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Method for simultaneously detecting two types of HIV DNA through DNA-directed color-variable silver nano-clusters

A silver nano-cluster and detection method technology, applied in the fields of molecular biology and nucleic acid chemistry, can solve the problems of complicated operation, high modification cost, increased difficulty, etc., and achieve the effects of simple preparation, large emission range, and economic simplicity.

Active Publication Date: 2018-12-25
XIANGTAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high cost of modification, the time-consuming separation and purification of materials, and the cumbersome operation increase the difficulty of this method.

Method used

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  • Method for simultaneously detecting two types of HIV DNA through DNA-directed color-variable silver nano-clusters
  • Method for simultaneously detecting two types of HIV DNA through DNA-directed color-variable silver nano-clusters
  • Method for simultaneously detecting two types of HIV DNA through DNA-directed color-variable silver nano-clusters

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Design of fluorescent probes based on color-changing silver nanoclusters

[0047] (1) The design of DNA probe in the present embodiment:

[0048] 1) The DNA probe is composed of two partially complementary ssDNAs, namely cDNA and P strand;

[0049] 2) The cDNA chain is composed of four parts: G-rich sequence, C-rich sequence for synthesizing AgNCs, sequence complementary to HIV-2 sequence and base sequence partially paired with HIV-1 complementary chain;

[0050] 3) The P chain consists of four parts: the sequence of AgNCs capable of synthesizing yellow-green fluorescence, the sequence of AgNCs capable of synthesizing orange-red light, the sequence complementary to the HIV-1 sequence, and the base sequence partially paired with the HIV-2 complementary chain .

[0051] The DNA sequence involved in this embodiment is as figure 2 shown.

[0052] (2) Synthesis of DNA-templated AgNCs:

[0053] 10 μL of Sequence P (500 nM) was dissolved in 170 μL of PBS buffer (20 mM, p...

Embodiment 2

[0055] Validation of the Feasibility of Simultaneously Detecting Two HIV DNAs Using Fluorescent Probes Based on Silver Nanoclusters

[0056]The present invention simultaneously uses luminescent AgNCs with 565nm and 630nm emission peaks as templates to detect HIV-1 and HIV-2, and the working principle is as follows figure 1 .

[0057] In this embodiment, we use fluorescence spectroscopy to verify the feasibility of the principle.

[0058] The conditions for fluorescence spectrometry detection are:

[0059] Concentration of analytes: 500nM for HIV-1 and HIV-2

[0060] Instrument: RF-5301PC Fluorescence Spectrophotometer

[0061] Instrument parameters: excitation wavelength: 500nm and 580nm, emission wavelength: 565nm and 630nm, excitation slit: 5nm, emission slit: 5nm

[0062] The specific experimental operation is:

[0063] (1) First, in the absence of target DNA (HIV-1 and HIV-2), mismatch hybridization of 10 μL of P (500 nM) and 10 μL of cDNA solution was dissolved in 15...

Embodiment 3

[0068] Optimization of Experimental Conditions

[0069] (1) Optimization of pH

[0070] Since the pH of the buffer has a certain influence on the synthesis of AgNCs, the pH environment of the experiment must be optimized. We chose five pH values ​​of 6.4, 6.8, 7.0, 7.2, and 7.6 to do a careful optimization. Figure 4 It can be clearly seen that the fluorescence difference obtained is the highest when the pH is 7.0, so in the following experiments, 7.0 is selected as the optimal pH of the system. (I 0 is the fluorescence intensity before adding the target DNA, and I is the fluorescence intensity after adding the target).

[0071] (2) Optimization of the dosage ratio of reagents and DNA templates

[0072] After the pH is determined, a series of optimization work is carried out on the ratio of reagents and DNA templates so that the synthesized AgNCs have the best fluorescence effect. In the experiment, DNA:Ag + : NaBH 4 Proportional usage optimization is shown in the Fig...

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Abstract

The invention discloses a method for simultaneously detecting two types of HIV DNA by using silver nano-clusters (AgNCs) as a label-free fluorescence biosensing platform. The fluorescence biosensing platform is based on two features of the silver nano-clusters: (1) fluorescence of the silver clusters can be enhanced in multiples by a rich guanine (G) sequence; and (2) the fluorescence can be remarkable enhanced by mutual extrusion of two neighboring AgNCs. Different templates of the synthetic AgNCs are designed at two ends of a single chain, and two different signals with different luminous peaks are obtained so as to avoid signal interference. A DNA chain mispaired with the single chain is designed as a template of which one end is a rich-G sequence, and the other end is a synthetic silver cluster extrusion pair. A target object is completely paired with complementary DNA, high fluorescence of the AgNCs is damaged, and a fluorescence signal is weakened. Only one type of the color-variable silver nano-clusters needs to be used as a fluorescence probe so that two wider detection ranges (0.2-700 nm) can be obtained. A limit of detection is as low as 0.2 nm, and high sensitivity detection of multiple types of DNA or MiRNA can be further realized, so accuracy and practicability of bioanalysis are improved.

Description

technical field [0001] The invention belongs to the fields of molecular biology and nucleic acid chemistry, and relates to a method for simultaneously detecting two kinds of HIV DNA by DNA-guided color-changing silver nanoclusters. Background technique [0002] When human immunodeficiency virus (HIV) infects and destroys the immune system of the host, the function of the immune system will gradually lose and be accompanied by the occurrence of many diseases, and even cause death in severe cases. Therefore, accurate detection of HIV genes is of great significance for early detection and timely treatment of infected persons. [0003] For decades, many methods for single-target HIV gene detection have been developed, including surface-enhanced Raman scattering (SERS), fluorescence, colorimetry, and electrochemical techniques. Among them, the fluorescence method is widely used because of its simple operation and intuitive observation. Das et al. designed a new type of fluoresc...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6813
CPCC12Q1/6813C12Q1/701C12Q2563/107C12Q2563/137
Inventor 蔡昌群邹容龚行韩云鹏
Owner XIANGTAN UNIV
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