Cadmium ion sensing method combining inducible allosteric probe with rolling circle amplification

A technology of rolling circle amplification and sensing method, which is applied in the field of cadmium ion sensing combined with inducible allosteric probes and rolling circle amplification, can solve the problems of time-consuming, cumbersome and the like, and achieves avoiding cumbersome, cost-effective, simple method effect

Active Publication Date: 2020-12-01
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Purpose of the invention: The purpose of the present invention is to provide an inducible allosteric probe combined with a rolling circle amplification cadmium ion sensing method, which improves the sensitivity and specificity of detecting cadmium ions, and overcomes the existing analytical detection method Disadvantages of cumbersome and time-consuming

Method used

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  • Cadmium ion sensing method combining inducible allosteric probe with rolling circle amplification
  • Cadmium ion sensing method combining inducible allosteric probe with rolling circle amplification
  • Cadmium ion sensing method combining inducible allosteric probe with rolling circle amplification

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1) Take 3 μL of sample solutions containing cadmium ions at different concentrations and incubate with 3 μL of the probe sequence. Then 3 μL of primers were added and incubated for 10 min. Then, add the mixture to 10×T4 DNA ligation buffer (400 mM Tris-HCl, 100 mM MgCl) containing 1 μL of T4 DNA ligase 2 , 10 mM DTT, 5 mM ATP, pH 7.8), add 4.8 μL of DEPC water and incubate at room temperature for 10 min. Ligation products can be obtained in varying amounts.

[0032] Table 1 DNA sequence

[0033]

[0034] 2) Take 20 μL of the ligation product obtained in 1) and 4 μL of buffer 2 (50 mM Tris-HCl, 10 mM MgCl 2, 10mM (NH 4 ) 2 SO 4 , 4mM DTT, pH 7.5@25℃), 1μL of BSA (10mg / mL), 5μL of dNTP (10mM), 1μL of Phi29 DNA polymerase (10U / μL) and 9μL of DEPC water were mixed to make the whole reaction at 37 Incubate at ℃ for 2h. Finally, different amounts of DNA strands are obtained.

[0035] 3) The fluorescent dye SYBR Green I solution was diluted with 10 mM Tris-HCl 10 m...

Embodiment 2

[0037] Sensitivity experiment

[0038] Select sample solutions with cadmium ion concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100ppb for verification, and the results are as follows figure 2 shown. It is determined that the detection linear range is 1-60ppb, and the detection limit reaches 0.08ppb.

Embodiment 3

[0040] The present invention is compared with the detection result based on Real-Time PCR

[0041] Since this method is based on the fluorescence of fluorescent dyes to detect the final fluorescence intensity, we used Real-Time PCR, the gold standard method for detection of fluorescence intensity, for verification experiments. Detect the sample of embodiment 2, Cd 2+ =50ppb, the result is as image 3 shown. Our method is more sensitive and time-saving than Real-Time PCR method.

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Abstract

The invention discloses a cadmium ion sensing method combining an inducible allosteric probe with rolling circle amplification. The method can realize high-sensitivity and specific detection of tracecadmium ions. Potential heavy metal pollution in air, water and soil exists everywhere, and life quality and body health of people are seriously affected. Heavy metal ion cadmium is a common heavy metal in grain pollution. Cadmium is easy to accumulate in kidneys, bone tissues, eyes and other organs and tissues of a human body. Particularly an accumulation phenomenon in the kidneys and the bone tissues is the most serious, diseases such as renal failure and fragile fractures can be caused, and human body health is threatened. The trace cadmium ions can cause serious damage to the human body, the method is of great significance to environmental protection and food safety.

Description

technical field [0001] The invention relates to an analysis and detection method, in particular to a cadmium ion sensing method using an inducible allosteric probe combined with rolling circle amplification. Background technique [0002] The U.S. Environmental Protection Agency (EPA) has set a maximum cadmium pollution value of 5ppb in drinking water. Considering its harmful effects, we need a sensitive and reliable cadmium ion detection technology. Common detection techniques for cadmium ions include atomic absorption spectrometry, atomic fluorescence spectrometry, and inductively coupled plasma spectrometry. Although the selectivity and accuracy of these methods are high, most of them require complex preprocessing, complex tools, and time-consuming tools. [0003] As a high-efficiency amplification technique, rolling circle amplification (RCA) is a simple and efficient isothermal enzymatic amplification technique that utilizes unique DNA and RNA polymerases such as Phi29,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844G01N21/64
CPCC12Q1/6844G01N21/643C12Q2531/125C12Q2521/501C12Q2563/107Y02A20/20
Inventor 黄和李昺之张幸锁缇莹张世林谢思盈夏安祺
Owner NANJING NORMAL UNIVERSITY
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