NL2031380B1 - Functional nucleic acid-based fluorescent sensor and use thereof in lead ion detection - Google Patents
Functional nucleic acid-based fluorescent sensor and use thereof in lead ion detection Download PDFInfo
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- NL2031380B1 NL2031380B1 NL2031380A NL2031380A NL2031380B1 NL 2031380 B1 NL2031380 B1 NL 2031380B1 NL 2031380 A NL2031380 A NL 2031380A NL 2031380 A NL2031380 A NL 2031380A NL 2031380 B1 NL2031380 B1 NL 2031380B1
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- nucleic acid
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- dnazyme
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- 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
Abstract
The present disclosure relates to a functional nucleic acid—based fluorescent sensor and use thereof in lead ion detection, belonging to the technical field of biochemistry. In the present disclosure, a DNAzyme and a molecular beacon are designed to be 5 used as lead ion recognition elements, and signal amplification mediator and signal emission elements. A detection method includes the following steps: hybridizing the DNAzyme and a ribozyme substrate into a buffer, adding the molecular beacon, a DNA polymerase, a restriction endonuclease, a polynucleotide kinase 10 (PNK), dTNPs and a test solution; incubating an obtained mixed system at a constant temperature under specific conditions, cooling to room temperature, followed by detection of a fluorescence intensity of the system to high—sensitively and rapidly detect lead ions. 15 (+ Fig. l)
Description
P1261 /NLpd
FUNCTIONAL NUCLEIC ACID-BASED FLUORESCENT SENSOR AND USE THEREOF
IN LEAD ION DETECTION
The present disclosure relates to a functional nucleic acid- based fluorescent sensor and use thereof in lead ion detection, belonging to the technical field of biochemistry.
GR-5 DNAzyme, as a single-stranded DNA obtained by in vitro screening technology, is a highly specific recognition element for lead ions. Molecular beacons are stem-loop oligonucleotides. A basic principle is that: a fluorophore and a quenching group are labeled at both ends of the oligonuclectide; when a target DNA and the molecular beacon hybridize to each other, an energy resonance transfer between the fluorophore and the quenching group of the molecular beacon is cut off to restore fluorescent signals.
An isothermal nucleic acid signal amplification strategy is an efficient nucleic acid sequence amplification strategy that does not require precise temperature control equipment and compli- cated operations during the signal amplification. Compared with
PCR technology, the isothermal nucleic acid signal amplification has great potential prospects and values for use in highly- sensitive detection of DNAs, RNAs, cells, proteins, small mole- cules and metal ions due to its simplicity, rapidity and low cost.
Lead ion, as one of the dangerous sources of heavy metal pol- lution, can seriously endanger human health, especially the physi- cal health and intellectual development of young children. In ad- dition, the lead ion (Pb*") pollution is also widely distributed in the natural environment, daily necessities and food, such that it is necessary to implement highly sensitive detection of the lead ions. Traditional detection methods require complex and expensive instruments, cumbersome detection processes and professional tech- nicians, which are difficult to meet people's urgent needs for highly-sensitive, low-cost, simple, real-time and rapid detection of the lead ions.
In view of the above defects in the prior art, the present disclosure proposes a functional nucleic acid-based fluorescent sensor and use thereof in lead ion detection.
To achieve the above purpose, in the present disclosure, a
DNAzyme, a ribozyme substrate and a molecular beacon are designed; a fluorescence analysis method is constructed based on an isother- mal cascade amplification strategy, which realizes highly sensi- tive and rapid detection of lead ions.
The present disclosure provides a functional nucleic acid- based fluorescent sensor, including a DNAzyme ‚ a ribozyme substrate, a molecular beacon, a DNA polymerase, a restriction endonuclease, a polynucleotide kinase (PNK) and dTNPs.
The DNAzyme has a sequence as shown in SEQ ID No. 1; for the convenience of description, the DNAzyme is named as GR-5 El in the present disclosure.
The ribozyme substrate has a sequence as shown in SEQ ID No. 2; for the convenience of description, the ribozyme substrate is named as GR-5 S1 in the present disclosure.
The molecular beacon has a sequence as shown in SEQ ID No. 3, where a 5'-end is labeled with a fluorophore, and a 3'-end is la- beled with a fluorescence quenching group; in the present disclo- sure, the fluorophore is a FAM, and the fluorescence quenching group is a Dabcyl; and for the convenience of description, the mo- lecular beacon is named as MB.
Further, the DNA polymerase may be a large-fragment Bsm DNA polymerase.
Further, the restriction endonuclease may be an Nb.Bpul0I.
Further, the PNK may be a T4 PNK.
Further, the dTNPs may be an equimolar mixture of dATP, dCTP, dGTP and dTTP, and a mixture including 10 mM for each of the above components may be used in the present disclosure.
The present disclosure further provides use of the functicnal nucleic acid-based fluorescent sensor in lead ion detection.
A method for detecting lead ions using the functional nucleic acid-based fluorescent sensor includes the following steps: (1) preparing a DNAzyme buffer, a ribozyme substrate buffer and a molecular beacon buffer:
Dissolving the DNAzyme, the ribozyme substrate and the molec- ular beacon in a buffer to obtain the DNAzyme buffer, the ribozyme substrate buffer and the molecular beacon buffer; (2) plotting a standard curve: 1) mixing the DNAzyme buffer and the ribozyme substrate buff- er, adding the molecular beacon buffer, the DNA polymerase, the restriction endonuclease, the PNK, a dTNPs solution and a lead ion standard solution of known concentration, followed by well mixing; 2) adding a buffer to dilute a mixed solution obtained in step 1); 3) conducting incubation on a mixed reaction system obtained in step 2) in a constant-temperature incubator; 4) subjecting a mixed reaction system obtained in step 3) to fluorescence detection, followed by recording a fluorescence in- tensity peak corresponding to the lead ion standard solution; and 5) replacing the lead ion standard solution in step 1) using a series of lead ion standard solutions with known concentrations, followed by repeating steps 1) to 4) to obtain a series of fluo- rescence intensity peaks of the lead ion standard solutions; plot- ting the standard curve with logarithms of lead ion concentrations of the lead ion standard solutions as abscissas and the fluores- cence intensity peaks of the lead ion standard solutions as ordi- nates; and (3) detection of a sample to be tested: replacing the lead ion standard solution in 1) of step (2) with the sample to be tested, followed by repeating 1) to 4) of step (2) to obtain a fluorescence intensity peak of the sample to be tested; substituting the fluorescence intensity peak of the sample to be tested into a regression equation of the standard curve to calculate a lead ion concentration in the sample to be tested.
Further, in step (1) and 2) of step (2), the buffer used may be a Bsm R buffer, including: 10 mM Tris-HCl (pH = 8.5), 10 mM
MgCl;, 100 mM KCl and 0.1 mg/ml bovine serum albumin (BSA).
Further, 3) of step (2) may specifically include: conducting incubation on the mixed reaction system obtained in step 2) in a constant-temperature incubator at 37°C for 40 min, and then at 80°C for 15 min.
Further, in 4) of step (2), when measuring the fluorescence intensity, an excitation wavelength and an emission wavelength used may be determined according to an excitation wavelength and an emission wavelength of the fluorophore. In the present disclo- sure, the molecular beacon used has the fluorophore FAM and the fluorescent quenching group Dabcyl; therefore, the fluorescence detection is conducted at an excitation wavelength of 492 nm and an emission wavelength of 520 nm.
A method for detecting lead ions using the functional nucleic acid-based fluorescent sensor includes the following steps:
When Pb“' exists in a system, GR-5 El cleaves a corresponding substrate strand into two parts. The Bsm DNA polymerase is sub- jected to replication using a 5'-end of a fragment as a primer and the GR-5 El as a template to form a double-stranded DNA. The re- striction endonuclease Nb.BpulOI cuts a specific recognition site of the double-stranded DNA, and the Bsm DNA polymerase conducts strand substitution replication using an obtained section as a starting point to form a double-stranded DNA. Meanwhile, an oligo- stranded Target DNA that can bind to MB was generated. Hybridiza- tion between the Target DNA and the MB opens a hairpin structure to release a fluorescent signal; at the same time, a free sub- strate fragment (a right 3'-end part P;) due to GR-5 El cleavage hybridizes with a left end of an MB-Target DNA complex to form a
MB-target-P:. The Bsm DNA polymerase conducts isothermal chain sub- stitution using P: as a primer and the MB as a template to form a double-stranded DNA, and conducts substitution to generate a new target DNA that hybridizes with a next MB. By conducting isother- mal cascade amplification in this way, a trace amount of Pb*’ can open a large number of fluorescence-quenched MB stem-loop struc- tures, releasing a strong fluorescent signal. Therefore, the sys- tem of isothermal feedback series amplification can detect the content of Pb?’ in the system with ultra-high sensitivity.
The present disclosure has the following beneficial effects:
In the present disclosure, an automatic isothermal cascade amplification strategy is constructed through the designed DNAzyme and the molecular beacon, and no separation is required; a process 5 of sample addition-incubation-detection is completed in a single tube, realizing high-sensitivity and rapid detection of the lead ions in a sample.
In the present disclosure, the designed DNAzyme and molecular beacon have stability, easy synthesis and modification, low cost and desirable biocompatibility.
Compared with other traditional detection methods, the detec- tion method of the present disclosure has simple operation and short detection time.
The detection method has a high sensitivity and low detection limit.
FIG. 1 shows a schematic diagram of principle of a functional nucleic acid-based fluorescent sensor in lead ion detection;
FIG. 2 shows a gel electrophoresis image of feasibility of the lead ion detection;
FIG. 3 shows an influence of various substances in a system on a fluorescence release of a molecular beacon;
FIG. 4 shows a standard curve of the functional nucleic acid- based fluorescent sensor in lead ion detection;
FIG.5 shows a graph of fluorescence intensities of lead ion standard solutions with different Pb*" concentrations; and
FIG. 6 shows a graph of fluorescence intensities of different interfering ions detected.
To make the objectives and technical solutions of the disclo- sure clearer, the disclosure is further described in detail below with reference to the accompanying drawings.
Example 1 Electrophoresis feasibility verification (1) Preparation of a 17% urea-denatured polyacrylamide gel
In a 50 mL centrifuge tube, 6.8 mL of a 30% acrylamide gel storage solution, 1.2 mL of a 10xTBE and 5.04 g of urea were add- ed, ultrapure water was added to dilute to 12 mL, followed by mix- ing well and sonication for 5 min to remove bubbles. 6 pL of tet- ramethylethylenediamine and 80 pL of a 10% ammonium persulfate so- lution were added, and mixed quickly; an obtained gel solution was immediately injected into a gel plate, followed by standing for about 1 h until the gel solidified.
The 10x TBE buffer included: 890 mM Tris boric acid, 20 mM ethylene diamine tetraacetic acid (EDTA), pH = 8.2-8.4 (at 25°C). (2) Preparation of a sample to be tested
A DNAzyme, a ribozyme substrate and a molecular beacon were prepared with a Bsm R buffer to obtain a DNAzyme buffer, a ribo- zyme substrate buffer and a molecular beacon buffer for later use.
The DNAzyme buffer and the ribozyme substrate buffer were mixed; a lead ion test solution, the molecular beacon buffer, 2 units of a DNA polymerase, 3 units of a restriction endonuclease, 4 units of a T4 PNK and dTNPs were added, and mixed well; the Bsm
R buffer was added to dilute an obtained reaction sclution to 50 pL, such that concentrations of the DNAzyme, the ribozyme sub- strate, the molecular beacon, the dTNPs and the lead ions were 1 aM, 1 uM, 1 pM, 250 uM and 1 uM, respectively. The above mixed re- action system was incubated in a constant-temperature incubator at 37°C for 40 min, and then at 80°C for 15 min, followed by cooling to room temperature for later use. (3) Electrophoresis analysis
The gel prepared in step (1) was pre-electrophoresed for 1 h at 90 V in an electrophoresis tank. 10 pL of the sample to be tested prepared in step (2) was mixed with 2 pL of a loading buff- er as a sample, and 6 pL of the above sample was loaded with a mi- cro-injection needle for loading. Electrophoresis was conducted at 90 V for 30 min. The electrophoresis was conducted at 160 V until a lowest blue indicator reached a bottom of the gel, followed by terminating the electrophoresis. A buffer used during electropho- resis was 1xTBE.
The loading buffer included: 1% SDS, 100 nM EDTA, 60% glycer- ol, 0.03% bromophenol blue, 0.03% xylene cyanol FF, pH = 7.6. (4) Silver staining
The gel after step (3) was washed in a 10% acetic acid solu- tion to remove the indicator until the gel was transparent and colorless, followed by rinsing with ultrapure water for 3 times with about 3 min in each time. A silver staining solution was add- ed for silver staining for 25 min, followed by rinsing once to re- move excessive silver. A developing solution was added to develop on a shaker for about 10 min. Gel electrophoresis results were collected by ChemiDoc XRS gel imaging.
The silver staining solution included: each 100 mL of the silver staining solution included 0.1 g of silver nitrate, 150 pL of a formaldehyde solution, and 20 pL of a 10% sodium thiosulfate solution; and the developing solution included: 3 g of sodium bicarbonate and 150 pL of the formaldehyde solution per 100 mL of the develop- ing solution.
FIG. 2 shows a gel electrophoresis chart for feasibility of lead ion detection, showing analysis results of the feasibility of urea-denatured gel electrophoresis. Lanes 1, 2, 3 and 4 correspond to GR-5E1, GR-5S1, Target DNA and MB, respectively; Lane 5 corre- sponds to a system of GR-5E1 (1 uM) + GR-531 (1 uM) + MB (1 pM) without Pb"; and Lane 6 corresponds to a system of the GR-5E1 (1 uM) + the GR-5S1 (1 uM) + the MB (1 uM) with Ph. Electrophoresis results show that the lead ions can trigger isothermal amplifica- tion, to achieve the synthesis and amplification of the target
DNA.
Example 2 Influences of various substances on fluorescence release of molecular beacon (1) A DNAzyme, a ribozyme substrate and a molecular beacon were prepared with a Bsm R buffer to obtain a DNAzyme buffer, a ribozyme substrate buffer and a molecular beacon buffer for later use. (2) The molecular beacon buffer was mixed with P, evenly; a
Bsm R buffer was added to dilute an obtained reaction solution to 100 pL, such that a final concentration of the molecular beacon was 200 nM; a mixed reaction system 1 was prepared. (3) The molecular beacon buffer, the P; and 2.5 pL of 10 mM dTNPs were mixed evenly; a Bsm R buffer was added to dilute an ob-
tained reaction solution to 100 uL, such that a final concentra- tion of the molecular beacon was 200 nM; a mixed reaction system 2 was prepared. (4) The molecular beacon buffer, the Ps, 2.5 pL of the 10 mM dTNPs, and 3 units of a restriction endonuclease were mixed even- ly; a Bsm R buffer was added to dilute an obtained reaction solu- tion to 100 pL, such that a final concentration of the molecular beacon was 200 nM; a mixed reaction system 3 was prepared. (5) The molecular beacon buffer, the P;, 2.5 HL of the 10 mM dTNPs, 3 units of the restriction endonuclease, and 2 units of a
DNA polymerase were mixed evenly; a Bsm R buffer was added to di- lute an obtained reaction solution to 100 pL, such that a final concentration of the molecular beacon was 200 nM; a mixed reaction system 4 was prepared. (6) The molecular beacon buffer, the P:, 2.5 pL of the 10 mM dTNPs, 3 units of the restriction endonuclease, 2 units of the DNA polymerase, and a ribozyme substrate buffer were mixed evenly; a
Bsm R buffer was added to dilute an obtained reaction solution to 100 pL, such that a final concentration of the molecular beacon and the ribozyme substrate was 200 nM and 9 nM, respectively; a mixed reaction system 5 was prepared. (7) The DNAzyme buffer and the ribozyme substrate buffer were mixed, and the molecular beacon buffer, the P;, 2.5 pL of the 10 mM dTNPs, 3 units of the restriction endonuclease, and 2 units of the
DNA polymerase were added; a Bsm R buffer was added to dilute an obtained reaction solution to 100 pL, such that a final concentra- tion of the molecular beacon, the ribozyme substrate, and the
DNAzyme was 200 nM, 9 nM, and 10 nM, respectively; a mixed reac- tion system 6 was prepared. (8) The molecular beacon buffer and a target DNA were mixed; a Bsm R buffer was added to dilute an obtained reaction solution to 100 pL, such that a final concentration of the molecular beacon and the target DNA was 200 nM and 50 nM, respectively; a mixed re- action system 7 was prepared. (9) The DNAzyme buffer and the ribozyme substrate buffer were mixed, and the molecular beacon buffer, the P:, 2.5 pL of the 10 mM dTNPs, 3 units of the restriction endonuclease, 2 units of the DNA polymerase and the Target DNA were added; a Bsm R buffer was added to dilute an obtained reaction solution to 100 pL, such that a fi- nal concentrations of the molecular beacon, the ribozyme sub- strate, the DNAzyme, and the Target DNA was 200 nM, 9 nM, 10 nM and 50 nM, respectively; a mixed reaction system 8 was prepared. (10) The above mixed reaction systems 1-8 were incubated in a constant-temperature incubator at 37°C for 40 min, and then at 80°C for 15 min. (11) The mixed reaction systems 1-8 were cooled to room tem- perature, and fluorescence intensities of the mixed reaction sys- tems were measured with an Edinburgh FS5 fluorescence spectrometer at an excitation wavelength of 492 nm and an emission wavelength of 520 nm.
FIG. 3 shows an influence of various substances in a system on a fluorescence release of a molecular beacon. It can be ob- served that only the system with the target DNA can detect a strong fluorescent signal at 520 nm.
SEQLTXT
<110> Shanghai Ocean University <120> FUNCTIONAL NUCLEIC ACID-BASED FLUORESCENT SENSOR AND USE THEREOF
IN LEAD ION DETECTION
<130> HKJP202112977 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the DNAzyme <400> 1 tggcgtcgca gaatatgcgc ccatcctcag ccgacgccat ctgaagtagc gccgccgtat 60 agtgactcgt gac 73 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the ribozyme substrate <400> 2 gtcacgagtc actatragga agatggcgtc gc 32 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the molecular beacon <400> 3 tggcgtcgca gaatatgcgc ccatcctcag ctgcgacgcc a 41
Pagina 1
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