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Label-free nuclease analysis method based on stable isotope detection

A stable isotope and analysis method technology, applied in the field of label-free nuclease analysis based on stable isotope detection, can solve the problems of low stability of CuNPs, long-term detection and real-time monitoring, etc.

Pending Publication Date: 2020-10-23
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the fluorescence spectrometry detection of CuNPs synthesized based on DNA template method has been successfully used in the label-free detection of various nucleases, the stability of the synthesized CuNPs is not high (the relative fluorescence intensity decays by more than 80% within 1 hour) is still a problem. The key shackles restricting the application of this method for long-term detection and real-time monitoring

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  • Label-free nuclease analysis method based on stable isotope detection
  • Label-free nuclease analysis method based on stable isotope detection
  • Label-free nuclease analysis method based on stable isotope detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 The label-free analysis method based on stable isotope detection to exonuclease (ExoI) detection

[0043] exonuclease (ExoI) is a nuclease that can specifically cut single-stranded DNA (ssDNA) from the 3' end to the 5' end. Meanwhile, single-stranded DNA (PolyT-ssDNA) composed entirely of thymines can be used as a DNA template to synthesize CuNPs natively. Therefore, stable isotope-based label-free detection of ExoI can be performed using PolyT-ssDNA.

[0044] In the experiment, the concentration of ExoⅠ ranged from 0.1 Unit / μL (U / μL) to 20U / L, depending onfigure 1 It can be seen that within this range, the common logarithm (X) of ExoI concentration is linearly correlated with the ICPMS intensity signal (Y) of 63Cu. The linear equation is Y=2.23E4X +1.07E5, the correlation coefficient R 2 =0.9814, the limit of detection (LOD) was 0.029U / μL.

[0045] This example fully proves that the present invention can realize highly sensitive and label-free analysis...

Embodiment 2

[0046] Example 2 Explore the specificity of the analytical method of the present invention to the detection of nuclease ExoI

[0047] In this example, taking Exo I as an example, several different common nucleases and proteins were detected by the label-free nuclease assay method based on PolyT-ssDNA template, and the specificity of the method was explored.

[0048] Using the above steps, use PolyT-ssDNA as a template to detect 5U / μL Exo I and 20U / μL Exo III, Exo VI, Bst, Thrombin, 0.05% m / v BSA respectively, keep the rest of the conditions exactly the same, and react at 37°C After 120 Min, 10 μL of 20 mM ascorbic acid solution and 35 μL of 1 mM copper sulfate solution were added and shaken at room temperature for 10 min. After magnetic separation, nitric acid digestion was performed, and stable isotope detection was performed by ICPMS. The result is as figure 2 As shown, except for Exo I, which can cause a significant decrease in signal, other common nucleases (exonuclease ...

Embodiment 3

[0049] Example 3 Explore the long-term stability of the analytical method of the present invention for the detection of nuclease ExoI

[0050] In this example, Exo I was taken as an example to compare the signal stability under long-term repeated detection with the traditional label-free fluorescence detection method.

[0051] In the fluorescence detection experiment, PolyT-ssDNA was directly reacted with 1U / μL ExoⅠ, reacted at 37°C for 120Min, then added 10μL of 20mM ascorbic acid solution and 35μL of 1mM copper sulfate solution and shaken at room temperature for 10min to generate CuNPs with fluorescent properties. Fluorescence detection at an emission wavelength of 660 nm was performed at the excitation wavelength. The total detection time is 3 hours, and the interval between two times is 5 minutes. The intensity of each time is recorded and the relationship between fluorescence intensity and time is drawn.

[0052] In the experiment of stable isotope detection, PolyT-ssDNA...

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Abstract

The invention provides a label-free analysis method based on stable isotope detection and application of the label-free analysis method in nuclease detection. In the presence of nuclease, a template DNA sequence for synthesizing CuNPs is subjected to enzyme digestion, DNA fragments obtained after enzyme digestion lose the template effect, and CuNPs cannot be synthesized under the action of ascorbic acid and copper ions. The synthesized CuNPs are digested by nitric acid to form copper ions, and ICPMS quantitative analysis can be carried out. The invention provides the analysis method capable ofrealizing long-time stable label-free detection on nuclease. According to the invention, an analysis signal can be kept stable for a long time without attenuation; the method has the advantages thatcost is low, tedious operation steps are omitted, and response is rapid; and meanwhile, the advantages of low detection limit and excellent stability of ICPMS for stable isotope detection are utilized, so long-time stability is greatly improved while nuclease detection sensitivity is improved, and long-time stable detection and real-time monitoring can be achieved.

Description

technical field [0001] The invention belongs to the detection field of analytical chemistry, relates to the field of nuclease stable isotope (Metal stableisotope) sensing, and specially designs a stable isotope produced by in situ synthesis of copper nanoparticles (CuNPs) based on deoxyribonucleic acid (DNA) as a template through Nuclease detection method by inductively coupled plasma mass spectrometry. Background technique [0002] Nucleases are a series of biological enzymes with high specificity to cut and manipulate deoxyribonucleic acid (DNA) structural changes, including nucleic acid ligase, telomerase, endonuclease, exonuclease, etc. The establishment of highly sensitive detection of nucleases is of great significance for DNA replication, recombination, repair, genotyping and sequencing. [0003] Among the currently established analytical methods for nucleases, the label-free analytical method based on the in situ synthesis of fluorescent copper nanoparticles based o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62C12Q1/44
CPCG01N27/62C12Q1/44
Inventor 刘睿胡建煜吕弋
Owner SICHUAN UNIV
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