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High-salinity-resistant nucleic acid sensor for copper and application thereof

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: 2018-05-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology describes a device that uses specialized molecules called metalloproteinase-2 or MOP2 to create a stable chemical bond between certain small parts of DNA. These tiny structures have unique properties such as being able to recognise different types of metals like iron, nickel, cobalt, etc., allowing them to react with other substances without affecting their function. By measuring this activity, researchers may use these techniques to study how biological processes work at various levels within cells.

Problems solved by technology

This patented technical problem addressed in this patent relates to detecting copperosion iron(II), nicklebdenanese zinkler blacksmithium ion batteries that contain copper, from both natural sources like sea water and animal feedings. These materials may contaminate environments where humans live, work product, equipment, and products made therefrom. Toxic levels of certain elements present pose challenges when analyzing them because their presence cannot be detected directly without physically touching any part of those objects being examined. Current techniques involve complicated procedures involving multiple steps and laborious operations, making analysis time-consuming and costly.

Method used

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  • High-salinity-resistant nucleic acid sensor for copper and application thereof
  • High-salinity-resistant nucleic acid sensor for copper and application thereof
  • High-salinity-resistant nucleic acid sensor for copper and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Preparation of copper ion deoxyribozyme and production of cleavage product

[0073] The substrate chain, enzyme chain and 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 cleaves the target product and amplifies The target products are 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 the 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 strand stock solution with 4 µL of 10 µM DNAzyme strand stock solution with ...

Embodiment 2

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

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

[0081] Part A System composition: 24.2 μL system

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

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

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

[0085] Ultrapure water: 9.2 μL

[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 nickase (10U / μL stock solution): 1.2μL (final concentration 0.37U / μL)

[0090] Nt.BstNBI nickase reaction buffer solution (10x stock solution): 1.5 μL (final concentration 0.5x).

[00...

Embodiment 3

[0094] Example 3G-Preparation of quadruplex functional nucleic acid color sensor

[0095] 80 μL of enzyme activity buffer (100 mM Tris, 120 mM NaCl, 10 mM MgCl 2 , 100mM KCl, pH8.4), 10μL of hemin dilution solution (mixed with 2μL of hemin stock solution (10μM) and 1mL of enzyme activity buffer) and 10μL of the substance to be developed (i.e. amplification product), mixed well After the reaction at 37°C for 30min, the amplified product was combined with hemin to form a G-quadruplex structure, 50μL of TMB chromogenic solution was added, mixed well, reacted at 37°C for 10min, and 50μL of 2M H was added. 2 SO 4 , and mixed to obtain a colored product, and a G-quadruplex functional nucleic acid color sensor was obtained.

[0096] 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 copper and application thereof. The sensor comprises a molecular recognition element, a signal multiplication element and a signal conversion element, wherein the molecular recognition element comprises copper ion deoxyribozyme; the copper ion deoxyribozyme consists of a substrate chain and an enzyme chain; the signal multiplication element comprises an isothermal amplification system; the isothermal amplification system comprises an amplification template; the signal conversion element comprises hemin and a color developing agent. The sensor provided by the invention is used for specifically recognizing a copper ion; the primary enlargement and conversion of a signal are carried out through an isothermal index multiplication reaction; a G-four-chain body structure with activity is formed under the induction of the hemin; the color developing agent is catalyzed to develop a color, thus, secondary multiplication and conversion are generated; the signal is converted into a visual signal, and the qualitative and quantitative detection in a high-salinity environment can be carried out.

Description

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Claims

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Application Information

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Owner CHINA AGRI UNIV
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