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Ratio type fluorescent probe for detecting microRNA-21 based on silver nanometer cluster pairs and G-triplex

A technology of silver nanoclusters and fluorescent probes, applied in the field of analysis and detection, can solve the problems of low preparation cost, short time, false positive signals, etc.

Active Publication Date: 2020-01-17
XIANGTAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to design a ratiometric fluorescent probe based on silver nanocluster pair and G-triplex detection microRNA-21, the preparation cost of the probe is low, the time is short, and it is expected to solve the problem of traditional single signal probe detection Problems such as false positive signals in the process can easily and quickly realize the high-sensitivity detection of microRNA-21

Method used

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  • Ratio type fluorescent probe for detecting microRNA-21 based on silver nanometer cluster pairs and G-triplex
  • Ratio type fluorescent probe for detecting microRNA-21 based on silver nanometer cluster pairs and G-triplex
  • Ratio type fluorescent probe for detecting microRNA-21 based on silver nanometer cluster pairs and G-triplex

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Experimental program
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Effect test

Embodiment 1

[0031] Preparation of ratiometric fluorescent probes

[0032] The DNA probe in the present embodiment, its base sequence is as follows:

[0033]

[0034] MB2:CCC TTA ATC CCC CAG ACT GAT GTT TGG GAT TAA CAT CAG TCT GCC CTAACT CCC C

[0035] The part in bold is the G-tripplex sequence, and the part in italics is the DNA template sequence for synthesizing the AgNCs pair.

[0036] MB1 was incubated and annealed at 95°C to obtain a hairpin structure locked with the G-triplet sequence.

[0037] Anneal MB2 by incubating at 95°C. Then, the AgNO 3 The solution was added to the above solution, then vigorously shaken for 1 min and incubated at 4°C in the dark for 30 min, and then the newly prepared NaBH 4 Add the solution to the above solution, shake for 1min, DNA:Ag + :NaBH 4 The ratio was 1:24:24. Finally, the above sample was placed in a dark room at 4°C for 4 hours to obtain a molecular beacon with a pair of silver nanoclusters at the 5' end and the 3' end.

Embodiment 2

[0039] Feasibility verification of fluorescent probe ratiometric detection of microRNA-21

[0040] (1) Qualitative analysis of chain structure

[0041] The conditions for circular dichroism (CD) detection are:

[0042] Instrument: ChirascanTM CD chiroptical spectrometer

[0043] Instrument parameters: path length: 0.1cm; wavelength range: 200-500nm; scanning speed: 200nm / min; response time: 0.5s; bandwidth: 1.0nm.

[0044] Circular dichroism was used to verify the formation of the G-triplet conformation. Such as image 3 As shown, MB1 has a positive peak at about 265 nm and a negative peak at 240 nm, which are characteristic peaks of ssDNA and dsDNA. In addition, there was no obvious change in the CD spectrum after adding THT to MB1 solution, indicating that the G-triplet sequence was effectively locked in MB1 and would not form a G-triple structure. However, when microRNA-21 was added to the mixture solution of MB1 and THT, an obvious positive peak appeared in the CD spe...

Embodiment 3

[0054] Optimization of Experimental Conditions

[0055] To achieve the best effect of an analytical method, it is necessary to optimize a series of reaction conditions to obtain an optimal experimental condition. Considering the concentration of THT, MB2, potassium ion, magnesium ion and DNA:Ag + :NaBH 4 The effects of factors such as ratio, pH, temperature, and time on the detection effect were studied respectively for these influencing factors. During the research process, only the research objects were changed, and other conditions were not changed to detect microRNA-21. When studying different factors, they are all based on the best conditions that have been obtained. The optimization effect is as follows Figure 8-11 .

[0056] Finally, the best detection condition is obtained: c THT = 1 μM, c MB2 =500nM, c K+ = 10mM, c Mg2+ =5mM, DNA:Ag + :NaBH 4 =1:24:24, pH=7.2, T=37°C, t=60min.

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Abstract

The invention provides a ratio type fluorescent probe for detecting microRNA-21 based on silver nanometer cluster pairs and G-triplex, and belongs to the field of analysis and detection. The probe adopts the silver nanometer cluster pairs and a G-triplex / THT compound as fluorescence output signals. When an object does not exist, one probe sequence (G-triplex) is locked up by a molecular beacon (MB1) to present weak fluorescence state, and another probe sequence (MB2) can synthesize the silver nanometer cluster pairs to present strong fluorescence state. After microRNA-21 is added, the object as an initiating chain triggers a hybridizing chain reaction (HCR) of MB1 and MB2, two hairgrips are continuously and mutually opened to cause release of massive G-triplex sequences and separation of the silver nanometer cluster pairs, the fluorescence of the silver nanometer cluster pairs is weakened, and after thioflavine (THT) is added, the G-triplex and the THT are combined to give out strong fluorescence. The ratio type fluorescent probe disclosed by the invention not only has high sensitivity and high selectivity, but also can effectively detect objects in complex samples of cell lysis solutions and the like.

Description

technical field [0001] The invention belongs to the field of analysis and detection, and more specifically relates to a ratio-type fluorescent probe for detecting microRNA-21 based on silver nanocluster pairs and G-triplexes. Background technique [0002] MicroRNA is an important marker in the early diagnosis of tumors, which can regulate the process of cell division and differentiation. Therefore, it is very valuable to propose an accurate and sensitive detection strategy for microRNA monitoring. In recent years, many fluorescent detection strategies have relied on the output of a single signaling channel to detect microRNAs. However, these methods may suffer from false positive signals and poor reproducibility due to light source stability, probe concentration, and instrument factors. Ratiometric fluorescence strategies can be internally corrected by dual fluorescence emission to avoid the influence of these confounding factors. Therefore, designing a ratiometric fluore...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6839C12N15/11
CPCC12Q1/6886C12Q1/6839C12Q2600/158C12Q2600/178C12Q2525/207C12Q2525/301C12Q2563/107Y02A50/30
Inventor 蔡昌群向灵王双陈小明
Owner XIANGTAN UNIV
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