A functional nucleic acid fluorescent sensor and its application in lead ion detection

A fluorescent sensor and functional nucleic acid technology, applied in fluorescence/phosphorescence, instruments, measuring devices, etc., can solve the problems of high detection limit, expensive instruments, high background, etc., achieve high temperature stability, fast and accurate detection, high detection The effect of sensitivity

Active Publication Date: 2020-12-22
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the detection methods of lead ions mainly include physical and chemical methods and biosensor methods. X-ray fluorescence spectrometry, etc. These methods have high operational requirements, expensive instruments, and high detection limits for lead ions. They can only achieve qualitative or semi-quantitative detection, and cannot meet the detection requirements of lower concentrations of lead ions.
The existing biosensor method is a method for qualitative and quantitative detection of lead ions through the specific combination between biomolecules and lead ions. Detection of environmental impacts and high detection limits
For example, the existing fluorescent sensors based on lead ion-dependent DNAzyme still have defects such as high background, low sensitivity, and susceptibility to detection temperature, which are not enough to achieve reliable detection in the field environment and low concentration of lead ions in the environment. Effective detection of

Method used

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  • A functional nucleic acid fluorescent sensor and its application in lead ion detection
  • A functional nucleic acid fluorescent sensor and its application in lead ion detection
  • A functional nucleic acid fluorescent sensor and its application in lead ion detection

Examples

Experimental program
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Effect test

Embodiment 1

[0050] In this embodiment, the preparation of lead ion-dependent deoxyribozyme solution, the steps are as follows:

[0051] Add 3 μL of 2 μM DNAzyme substrate chain stock solution and 6 μL of 0.5 μM DNAzyme enzyme chain stock solution to 2 μL buffer (500 mM HEPES, 500 mM NaCl, 50 mM MgCl 2 ,pH=7.26), then dilute to 18μL with ultrapure water, shake well, heat at 80°C for 3min, then slowly cool down to room temperature, the whole cooling process lasts for 40min, and then stand at room temperature for 25min to obtain lead ion-dependent deoxidation Ribozyme solution. The nucleotide sequence of the DNAzyme substrate chain is shown in SEQ ID No.1 in the sequence listing, and the nucleotide sequence of the DNAzyme enzyme chain is shown in SEQ ID No.2 in the sequence listing.

[0052] Repeat the above operations to prepare several portions of lead ion-dependent DNAzyme solutions, each 18 μL.

Embodiment 2

[0054] In the present embodiment, the standard solution curve is drawn, and the steps are as follows:

[0055] (1) Prepare lead ion standard solution

[0056] Preparation of Pb 2+ Lead acetate standard solutions with concentrations of 0, 0.8, 4, 8, 40, and 80 nM, respectively.

[0057] (2) Draw a standard curve

[0058] ① Get a part (18 μL) of the lead ion-dependent deoxyribozyme solution prepared in Example 1, add 2 μL Pb to it 2+ Lead acetate standard solution with a concentration of 0nM, placed at room temperature (25°C) for 80min for cleavage reaction, the cleavage reaction will give two cleavage products with nucleotide sequences as shown in 7 and 8, and then add 2μL of 5' with a concentration of 2μM Sub F labeled with a fluorescent dye group at the end and 4 μL of Sub Q labeled with a fluorescent quencher at the 3’ end at a concentration of 2 μM were mixed and incubated at 37°C for 35 min to terminate the cleavage reaction to obtain a reaction mixture, which was then ...

Embodiment 3

[0061] In this example, adding Pb to tap water 2+ And detect Pb 2+ Concentration, the steps are as follows:

[0062] (1) Prepare Pb from tap water 2+ Concentrations of 0, 4, 8, 40, 80nM Pb 2+ The sample solution is recorded as 1#~5# sample solution in turn.

[0063] (2)Pb 2+ Sample solution detection

[0064] ① Take a portion (18 μL) of the lead ion-dependent deoxyribozyme solution prepared in Example 1, add 1# sample solution to it, and place it at room temperature (25° C.) for 80 minutes to carry out the cleavage reaction. The cleavage reaction will give a nucleotide sequence such as The two cleavage products shown in 7 and 8, and then add 2 μL of 2 μM Sub F labeled with a fluorescent dye at the 5’ end and 4 μL of 2 μM Sub Q labeled with a fluorescent quencher at the 3’ end, After mixing, incubate at 37°C for 35 min to terminate the cleavage reaction to obtain a reaction mixture, and then use a fluorescence spectrometer to measure the fluorescence intensity of the obta...

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Abstract

The invention provides a functional nucleic acid fluorescence sensor with low background and stable temperature. The sensor comprises a molecular recognition component and a signal conversion component, wherein the molecular recognition component is a lead ion dependent deoxyribozyme, which consists of a deoxyribozyme substrate chain and a deoxyribozyme enzyme chain, a nucleotide sequence of the deoxyribozyme substrate chain is shown in SEQ ID NO.1 in a sequence table, and a nucleotide sequence of the deoxyribozyme enzyme chain is shown in SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 in the sequence table; and the signal conversion component consists of a Sub F whose 5' end is marked by a fluorescent staining group and a Sub Q whose 3' end is marked by a fluorescent quenching group, nucleotidesequences of Sub F and Sub Q are shown in SEQ ID NO. 5 and SEQ ID NO. 6 in the sequence table. The invention further provides an application of the fluorescence sensor in lead ion detection. The fluorescence sensor can improve the sensitivity of the existing biosensor method for detecting lead ions and the tolerance to the detection environment.

Description

technical field [0001] The invention belongs to the technical field of metal ion detection, and relates to a fluorescent sensor based on functional nucleic acid and its application in lead ion detection. Background technique [0002] Lead is an accumulative toxic heavy metal element and one of the earliest metals used by humans. It exists in the form of stable lead sulfide (galena) in the earth's crust, with a content of 0.0016%. Lead is a silver-white metal, very soft and easy to oxidize, so it is often gray. Its chemical properties are relatively stable, it is not easy to be corroded, and it has good ductility and other processing characteristics. Therefore, lead is mostly used in construction, lead batteries, warheads, welding Materials, fishing equipment and radiation protection materials and other industries. [0003] The problem of lead exceeding the standard in my country is relatively serious, mostly from industrial pollution. There is a phenomenon of generally high...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6432
Inventor 邓锐杰赵志峰杨淏何强夏许寒
Owner SICHUAN UNIV
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