A method for the simultaneous detection of two HIV DNAs by a DNA-directed color-changing silver nanocluster

A technology of silver nanoclusters and DNA probes, which is applied in the fields of molecular biology and nucleic acid chemistry, can solve the problems of long time-consuming separation and purification of materials, high modification costs, cumbersome operations, etc., and achieve high cost, large emission range, and economical simple effect

Active Publication Date: 2021-10-19
XIANGTAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A method for the simultaneous detection of two HIV DNAs by a DNA-directed color-changing silver nanocluster
  • A method for the simultaneous detection of two HIV DNAs by a DNA-directed color-changing silver nanocluster
  • A method for the simultaneous detection of two HIV DNAs by a DNA-directed color-changing silver nanocluster

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Experimental program
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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 present invention utilizes silver nanoclusters (AgNCs) as a label-free fluorescent biosensing platform for simultaneous detection of two HIV DNAs. The fluorescent sensing platform is based on two characteristics of silver nanoclusters: (1) the guanine (G)-rich sequence can exponentially enhance the fluorescence of silver clusters; (2) the mutual extrusion of two adjacent AgNCs can significantly enhance the fluorescence. Design different synthetic AgNCs templates at both ends of the single strand to obtain signals with two different luminescent peaks to avoid signal interference. The mismatched DNA strand with the single strand is designed to have a G-rich sequence at one end and a synthetic silver cluster at the other end Extrusion pair template. The target is completely paired with the complementary DNA, which destroys the high fluorescence of AgNCs and weakens the fluorescent signal. Only one color-changing silver nanocluster is used as a fluorescent probe to obtain two wide detection ranges (0.2-700nm), the detection limit is as low as 0.2nM, and it can also achieve high-sensitivity detection of various DNA or miRNA, Thereby improving the accuracy and practicability of biological analysis.

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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Patent Type & Authority Patents(China)
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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