阅读说明：本技术 一种功能核酸荧光传感器及其在铅离子检测中的应用 (Functional nucleic acid fluorescence sensor and application thereof in lead ion detection ) 是由 吴继魁 贾敏 张俊玲 于 2020-06-24 设计创作，主要内容包括：本发明涉及一种功能核酸荧光传感器及其在铅离子检测中的应用,属于生物化学技术领域。本发明设计的脱氧核酶和分子信标可作为铅离子的识别元件、信号扩增介导和信号发射元件。方法具体步骤如下：将脱氧核酶、核酶底物杂交混合于缓冲液中,加入分子信标、DNA聚合酶、限制性内切酶、多聚核苷酸激酶、dTNPs和待测液；将混合体系在特定条件下恒温孵育,冷却至室温,通过检测体系荧光强度,实现对铅离子的高灵敏快速检测。本发明通过设计的脱氧核酶和分子信标构建了自动等温级数扩增策略,无需分离操作,在单管中完成加样-孵育-检测过程,实现了对样品中铅离子高灵敏快速检测。(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.)

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.

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.