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

A sensor, salt nucleic acid technology, applied in the field of heavy metal detection, can solve the problems of lack of universal applicability and cumbersome sample processing, and achieve the effects of rapid detection, high specificity and sensitivity, and rapid response

Active Publication Date: 2021-04-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Common detection methods for copper ions mainly include atomic spectroscopy, including atomic absorption spectroscopy, inductively coupled plasma emission spectrometry and fluorescence spectroscopy, visible spectrophotometry, flow injection chemiluminescence and differential potential stripping methods, etc., but these The detection methods all require expensive instruments and professionally trained operators, and the sample pretreatment is also relatively cumbersome, which is not universally applicable

Method used

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

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Effect test

Embodiment 1

[0072] The preparation of embodiment 1 copper ion deoxyribozyme and the production of cleavage product

[0073] The substrate chain, enzyme chain and DNAzyme cleavage products of the DNAzyme designed for copper ions are as follows:

[0074]

[0075] Note: GACTC in the amplification template 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; the ribozyme cuts the target product and amplifies The target products are all completely complementary to the amplification template; the TTGGGGGGT sequence at the end of the ribozyme substrate chain A is added to increase the Tm value of binding to the amplification template D; the copper ion cleavage site is after the -G of the ribozyme substrate chain A .

[0076] The preparation method of copper ion deoxyribozyme:

[0077] Mix 4 μL of 10 μM DNAzyme substrate chain stock solution with 4 μL of 10 μM DNAzyme enzyme chain ...

Embodiment 2

[0079] Embodiment 2 Amplification of copper ion deoxyribozyme cleavage product

[0080] The system for isothermal amplification reaction consists of two parts (system A and system B). Amplification reaction system composition: 30 μL system.

[0081] Part A system composition: 24.2μL system

[0082] Amplification template (1μM stock solution): 6μL (final concentration 0.2μM)

[0083] dNTPs (2.5mM stock solution): 3μL

[0084] Cleavage product of copper ion deoxyribozyme (1 μM): 6 μL, final concentration 0.2 μM

[0085] Ultrapure water: 9.2μL

[0086] Part B system composition: 5.8 μL

[0087] Bst DNA polymerase (8U / μL stock solution): 0.1μL (final concentration 0.02U / μL)

[0088] Polymerase reaction buffer solution (10x stock solution): 3 μL (final concentration 1x)

[0089] Nt.BstNBI nicking endonuclease (10U / μL stock solution): 1.2μL (final concentration 0.37U / μL)

[0090] Nt.BstNBI nicking endonuclease reaction buffer solution (10x stock solution): 1.5 μL (final conc...

Embodiment 3

[0094] Embodiment 3G-Preparation of Quadruplex Functional Nucleic Acid Colorimetric Sensor

[0095] 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 amplification product), and mixed Afterwards, react at 37°C for 30 minutes to make the amplified product 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 SO 4 , and mix well to obtain a chromogenic product, and obtain a G-quadruplex functional nucleic acid chromogenic sensor.

[0096] Then carry out microplate reader to measure OD 450 .

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Abstract

The invention discloses a copper high-salt-resistant nucleic acid sensor and an application thereof. The sensor includes a molecular recognition element, a signal amplification element and a signal conversion element, the molecular recognition element includes a copper ion deoxyribozyme; the copper ion deoxyribozyme is composed of a substrate chain and an enzyme chain; the signal amplification element It includes an isothermal amplification system, and the isothermal amplification system includes an amplification template; the signal conversion element includes hemin and a chromogen. The sensor of the present invention specifically recognizes copper ions, and performs signal-level amplification and conversion through an isothermal exponential amplification reaction; under the induction of hemin, an active G-quadruplex structure is formed, and the chromogenic agent is catalyzed for color development. As a result, secondary amplification and conversion are generated, and converted into visualized signals, which can be used for qualitative and quantitative detection in high-salt environments.

Description

technical field [0001] The invention belongs to the technical field of heavy metal detection, and in particular relates to a copper high-salt-resistant nucleic acid sensor and an application thereof. . Background technique [0002] Copper is a transition element. Pure copper is a soft metal with good ductility, high thermal conductivity and electrical conductivity. Therefore, it is the most commonly used material in cables and electrical and electronic components. It can also be used as a building material and can be composed of many kinds. alloy. The pollution caused by copper (Cu) and its compounds in the environment is mainly caused by the mining and smelting of copper-zinc mines, metal processing, machinery manufacturing, and steel production, among which the smoke and dust emitted by smelting is the main source of atmospheric copper pollution. [0003] For organisms, whether animals or plants, copper is an essential trace element. The amount of copper in a normal hum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78G01N21/25
CPCG01N21/25G01N21/78
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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