional nucleic acid fluorescence sensor and application thereof in lead ion detection, belonging to the technical field of biochemistry.
Background
GR-5 deoxyribozymes are single-stranded DNA obtained by in vitro screening techniques, and are recognition elements with high specificity for lead ions. The molecular beacon is stem-loop oligonucleotide, the basic principle is that the fluorescent group and the quenching group are respectively marked at two ends of the oligonucleotide, and when the 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 as to restore the fluorescent signal.
Isothermal nucleic acid signal amplification strategies are highly efficient nucleic acid sequence amplification strategies that do not require elaborate temperature control equipment and complex procedures during signal amplification. Compared with the PCR technology, isothermal nucleic acid signal amplification has the advantages of simplicity, rapidness, low cost and the like, and has great 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 sources of heavy metal contamination, which severely endanger humansThe physical health, especially the physical health and the intelligence development of young children are seriously affected. And, lead ions (Pb) 2+ ) Pollution is also widely distributed in natural environments, living goods, and foods, and thus it is required to perform highly sensitive detection thereof. The traditional detection method needs complex and expensive instruments, complicated detection processes and professional technicians, and is difficult to meet the urgent demands of people for detecting the content of lead ions in a high-sensitivity, low-cost, simple, real-time and rapid manner.
Disclosure of Invention
Aiming at the defects in the prior art, the utility model provides a functional nucleic acid fluorescence sensor and application of the fluorescence sensor in lead ion detection.
In order to achieve the purpose, the utility model 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 of lead ions.
The functional nucleic acid fluorescence sensor provided by the utility model comprises deoxyribozyme, ribozyme substrate, molecular beacon, DNA polymerase, restriction endonuclease, polynucleotide kinase and dNPs.
The sequence of the deoxyribozyme is shown in SEQ No. 1; for convenience of description, it is designated as GR-5E1 in the present utility model.
The sequence of the ribozyme substrate is shown as SEQ No.2, wherein rA is adenine ribonucleotide; for convenience of description, it is designated as GR-5S1 in the present utility model.
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, the fluorescent group used in the utility model is FAM, and the fluorescence quenching group is Dabcyl; the molecular beacon is designated as MB in the present utility model for convenience of description.
Further, the DNA polymerase is a large fragment Bsm DNA polymerase.
Further, the restriction enzyme is nb.bpu10i.
Further, the polynucleotide kinase is T4PNK.
Further, the dNPs were an equimolar mixture of dATP, dCTP, dGTP, dTTP, and 10mM each was used in the present utility model.
The utility model also provides application of the functional nucleic acid fluorescence sensor in lead ion detection.
The lead ion detection method by using the functional nucleic acid fluorescence sensor comprises the following steps:
(1) Preparation of deoxyribozyme buffer, ribozyme substrate buffer, and molecular beacon buffer
Dissolving deoxyribozyme, a ribozyme substrate and a 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
(1) Mixing a deoxyribozyme buffer solution and a ribozyme substrate buffer solution, adding a molecular beacon buffer solution, DNA polymerase, restriction endonuclease, polynucleotide kinase, dNPs solution and lead ion standard solution with known concentration, and uniformly mixing;
(2) adding buffer solution to fix the volume of the mixed solution obtained in the step (1);
(3) placing the mixed reaction system obtained in the step (2) in a constant temperature incubator for incubation;
(4) performing fluorescence detection on the mixed reaction system obtained in the step (3), and recording a fluorescence intensity peak value corresponding to the lead ion standard solution;
(5) using a series of lead ion standard solutions with known concentrations to replace the lead ion standard solution in the step (1), repeating the operations of the steps (1) (2) (3) (4) to obtain a series of fluorescence intensity peaks of the lead ion standard solution, and drawing a standard curve by taking the lead ion concentration logarithmic value of the lead ion standard solution as an abscissa and taking the fluorescence intensity peaks of the lead ion standard solution as an ordinate;
(3) Sample detection to be tested
And (3) replacing the lead ion standard solution in the step (2) (1) with a sample to be detected, repeating the operations of the steps (2) (1) (2) (3) (4) to obtain a fluorescence intensity peak value of the sample to be detected, bringing 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) is Bsm R buffer, and the composition thereof is: 10mM Tris-HCl (pH=8.5), 10mM MgCl 2 、100mM KCl、0.1mg/ml BSA。
Further, the steps (2) and (3) are that the mixed reaction system obtained in the step (2) is placed in a constant temperature incubator at 37 ℃ for 40min, and then incubated at 80 ℃ for 15min.
Further, when the fluorescence intensity is measured in the steps (2) and (4), the excitation wavelength and the emission wavelength used are determined according to the excitation wavelength and the emission wavelength of the fluorescent group. The fluorescent group based on the molecular beacon used in the utility model is FAM, and the fluorescence quenching group is Dabcyl; the conditions for fluorescence detection are: the excitation wavelength was 492nm and the emission wavelength was 520nm.
The principle of lead ion detection by applying the functional nucleic acid fluorescence sensor provided by the utility model is as follows:
as shown in FIG. 1, pb is present in the system 2+ GR-5E1 cleaves its corresponding substrate strand into two parts. Then, bsm DNA polymerase uses the 5' end part of the fragment as a primer and GR-5E1 as a template to replicate so as to form double-stranded DNA. Next, the restriction enzyme Nb.Bpu10I nicks the specific recognition site of the double-stranded DNA, and then Bsm DNA polymerase performs strand-displacement replication again starting from the nick to form double-stranded DNA. At the same time, oligo-chain Target DNA capable of binding to MB is generated. Hybridization of Target DNA with MB opens the hairpin structure to release the fluorescent signal, while the substrate fragment (right 3' -end portion P 2 ) Hybridization with the left end of the MB-Target DNA complex to form MB-Target-P 2 . Bsm DNA polymerase then uses P 2 Isothermal strand substitution with MB as template for primer forms double stranded DNA while substitution generates new Target DNA which again hybridizes to the next MB. Isothermal cascade amplification was performed in this way, trace amounts of Pb 2+ A large number of fluorescence quenched MB stem-loop structures can be opened, releasing a strong fluorescent signal. Therefore, pb in the ultra-sensitive detection system can be detected by the isothermal feedback series amplification system 2+ Content of (3)。
The beneficial effects of the utility model are as follows:
(1) According to the utility model, an automatic isothermal cascade amplification strategy is constructed through the designed deoxyribozyme and molecular beacon, separation operation is not needed, and the sample adding-incubation-detection process is completed in a single tube, so that high-sensitivity rapid detection of lead ions in a sample is realized.
(2) The deoxyribozyme and the molecular beacon designed by the utility model are stable, easy to synthesize and modify, low in cost and good in biocompatibility.
(3) Compared with other traditional detection methods, the detection method 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 a functional nucleic acid fluorescence sensor for detecting lead ions.
FIG. 2 is a gel electrophoresis chart showing the feasibility of detecting lead ions.
FIG. 3 is a graph showing the effect of various substances in the system on the release of fluorescence from molecular beacons.
FIG. 4 is a standard curve of a functional nucleic acid fluorescence sensor for detecting lead ions.
FIG. 5 shows a different Pb 2+ Fluorescence intensity profile of a standard solution of lead ions at concentration.
FIG. 6 is a graph of fluorescence intensity for detection of different interfering ions.
Detailed Description
The present utility model will be described in further detail with reference to the accompanying drawings, in order to make the objects and technical solutions of the present utility model more apparent.
Example 1: electrophoretic feasibility verification
(1) Preparation of 17% urea denatured polyacrylamide gel
6.8mL of 30% acrylamide gel storage solution, 10 XTBE 1.2mL and 5.04g 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 5min. 6 mu L of tetramethyl ethylenediamine and 80 mu L of 10% ammonium persulfate solution are added, the mixture is quickly and evenly mixed, the glue solution is immediately injected into a glue plate, and the glue plate is left for about 1h to solidify the gel.
The composition of the 10 XTBE buffer was: 890mM Tris boric acid, 20mM EDTA, pH=8.2-8.4 (25 ℃ C.)
(2) Preparation of the sample to be tested
Preparing a deoxyribozyme, a ribozyme substrate and a molecular beacon into a deoxyribozyme buffer solution, a ribozyme substrate buffer solution and a molecular beacon buffer solution for standby by using Bsm R buffer solution;
mixing a deoxyribozyme buffer solution and a ribozyme substrate buffer solution, adding a lead ion to-be-detected solution, a molecular beacon buffer solution, 2 units of DNA polymerase, 3 units of restriction endonuclease, 4 units of T4 polynucleotide kinase and dTMPS, uniformly mixing, adding a Bsm R buffer solution to fix the volume of the reaction solution to 50 mu L, so that the concentration of the deoxyribozyme, the ribozyme substrate, the molecular beacon, the dTMPS and the lead ion is 1 mu M, 250 mu M and 1 mu M respectively. And placing the mixed reaction system in a constant temperature incubator at 37 ℃ for incubation for 40min, then adjusting the temperature of the constant temperature incubator to 80 ℃ for incubation for 15min, and cooling to room temperature for standby.
(3) Electrophoretic analysis process
Firstly, pre-electrophoresis is carried out on the gel prepared in the step (1) in an electrophoresis tank at 90V for 1h. And (2) uniformly mixing 10 mu L of the sample to be detected prepared in the step (2) with 2 mu L of the loading buffer solution to obtain a sample, and taking 6 mu L of the sample by using a microsyringe needle to load the sample. Next, electrophoresis was performed at 90V for 30min. Electrophoresis was then stopped at 160V until the lowest blue indicator reached the bottom of the gel. The buffer used in the electrophoresis was 1 XTBE.
The composition of the loading buffer solution is as follows: 1%SDS,100nM EDTA,60% glycerol, 0.03% bromophenol blue, 0.03% xylene cyanoff, ph=7.6.
(4) Silver dyeing process
The gel after the step (3) is washed away with an indicator in 10% acetic acid solution until the gel is transparent and colorless, and then rinsed with ultrapure water for 3 times, each time for about 3 minutes. Then adding silver dye liquor to carry out silver dyeing for 25min, and rapidly rinsing for one time to wash out superfluous silver. Finally, the developer is added and developed on a shaker for about 10min. Gel electrophoresis results were collected using ChemiDoc XRS gel imaging.
The silver dye solution comprises the following components: each 100mL of silver dye liquor contains 0.1g of silver nitrate, 150 mu L of formaldehyde solution and 20 mu L of 10% sodium thiosulfate solution;
the composition of the developing solution is as follows: each 100mL of the developer contains 3g of sodium bicarbonate and 150. Mu.L of formaldehyde solution.
FIG. 2 is a gel electrophoresis chart for detecting feasibility of lead ions, and shows the analysis result of urea denaturation gel electrophoresis feasibility. 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) and no Pb was added 2+ Is a system of (2); lane 6 corresponds to GR-5E1 (1. Mu.M) +GR-5S1 (1. Mu.M) +MB (1. Mu.M) and Pb was added 2+ Is a system of (3). The electrophoresis result shows that the lead ions can trigger the isothermal amplification process, thereby realizing the synthesis and amplification of target DNA.
Example 2: effect of substances on fluorescence Release from 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 Bsm R buffer solution.
(2) Buffer of molecular beacon and P 2 Uniformly mixing; 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 is 200nM; the mixed reaction system 1 is prepared.
(3) Buffer solution of molecular beacon, P 2 And 2.5. Mu.L of 10mM dTMPS were mixed well; 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 is 200nM; the mixed reaction system 2 is prepared.
(4) Buffer solution of molecular beacon, P 2 2.5. Mu.L of 10mM dTMPs, 3 units of restriction enzyme were mixed well; 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 is 200nM; the mixed reaction system 3 is prepared.
(5) Buffer solution of molecular beacon, P 2 2.5. Mu.L of 10mM dTMPs, 3 units of restriction enzyme, 2 units of DNA polymerase; adding Bsm R buffer solution to make the volume of the reaction solution constant at 100 mu L so as to scoreThe final concentration of sub-beacons was 200nM; the mixed reaction system 4 was prepared.
(6) Buffer solution of molecular beacon, P 2 2.5. Mu.L of 10mM dTMPs, 3 units of restriction enzyme, 2 units of DNA polymerase, and ribozyme substrate buffer; 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 9nM; the mixed reaction system 5 is prepared.
(7) Mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, and then adding molecular beacon buffer solution and P 2 2.5. Mu.L of 10mM dTMPs, 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 molecular beacons, ribozyme substrates and deoxyribozymes is 200nM, 9nM and 10nM; the mixed reaction system 6 was prepared.
(8) Mixing the molecular beacon buffer solution and Target DNA, 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 Target DNA is 200nM and 50nM; a mixed reaction system 7 was prepared.
(9) Mixing deoxyribozyme buffer solution and ribozyme substrate buffer solution, and then adding molecular beacon buffer solution and P 2 2.5. Mu.L of 10mM dNPs, 3 units of restriction enzyme, 2 units of DNA polymerase, target DNA; adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L so that the final concentration of molecular beacons, ribozyme substrates, deoxyribozymes and Target DNA is 200nM, 9nM, 10nM and 50nM; the mixed reaction system 8 was prepared.
(10) The mixed reaction systems 1-8 are placed in a constant temperature incubator at 37 ℃ for incubation for 40min, and then the temperature of the constant temperature incubator is adjusted to 80 ℃ for incubation for 15min.
(11) The mixed reaction systems 1 to 8 were cooled to room temperature, and the fluorescence intensity of the mixed reaction systems was measured by using an Edinburgh FS5 type fluorescence spectrometer under the conditions of an excitation wavelength of 492nm and an emission wavelength of 520nm.
FIG. 3 is a graph showing the effect of various substances in the system on the release of fluorescence from molecular beacons. It was observed that only the system in which the Target DNA was present was able to detect a strong fluorescent signal at 520nm.
Example 3: drawing standard solution curve
(1) Preparing lead ion standard solution
Preparing Pb 2+ Lead ion standard solutions with concentrations of 1pM, 10pM, 100pM, 500pM, 1nM, 5nM, 10nM, 50nM, 100nM, respectively.
(2) Drawing a standard curve
(1) Preparing a deoxyribozyme, a ribozyme substrate and a molecular beacon into a deoxyribozyme buffer solution, a ribozyme substrate buffer solution and a molecular beacon buffer solution for standby by using Bsm R buffer solution;
(2) mixing a deoxyribozyme buffer solution and a ribozyme substrate buffer solution, adding a 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 1pM lead ion to-be-detected solution, and uniformly mixing;
(3) adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L so that the final concentration of deoxyribozyme, ribozyme substrate and molecular beacon is 10nM, 9nM and 200nM;
(4) the mixed reaction system is placed in a constant temperature incubator at 37 ℃ for incubation for 40min, and then the temperature of the constant temperature incubator is adjusted to 80 ℃ for incubation for 15min.
(5) The mixed reaction system 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 520nm, and the fluorescence intensity peak of the lead ion standard solution was recorded.
(6) Sequentially using Pb 2+ The lead ion standard solution with the concentration of 10pM, 100pM, 500pM, 1nM, 5nM, 10nM, 50nM and 100nM replaces the lead ion standard solution in the step (2), and the operations of the steps (2) (3) (4) (5) are repeated to obtain a series of fluorescence intensity peaks of the lead ion standard solution, the fluorescence intensity peak of the lead ion standard solution is used as an abscissa, the fluorescence intensity peak of the lead ion standard solution is used as an ordinate to draw a standard curve, and a regression equation of the fitted standard curve is F=81.10+22.87×lgC and R 2 =0.986, where F represents fluorescence intensity, C represents Pb 2+ The concentration, standard curve is shown in figure 4.
FIG. 5 shows a different Pb 2+ Fluorescence emission profile of a standard solution of lead ions at concentration. It can be observed that the fluorescence intensity of the system increases rapidly with increasing lead ion concentration.
Example 4: selectivity for other metal ions
(1) Zn of 10nM each 2+ 、Co 2+ 、Mn 2+ 、Cu 2+ 、Ni 2+ 、Fe 2+ 、Ca 2+ 、Pb 2+ The solution is ready for use; configuration of Zn 2+ 、Co 2+ 、Mn 2+ 、Cu 2+ 、Ni 2+ 、Fe 2+ 、Ca 2+ Wherein the concentration of each ion is 10nM each; configuration of Zn 2+ 、Co 2 + 、Mn 2+ 、Cu 2+ 、Ni 2+ 、Fe 2+ 、Ca 2+ 、Pb 2+ For use, wherein the concentration of each ion is 10nM.
(2) Preparing a deoxyribozyme, a ribozyme substrate and a molecular beacon into a deoxyribozyme buffer solution, a ribozyme substrate buffer solution and a molecular beacon buffer solution for standby by using Bsm R buffer solution;
(3) mixing a deoxyribozyme buffer solution and a ribozyme substrate buffer solution, adding a 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 dTMPs and 10 mu L of ultrapure water, and uniformly mixing;
(4) adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L so that the final concentration of deoxyribozyme, ribozyme substrate and molecular beacon is 10nM, 9nM and 200nM;
(5) the mixed reaction system is placed in a constant temperature incubator at 37 ℃ for incubation for 40min, and then the temperature of the constant temperature incubator is adjusted to 80 ℃ for incubation for 15min.
(6) The mixed reaction system was cooled to room temperature, 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 520nm, and the fluorescence intensity peak value of the sample was recorded.
(7) Sequentially using single Zn 2+ 、Co 2+ 、Mn 2+ 、Cu 2+ 、Ni 2+ 、Fe 2+ 、Ca 2+ 、Pb 2+ Solution and Zn 2+ 、Co 2+ 、Mn 2+ 、Cu 2 + 、Ni 2+ 、Fe 2+ 、Ca 2+ Is a mixed solution of Zn and (B) 2+ 、Co 2+ 、Mn 2+ 、Cu 2+ 、Ni 2+ 、Fe 2+ 、Ca 2+ 、Pb 2+ The operations of steps (3), (4), (5) and (6) were repeated instead of ultrapure water in step (3), and the fluorescence intensity peaks of the respective samples were recorded.
FIG. 6 is a graph of fluorescence intensity for detection of different interfering ions. It was observed that the detection system showed a significant increase in fluorescence intensity only when lead ions were present, whereas ultrapure water, single other metal ions or mixtures thereof hardly caused an increase in fluorescence intensity of the system. This means that other metal ions do not interfere with Pb 2+ Is detected.
Example 5:
(1) collecting tap water of public experiment buildings of Shanghai ocean university and lake water of the Shanghai ocean university garden lake, filtering the water sample through a sterile microporous ultrafiltration membrane (0.22 mu m), and removing particle impurities. Mixing 0.9mL of tap water or lake water sample with 0.1mL of Pb 2+ Labeling the stock solution so that Pb in the sample 2+ The concentrations were 5nM, 10nM, 20nM, and were designated 1# to 6# sample solutions in this order.
(2) Preparing a deoxyribozyme, a ribozyme substrate and a molecular beacon into a deoxyribozyme buffer solution, a ribozyme substrate buffer solution and a molecular beacon buffer solution for standby by using Bsm R buffer solution;
(3) mixing a deoxyribozyme buffer solution and a ribozyme substrate buffer solution, adding a 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 dTMPs and 10 mu L of 1# sample solution, and uniformly mixing;
(4) adding Bsm R buffer solution to fix the volume of the reaction solution to 100 mu L so that the final concentration of deoxyribozyme, ribozyme substrate and molecular beacon is 10nM, 9nM and 200nM;
(5) the mixed reaction system is placed in a constant temperature incubator at 37 ℃ for incubation for 40min, and then the temperature of the constant temperature incubator is adjusted to 80 ℃ for incubation for 15min.
(6) The mixed reaction system was cooled to room temperature, 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 520nm, and the fluorescence intensity peak value of the sample was recorded.
(7) And (3) replacing the 1# sample solution in the step (3) with the 2# to 6# sample solution in sequence, repeating the operations of the steps (3), (4), (5) and (6), and recording the fluorescence intensity peak value of each sample.
(8) Substituting the fluorescence intensity peak values of the 1# to 6# sample solutions into the regression equation of the standard curve obtained in the example 3 respectively, and calculating Pb in each sample solution 2+ The concentrations and results are shown in Table 1.
TABLE 1
The foregoing description of the preferred embodiments of the utility model is not intended to be limiting, but rather is to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the utility model.
Sequence listing
<110> Shanghai university of ocean
<120> a functional nucleic acid fluorescence sensor and its application in lead ion detection
<130> 2020.6.24
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<211> 73
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<213> DNAzyme
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agtgactcgt gac 73
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Claims (6)
1. A functional nucleic acid fluorescence sensor, comprising a deoxyribozyme, a ribozyme substrate, a molecular beacon, a DNA polymerase, a restriction endonuclease, a polynucleotide kinase and dTNPs;
the sequence of the deoxyribozyme is shown in SEQ No. 1;
the sequence of the ribozyme substrate is shown as 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;
the DNA polymerase is Bsm DNA polymerase;
the restriction endonuclease is nb.bpu10i;
the polynucleotide kinase is T4 PNK;
dTNPs is an equimolar mixture of dATP, dCTP, dGTP, dTTP.
2. The use of the functional nucleic acid fluorescence sensor according to claim 1 for lead ion detection.
3. The use according to claim 2, characterized by the steps of:
(1) Preparation of deoxyribozyme buffer, ribozyme substrate buffer, and molecular beacon buffer
Dissolving deoxyribozyme, a ribozyme substrate and a 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
(1) Mixing a deoxyribozyme buffer solution and a ribozyme substrate buffer solution, adding a molecular beacon buffer solution, DNA polymerase, restriction endonuclease, polynucleotide kinase, dNPs solution and lead ion standard solution with known concentration, and uniformly mixing;
(2) adding buffer solution to fix the volume of the mixed solution obtained in the step (1);
(3) placing the mixed reaction system obtained in the step (2) in a constant temperature incubator for incubation;
(4) performing fluorescence detection on the mixed reaction system obtained in the step (3), and recording a fluorescence intensity peak value corresponding to the lead ion standard solution;
(5) using a series of lead ion standard solutions with known concentrations to replace the lead ion standard solution in the step (1), repeating the operations of the steps (1) (2) (3) (4) to obtain a series of fluorescence intensity peaks of the lead ion standard solution, and drawing a standard curve by taking the lead ion concentration logarithmic value of the lead ion standard solution as an abscissa and taking the fluorescence intensity peaks of the lead ion standard solution as an ordinate;
(3) Sample detection to be tested
And (3) replacing the lead ion standard solution in the step (2) (1) with a sample to be detected, repeating the operations of the steps (2) (1) (2) (3) (4) to obtain a fluorescence intensity peak value of the sample to be detected, bringing 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.
4. The method of claim 3, wherein the buffer used in step (1) and step (2) (2) is Bsm R buffer.
5. The method according to claim 3, wherein the step (2) (3) is performed by placing the mixed reaction system obtained in the step (2) in a constant temperature incubator at 37 ℃ for 40min, and then incubating at 80 ℃ for 15min.
6. The use according to claim 3, wherein the excitation wavelength and emission wavelength used in determining the fluorescence intensity in step (2) (4) are determined based on the excitation wavelength and emission wavelength of the fluorophore.
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