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A Metal Complex Nucleic Acid Fluorescent Probe

A nucleic acid and compound technology, applied in the field of nucleic acid fluorescent probes, can solve the problems of reducing the cost of nucleic acid detection, complex operation, single-stranded nucleic acid color development, etc., and achieve the effect of inhibiting the relaxation of excited states and enhancing fluorescence

Active Publication Date: 2015-09-30
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This type of molecule has two main disadvantages: firstly, the electrostatic interaction is a relatively weak intermolecular interaction, which directly leads to the low sensitivity of the probe, which is difficult to compare with traditional double-stranded probes; secondly, the probe molecule is easily affected by other electrolytes. Interference, such as negatively charged proteins can also bind the probe electrostatically, interfering with DNA detection
These two shortcomings limit the practical application of these probe molecules, so there are no fluorescent dyes sensitive enough to single-stranded DNA to replace fluorescently labeled or isotope-labeled DNA probes, especially in the application of nucleic acid electrophoresis, fluorescently labeled Or isotope-labeled nucleic acid probes are expensive and complicated to operate, and traditional nucleic acid fluorescent dyes are difficult to effectively color single-stranded nucleic acids. Therefore, the development of single-stranded nucleic acid fluorescent dyes can greatly reduce the cost of nucleic acid detection, which is of great significance

Method used

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  • A Metal Complex Nucleic Acid Fluorescent Probe
  • A Metal Complex Nucleic Acid Fluorescent Probe
  • A Metal Complex Nucleic Acid Fluorescent Probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The synthesis of embodiment 1 ligand TD1 and complex TD1Zn

[0051]

[0052] (1) Synthesis of intermediate b1

[0053] Add 3g of a1 and 0.92g of zinc powder into a 150mL flask, under the protection of argon, add 100mL of anhydrous THF, stir in an ice-water bath, slowly add 0.8mL of titanium tetrachloride, and reflux for 12 hours after the addition is complete. After cooling, add saturated sodium bicarbonate solution to quench the reaction, filter, and concentrate the filtrate under reduced pressure to obtain a crude product. Use chloroform / petroleum ether=1:1 column chromatography to separate the product, and remove the solvent under reduced pressure to obtain 1.56 g of b1 , yield 54%. 1 H NMR (300MHz, CDCl 3 )δ[ppm]: 6.92(d,8H); 6.65(d,8H); 4.23(t,8H); 3.62(t,8H).

[0054] (2) Synthesis of Ligand TD1

[0055] Add 825mg of b1, 2.96g of bis(2-pyridylmethyl)amine (DPA for short), 2.11g of potassium carbonate, and 100ml of acetonitrile into a 150mL flask, reflux for...

Embodiment 2

[0058] Example 2 Ligand TD1 Temporarily Coordinates with Zinc Ions in Solution to Detect DNA

[0059] The ligand TD1 was made into 1 μM acetonitrile solution, the fluorescence was very weak, and 4 μM zinc perchlorate was added to it, the fluorescence enhancement was not significant, and single-stranded DNA was added dropwise, the fluorescence was significantly enhanced. The test results are in figure 1 middle. The fluorescence instrument used was Hitachi F-4500, and the excitation wavelength was 330 nm. The selected single-stranded DNA sequence is: 5'-TTTTTTTTTT-3'.

Embodiment 3

[0060] Example 3 Complex TD1Zn detects DNA in high-concentration electrolyte

[0061] Many single-stranded DNA probes in the literature mainly bind to DNA through electrostatic interactions (see references: Chem. 2008,80,6443–6448), because other negatively charged substances can compete with this type of probe for DNA binding, the detection effect of this type of probe is easily affected by other electrolytes, in order to test whether TD1Zn is also interfered by electrolytes , we did the following test.

[0062] The ligand TD1Zn was made into 5 μM acetonitrile water buffer solution (acetonitrile / water=1:1, v / v; HEPES10mM, pH=7.2), and 30mM tetrabutylammonium bromide was added to the buffer solution in advance, and When single-stranded DNA was added dropwise, the fluorescence was significantly enhanced. Test results such as figure 2 shown. At the same time, when tetrabutylammonium bromide was not added to the buffer solution, the same single-stranded DNA was gradually add...

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Abstract

The invention discloses a novel nucleic acid fluorescent probe, and the structure is that a DPA (dipicolylamine) or DPA-Zn (II) group is connected with tetraphenylethylene. DPA-Zn (II) can be combined with nucleic acid and other phosphoric acid derivatives in a solution to inhibit non-radiative transition of molecules so as to emit florescence. The fluorescent probe can be used for detecting the nucleic acid and has higher sensitivity in the detection of double-stranded DNA (deoxyribonucleic acid) and single-stranded DNA. The probe has better application prospects when applied in gel electrophoresis or detection and color development of the nucleic acid in biological samples.

Description

technical field [0001] The invention relates to a nucleic acid fluorescent probe, which can be used for fluorescent detection of phosphoric acid derivatives such as nucleic acid, and belongs to the technical field of biological detection. Background technique [0002] Nucleic acid is the genetic material of life and plays an important role in life activities. Nucleic acid detection has important applications in the fields of disease diagnosis, environmental microbial identification, and aptamers. Therefore, the development of fluorescent detection probes for nucleic acids is of great significance. Traditional nucleic acid fluorescent probes include EB, YOYO, TOTO, Sybr Green, DAPI, and Hoechst dyes, which are used for the detection of double-stranded DNA with high sensitivity, but the detection effect for single-stranded DNA, especially short-chain single-stranded DNA very bad. [0003] At present, there are some reports of single-stranded DNA fluorescent probes in the li...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07F3/06C12Q1/68G01N21/64
Inventor 杨楚罗朱策泽
Owner WUHAN UNIV
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