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A lead high-salt-resistant nucleic acid sensor and its application

A salt nucleic acid and sensor technology, which is applied in the field of lead high-salt-resistant nucleic acid sensors, can solve the problems of low sensitivity, limitation, and poor repeatability, and achieve high specificity and sensitivity, rapid detection, and rapid response

Active Publication Date: 2020-05-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods rely on large-scale instruments and special personnel to operate, and are limited in field and field detection
Chemical sensing methods represented by organic small molecules such as ethers, polyamines, and aromatic hydrocarbons have also developed to a certain extent. At present, there are shortcomings such as low sensitivity, poor repeatability, and detection in organic solvents. The detection is reliable. Sexuality and practicality are not high

Method used

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  • A lead high-salt-resistant nucleic acid sensor and its application
  • A lead high-salt-resistant nucleic acid sensor and its application
  • A lead high-salt-resistant nucleic acid sensor and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] The preparation of embodiment 1 lead ion deoxyribozyme and the generation of cleavage product

[0070] The substrate chain, enzyme chain and DNAzyme cleavage products of the DNAzyme designed for lead ions are as follows:

[0071]

[0072]

[0073] Note: GACTC in the amplified template D is the Nt.BstNBI nicking endonuclease recognition sequence, and the first four base pairs of the sequence (between C and A) are the synthetic strand cleavage sites; DNAzyme cleavage products C and The amplification product F is completely complementary to the amplification template D; the TTGGGGGGT sequence at the end of the DNAzyme substrate chain A is added to increase the Tm value of the template binding; the lead ion cleavage site is at the rA of the DNAzyme substrate chain A after.

[0074] The preparation method of lead ion deoxyribozyme:

[0075] Mix 4 μL of 10 μM DNAzyme substrate chain stock solution with 4 μL of 10 μM DNAzyme enzyme chain stock buffer (final concentrati...

Embodiment 2

[0077] The amplification of embodiment 2 lead ion deoxyribozyme cleavage products

[0078] The system for isothermal amplification reaction consists of two parts (system A and system B). Amplification reaction system composition: 30 μL system.

[0079] Part A system composition: 24.2μL system

[0080] Amplification template (1μM stock solution): 6μL (final concentration 0.2μM)

[0081] dNTPs (2.5mM stock solution): 3μL

[0082] Cutting product of lead ion DNAzyme (1 μM): 6 μL, final concentration 0.2 μM

[0083] Ultrapure water: 9.2μL

[0084] Part B system composition: 5.8 μL

[0085] Bst DNA polymerase (8U / μL stock solution): 0.1μL (final concentration 0.02U / μL)

[0086] Polymerase reaction buffer solution (10x stock solution): 3 μL (final concentration 1x)

[0087] Nt.BstNBI nicking endonuclease (10U / μL stock solution): 1.2μL (final concentration 0.37U / μL)

[0088] Nt.BstNBI nicking endonuclease reaction buffer solution (10x stock solution): 1.5 μL (final concentrat...

Embodiment 3

[0092] Example 3 Preparation of G-quadruplex functional nucleic acid chromogenic sensor

[0093] 80 μL enzyme activity buffer (100mM Tris, 120mM NaCl, 10mM MgCl 2 , 100mM KCl, pH8.4), 10μL hemin dilution solution (2μL hemin stock solution (10μM) mixed with 1mL enzyme activity buffer) mixed with 10μL of the material to be developed (i.e. the amplified target product), mixed After homogenization, react at 37°C for 30 minutes to make the amplified target product F combine with hemin to form a G-quadruplex structure, add 50 μL TMB chromogenic solution, mix well, react at 37°C for 10 minutes, add 50 μL 2M H 2 SO4, mix well to obtain a color-developed product, and obtain a G-quadruplex functional nucleic acid color sensor.

[0094] Then carry out microplate reader to measure OD 450 .

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Abstract

The invention discloses a high-salinity-resistant nucleic acid sensor for lead and an application of the nucleic acid sensor. The sensor comprises a molecular recognition element, a signal amplification element and a signal conversion element, wherein the molecular recognition element comprises lead ion deoxyribozyme; the lead ion deoxyribozyme consists of a substrate chain and an enzyme chain; the signal amplification element comprises an isothermal amplification system which comprises an amplification template; the signal conversion element comprises hemin and a color developing agent. The sensor can specifically recognize lead ions and first amplification and conversion of signals are performed through an equivalent temperature index amplification reaction; a G-quadruplex structure withactivity is formed under the induction of hemin, the color developing agent is catalyzed to develop colors, so that second amplification and conversion are produced, the signals are converted into visual signals, and qualitative and quantitative detection in a high-salinity environment can be performed.

Description

technical field [0001] The invention belongs to the technical field of heavy metal detection, and in particular relates to a lead high-salt-resistant nucleic acid sensor and an application thereof. Background technique [0002] Lead ion is an environmental pollutant with wide distribution and accumulation toxicity. It is stable in nature and can cause harm to the human body at extremely low concentrations, causing multiple damages to the nervous system, blood circulation system, digestive system, and urinary system. Lead compounds in the environment mainly enter the human body through the digestive tract and respiratory tract, but liquid lead compounds can also enter the human body through skin contact. Lead absorbed by the human body enters the blood first, forming organic lead compounds such as soluble lead phosphate and lead glycerophosphate. Lead is combined with protein, circulates in the body, and can be absorbed by liver, kidney, brain, pancreas and other internal o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12Q1/44G01N21/78
CPCC12Q1/005C12Q1/44C12Q1/6844C12Q2326/12G01N21/78C12Q2521/301C12Q2521/101
Inventor 许文涛罗云波黄昆仑田晶晶杜再慧肖冰董凯
Owner CHINA AGRI UNIV
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