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Colorimetric sensor assembled based on magnetic particles and isothermal nucleic acid amplification method as well as preparation method and application of colorimetric sensor

An isothermal nucleic acid amplification and colorimetric sensor technology, which is applied in the field of colorimetric biosensing and nucleic acid detection, can solve the problems of limited by large instruments, single method, and long time consumption, so as to achieve improved sensitivity and robustness, simple operation, low cost effect

Active Publication Date: 2021-08-03
NANJING UNIV OF POSTS & TELECOMM
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the shortcomings of the current method for detecting pathogenic nucleic acid, such as single, time-consuming, and limited by large-scale instruments, the present invention provides a colorimetric sensor assembled based on magnetic particles and isothermal nucleic acid amplification method and its preparation method and application, which can be used for various nucleic acid Molecular detection to achieve rapid, sensitive and instant detection of infectious viral nucleic acids

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  • Colorimetric sensor assembled based on magnetic particles and isothermal nucleic acid amplification method as well as preparation method and application of colorimetric sensor
  • Colorimetric sensor assembled based on magnetic particles and isothermal nucleic acid amplification method as well as preparation method and application of colorimetric sensor
  • Colorimetric sensor assembled based on magnetic particles and isothermal nucleic acid amplification method as well as preparation method and application of colorimetric sensor

Examples

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Embodiment 1

[0064] A method for preparing a colorimetric sensor assembled based on magnetic particles and an isothermal nucleic acid amplification method, the specific steps are as follows:

[0065] (1) Disperse 10 μL of 10 mg / mL streptavidin-modified magnetic particles in Tris buffer (5mM Tris-HCl, 0.5mM EDTA, 1M NaCl, pH=7.4), wash 3 times to remove Protective agent. Add DNA anchor strands to the streptavidin-modified magnetic particles so that the final concentration of DNA anchor strands is 0.5 μM, incubate at 37°C for 1 h, magnetically separate and wash 3 times to remove the supernatant, and wash the The magnetic particles are dispersed in the reaction solution (50mM Tris-HCl, 140mM NaCl, 1mMMgCl 2 , pH=7.4).

[0066] The DNA anchor chain is modified by biotin or amino, and its sequence is shown in SEQ ID No.1, specifically: 5'-biotin-TTTTTTTTTTTCTCACTAACTGCATA-3'.

[0067] DNA anchor strands are assembled to the surface of magnetic particles by biotin-streptavidin or aminocarboxy...

Embodiment 2

[0077] The colorimetric sensor prepared in Example 1 is used for nucleic acid detection, and the specific steps are as follows:

[0078] (1) Take the colorimetric sensor prepared in Example 1, add the target nucleic acid (Ebola nucleic acid) with a final concentration of 100 nM, perform an isothermal nucleic acid amplification reaction at 37° C. for 45 minutes, and collect 50 μL of the supernatant by magnetic separation after the reaction.

[0079] The sequence of Ebola nucleic acid is as shown in SEQ ID No.5, specifically:

[0080] 5'-GTCTTTTTCCCTCAACTATCGGC-3'.

[0081] (2) Add 14 μL of reaction solution (50 mM HEPES, 0.4 M NaCl, 40 mM KCl, 2% dimethylsulfoxide (DMSO), 0.1% Triton X-100, pH 8.0) and heme (final The concentration is 1 μM), react at room temperature for 1 h, and form a DNase / heme complex. Finally, ABTS dilution at a final concentration of 6 mM and HO at a final concentration of 1 mM were sequentially added 2 o 2 The solution was incubated at room temperatu...

Embodiment 3

[0084] The DNA fuel chain concentration, isothermal nucleic acid amplification reaction temperature, and isothermal nucleic acid amplification reaction time were respectively optimized in the preparation method of the colorimetric sensor described in Example 1.

[0085] (1) Optimization of DNA fuel chain concentration:

[0086]In step (3) of Example 1, DNA fuel chains with final concentrations of 0nM, 250μM, 500nM, 750nM, and 1000nM were added to assemble the colorimetric sensors, and then 50μL of the colorimetric sensors were taken respectively, and the target concentration of 100nM was added to them respectively. Nucleic acid (Ebola nucleic acid). The isothermal nucleic acid amplification reaction was carried out at 37° C. for 45 minutes. After the reaction, 50 μL of the supernatant was separated by magnetic separation. Subsequently, 14 μL of reaction solution (50 mM HEPES, 0.4 M NaCl, 40 mM KCl, 2% dimethylsulfoxide (DMSO), 0.1% Triton X-100, pH 8.0) and heme (final concen...

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Abstract

The invention discloses a colorimetric sensor assembled based on magnetic particles and an isothermal nucleic acid amplification method and a preparation method and application of the colorimetric sensor. The colorimetric sensor comprises a molecular recognition element, a signal amplification element and a signal conversion element, and the molecular recognition element is composed of a DNA anchoring chain modified by the surfaces of the magnetic particles and a DNA recognition chain; the signal amplification element comprises an isothermal amplification system and a DNA fuel chain; the signal conversion element comprises a DNA enzyme chain, ABTS and hydrogen peroxide. When the target nucleic acid exists, the target nucleic acid and the DNA fuel chain jointly start a circulating chain replacement reaction, and a large number of DNA enzyme chains with catalytic performance are released. After magnetic separation, a DNA enzyme chain in supernate can be combined with heme to form a structure with peroxidase-like catalytic activity, in the presence of hydrogen peroxide, nearly colorless ABTS can be catalyzed to be oxidized into a green oxidation product, and on-site in-situ rapid detection of target nucleic acid can be achieved through naked eye observation or colorimetric spectrum.

Description

technical field [0001] The invention belongs to the technical field of colorimetric biosensing and nucleic acid detection, and in particular relates to a colorimetric sensor assembled based on magnetic particles and an isothermal nucleic acid amplification method, and a preparation method and application thereof. Background technique [0002] The spread and outbreak of infectious diseases is one of the important concerns in the world today. Infectious diseases are usually caused by microorganisms such as bacteria, viruses, fungi and parasites, which pose a huge threat to human health and economic development. Accurate and timely infection detection and pathogen identification are the keys to disease prevention, treatment and monitoring. Nucleic acid detection for pathogens is of great significance in medical diagnosis and epidemic prevention and control, and has received high attention. At present, the main method for detecting pathogenic nucleic acid is polymerase chain r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/682C12Q1/6825
CPCC12Q1/6844C12Q1/682C12Q1/6825Y02A50/30
Inventor 朱丹马子豪汪联辉晁洁
Owner NANJING UNIV OF POSTS & TELECOMM
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