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Fluorescent probe for detecting Tl<+> based on nucleic acid aptamer

A technology of nucleic acid aptamers and fluorescent probes, applied in the field of fluorescent probes for detecting Tl+, can solve the problems of aptamer development and screening, difficulty in specific detection, difficulty in eliminating sensors, etc., and achieve background fluorescence Reduced, high fluorescence intensity and anti-interference ability, the effect of reduced fluorescence energy transfer efficiency

Active Publication Date: 2021-08-10
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014]Firstly, the development and screening of aptamers is very difficult, and due to the Tl+ ionic radius, charge and K +Similarly, it is difficult for nucleic acid aptamer-based sensors to exclude the interference of K+ and Na+, although some people use hemin aptamers to detect Tl +, but it is still difficult to achieve true specificity detection
Second, other ions in the solution will also quench the sensor based on fluorescence technology

Method used

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  • Fluorescent probe for detecting Tl&lt;+&gt; based on nucleic acid aptamer
  • Fluorescent probe for detecting Tl&lt;+&gt; based on nucleic acid aptamer
  • Fluorescent probe for detecting Tl&lt;+&gt; based on nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Screening the length of nucleic acid chain using fluorescence energy transfer efficiency

[0047] Objectives: By screening and optimization, low spontaneous fluorescence energy transfer nucleic acid chains are obtained to achieve lower detection background values.

[0048] In a 5 mM pH 7.6 Tris-HCl buffer solution, GACT-N mixed was mixed, allowing GACT-N in the buffer solution to 1 μm, and the stock solution was placed at a low temperature (4 ° C) stationary preservation.

[0049] Double-marked nucleic acid chain fluorescence detection. The excitation wavelength is set to 545 nm, and the base extension of Poly-T is selected based on the ratio of the detection signal (568 nm / 667 nm) to reduce the fluorescence energy transfer efficiency of both end fluorophores between the nucleic acid system when the target material is not added. Such as figure 2 As shown, i.e., when the STAG-N base group is ≥ 32, the spontaneous fluorescent energy transfer efficiency reaches a ...

Embodiment 2

[0051] Example 2: Comparison of detection ranges of different lengths GACT-N

[0052] Experimental purpose: based on TL + The detected linear range is compared to test the sensing performance of GACT24, GACT28, GACT32.

[0053] Compare the performance of GACT24, GACT28, and GACT32 to detect the performance of 铊 子. Formulated different concentrations TL + Standard solution, then different concentrations TL + The standard solution is mixed with GACT24, GACT28, GACT32 solution, incubated for 5 min at room temperature, and TL is carried out. + The specific binding reaction with the nucleic acid adapter, then fluorescence detection, the excitation wavelength is set to 545 nm, according to the ratio of the detection signal (568 nm / 667 nm), establish TL + The concentration is the standard curve of the conventional wave, the result is as Figure 4 Description, TL + When the concentration range is 0.2mm-1.0mm: GACT24, GACT28, GACT32 detection system is linear, corresponding R 2 From: 0.99...

Embodiment 3

[0055] Example 3: Establish TL + Standard curve of concentration and fluorescence ratio signal

[0056] Experimental purpose: establish TL + Standard curve of concentration and fluorescence ratio signal for quantitative detection TL + .

[0057] In a 5 mM pH 7.6 Tris-HCl buffer solution, the GACT32 mixed was mixed. The concentration of GACT32 was 1 μm in the buffer solution, which was placed at a low temperature (4 ° C) stationary, spare.

[0058] Establish TL + Standard curve of concentration and fluorescence ratio signal: TL of 0.01 ~ 10 mmol / L in the buffer system + The solution was added to each solution, 50 nm of GACT32 probe was added. Het the room temperature for 5 min, TL + The specifically binding reaction with the nucleic acid fitting, so that the fluorescent resonance energy between the fluorophores is transferred to produce a variation of the fluorescent signal, then perform fluorescence detection (fluorescence spectrum, such as Figure 5 As shown), the excitation wav...

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Abstract

The invention belongs to the technical field of chemical analysis and detection, and aims at solving problems that in recent years, thallium environment detection and poisoning identification and troubleshooting requirements are met. Portable detection means are lacked, a G-rich nucleic acid aptamer fragment capable of specifically capturing Tl<+> is designed, and a double-label fluorescence technology and a fluorescence resonance energy transfer principle are combined, the fluorescent probe for detecting Tl<+> based on the nucleic acid aptamer is constructed. The nucleic acid aptamer is obtained by screening and optimizing a G-rich nucleic acid aptamer by using a Poly-T extension strategy, and is named as GACT32. cyanine dyes Cy3 and Cy5 are adopted as fluorescence signal indicating groups to be modified at the 5' end and the 3' end of the nucleic acid aptamer respectively. According to the probe, background fluorescence of a traditional fluorescent probe is greatly reduced, interference of K<+>, Na<+> and other ions in a solution is eliminated, specific detection of Tl<+> is achieved, the linear range is 10 mu M-10 mM, and a portable detection method is provided for thallium ion environment detection and poisoning investigation.

Description

Technical field [0001] The present invention belongs to the detection technology, and it is specifically related to the principle of fluorescent resonance energy transfer, based on TL + It has specific captured G nucleic acid fittings and double-labeled fluorescent technology, and constructs detection TL + Fluorescent probe. Background technique [0002] In recent years, with the rapid development of the industry, it has been widely used in the field of optical and high temperature superconducting. However, due to the poisonous, professional contact or environmental pollution threaten human health; at the same time, the case of poisoning, poisoning, such as the American Chinese female scientist, Tsinghua University Zhu Ling poisoning, etc. After entering the blood through the digestive system or the respiratory system, it will be absorbed by each organ and tissue due to the similarity of the electric charge and ion radius, which interferes with potassium ions and other metal ions...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/53G01N33/84
CPCG01N21/6428G01N33/84G01N33/5308
Inventor 聂志勇李彤彤王永安赵瑾孙晓红乔思默
Owner ACADEMY OF MILITARY MEDICAL SCI
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