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High salt resistant nucleic acid sensor of silver and application thereof

A sensor and salt nucleic acid technology, applied in the field of silver high-salt-resistant nucleic acid sensors, can solve the problems of expensive instruments, high cost, complicated processing methods, etc., and achieve the effects of rapid response, rapid detection, high specificity and sensitivity

Active Publication Date: 2018-04-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pretreatment methods of these methods are complicated and require analysis after sampling. They are not suitable for rapid in-situ detection of silver ion concentration. Although these methods have high sensitivity, they all require expensive instruments and high cost. Therefore, it is urgent to develop pollution-free methods. , simple, rapid, high sensitivity and high specificity method to meet the needs of trace metal silver detection

Method used

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  • High salt resistant nucleic acid sensor of silver and application thereof
  • High salt resistant nucleic acid sensor of silver and application thereof
  • High salt resistant nucleic acid sensor of silver and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0074]

[0075] 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; the ribozyme cuts the target product C and The amplified target product F is completely complementary to the amplified template D; the GGAGGCGGA sequence at the end of the ribozyme substrate chain A is added to increase the Tm value of binding to the template; the sodium ion cleavage site is after the rA of the ribozyme substrate chain A .

[0076] The preparation method of silver ion deoxyribozyme:

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

Embodiment 2

[0079] Embodiment 2 The amplification of silver 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 silver ion DNAzyme (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 concen...

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 high salt resistant nucleic acid sensor of silver and application thereof. The sensor includes a molecular recognition element, a signal amplification element, and a signal conversion element. The molecular recognition element includes silver ion deoxyribozyme, which is composed of a substrate chain and an enzyme chain. The signal amplification element includes an isothermal amplification system, and the isothermal amplification system includes an amplification template. The signal conversion element includes a color developing agent and hemin. The sensor provided bythe invention can specifically recognize silver ions, carries out primary amplification and conversion of signal through isothermal exponential amplification reaction, an active G-quadruplex structurecan be formed under the induction of hemin to catalyze color development of the color developing agent, thus generating secondary amplification and conversion, and the signal can be converted into avisual signal, therefore the sensor provided by the invention can achieve qualitative and quantitative detection.

Description

technical field [0001] The invention belongs to the technical field of heavy metal detection, and in particular relates to a silver high-salt-resistant nucleic acid sensor and an application thereof. Background technique [0002] Silver widely exists in nature. It can be used as a metal catalyst, antibacterial agent, photosensitizer, etc. Silver has special bactericidal, catalytic and optical properties, and is widely used as an anti-inflammatory and antibacterial agent in clinical medicine, such as burn dressings. Its application form includes silver salt (AgNO 3 ), silver compounds such as (SD-Ag) and nano-silver are widely used in electronics, electroplating, photosensitive and other industries. [0003] At the same time, the production and application of these silver-related industries will produce silver-containing industrial wastewater. This silver-containing wastewater pollutes the environment very seriously. Compared with other forms of silver, silver in the form o...

Claims

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Application Information

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IPC IPC(8): G01N21/78G01N33/53G01N33/535C12Q1/6844C12Q1/6825C12N15/113
CPCC12N15/113C12N2310/12C12Q1/6825C12Q1/6844G01N21/78G01N33/5308G01N33/535C12Q2521/345
Inventor 许文涛罗云波黄昆仑田晶晶肖冰杜再慧董凯
Owner CHINA AGRI UNIV
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