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An ultrasensitive closed-tube colorimetric circle-mediated isothermal amplification method using carboxyl-modified gold nanoparticles

A gold nanoparticle and particle technology, which is applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high sensitivity and carry pollution, and achieve the effect of high robustness

Active Publication Date: 2021-01-12
THE HONG KONG POLYTECHNIC UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the loop-mediated isothermal amplification method also has disadvantages. Due to its high sensitivity, once the lid is opened, it is easy to form carryover contamination.

Method used

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  • An ultrasensitive closed-tube colorimetric circle-mediated isothermal amplification method using carboxyl-modified gold nanoparticles
  • An ultrasensitive closed-tube colorimetric circle-mediated isothermal amplification method using carboxyl-modified gold nanoparticles
  • An ultrasensitive closed-tube colorimetric circle-mediated isothermal amplification method using carboxyl-modified gold nanoparticles

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Experimental program
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Effect test

Embodiment 1

[0065]The synthesis of embodiment 1MUA-AuNP

[0066] The synthesis of 15-nm AuNPs was based on a process previously reported by the inventors. [6b] All glassware and magnetic stir bars used for AuNP synthesis were cleaned with aqua regia (a mixture of concentrated hydrochloric acid and nitric acid in a volume ratio of 3:1; note: aqua regia is hazardous and highly corrosive and must be carefully ventilated cabinet), rinse with water, and dry in an oven. Hydrogen tetrachloroaurate (III) solution (30 mL, 0.01 wt %) was boiled under reflux with vigorous stirring. Additional sodium citrate (3 mL, 1 wt %) was added rapidly. The color of the solution changed from light yellow to dark red within a few minutes. Continue heating and stirring for 10 min, then cool to room temperature under stirring, and then filter the solution through a 0.8-μm nitrocellulose membrane (Millipore). The UV-Vis spectra of the synthesized AuNPs were measured using Ultrospec2100pro UV / visible spectrophoto...

Embodiment 2

[0070] The aggregation and disaggregation test of embodiment 2MUA-AuNP

[0071] Aggregation and disaggregation assays for MUA-AuNPs: For aggregation assays, prepare isothermal amplification buffer containing MUA-AuNPs (6 nM) and 1× (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 , 0.1% Tween 20, pH 8.8; New England Biolabs) mixture. For disaggregation experiments, after incubation for 1 min, add K 4 P 2 o 7 (1.4mM) or EDTA (2.8mM) or dNTP (each 0.35mM) in the above mixture. These solutions were incubated at 65°C for 1 h (GeneAmp PCRSystem 9700, Applied Biosystems). Colorimetric results were recorded before and after gentle ultrasound for 10 s (WiseCleanWUC-A01H ultrasonic cleaner, Daihan Scientific). for Mg 2+ Concentration-dependent aggregation assay, different amounts of MgSO 4 (0, 0.4, 0.8 and 1.2 mM) were added to MUA-AuNP (6 nM). These solutions were incubated at room temperature for 10 min. Colorimetric results were recorded after gentle ultrasou...

Embodiment 3

[0080] Example 3Mg 2+ and P 2 o 7 4- Effect on the color of MUA-AuNP solution

[0081] AuNPs with an average diameter of 15 nm were synthesized as described above or by the citrate reduction method [6b,13] and functionalized it with carboxyl groups by simple incubation with MUA. Carboxyl group and Mg 2+ Chelation in figure 1 Schematically shown in a. The obtained aggregated MUA-AuNPs can be extracted by adding chelated Mg 2+ P 2 o 7 4- And depolymerization. The conditions of the LAMP reaction were simulated, and the aggregation and depolymerization experiments were carried out at 65°C. After adding Mg 2+ (2mM) to MUA-AuNP (6nM), the solution color changed from red to purple immediately. After standing for 1h, a dark purple precipitate was observed and the supernatant became clear ( Figure 4 a). On the other hand, when adding Mg 2+ , then add P 2 o 7 4- (1.4mM), a dark red precipitate was observed ( Figure 4 a). After mild sonication, the precipitate dep...

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Abstract

The invention relates to a hypersensitive closed tube-type colorimetric loop-mediated isothermal amplification method utilizing carboxyl-modified gold nanometer particles. The method comprises that through combination of LAMP and a MUA-AuNP novel detection mechanism utilizing Mg<2+> and P2O7<4-> to control a color change, an AuNP-based colorimetric operation-closed tube type isothermal amplication combined nucleic acid detection platform is built and realizes visual detection of 200 copies of target DNA sequences. A color is controlled by magnesium ion template aggregation, and in the presence of a target, LAMP reaction by-product pyrophosphoric acid ions cause particle depolymerization. The platform has the advantages of high sensitivity, no residual pollutes and low cost.

Description

technical field [0001] The present invention relates to simple detection methods of specific nucleic acid sequences. In particular, the present invention relates to the use of functionalized particles, such as carboxyl-modified gold nanoparticles, in nucleic acid amplification systems capable of generating pyrophosphate ions, such as in loop-mediated isothermal amplification methods, at high Sensitive, low-cost method for detecting specific nucleic acid sequences. Background technique [0002] Detection of specific nucleic acid sequences in a simple manner can add greater value to point-of-care diagnostics and point-of-care pathogen detection. [1] Gold nanoparticles (AuNPs) with unique colorimetric properties are well suited for this task. [2] AuNPs exhibit characteristic surface plasmon resonance (SPR) absorption bands in the visible region and the exact spectrum depends on the distance between particles. Specifically, particle agglutination causes a red shift in the SPR...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844
Inventor 李铭鸿黃冠锋叶社平
Owner THE HONG KONG POLYTECHNIC UNIV
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