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Cis-platinum complex and application thereof

A complex and fluorescence technology, applied in the field of biochemistry, can solve the problems of unstable fluorescence intensity, low labeling efficiency, long labeling time, etc., and achieve the effect of remarkable labeling effect, excellent labeling effect and fast labeling speed.

Inactive Publication Date: 2017-05-31
SHANGHAI TONGREN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the labeling methods in the prior art still have defects such as low labeling efficiency, long labeling time and unstable fluorescence intensity.

Method used

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  • Cis-platinum complex and application thereof
  • Cis-platinum complex and application thereof
  • Cis-platinum complex and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Synthesis of the cisplatin fluorescent complex of the present invention when n=6

[0036] Such as figure 2 Shown synthetic route, concrete steps are as follows:

[0037] (1) with 1,6-diaminohexane as the starting reactant; first (Boc) 2 O (12.3g, 0.056mol) was dissolved in 100mL of dichloromethane, then slowly added dropwise into 150mL of dichloromethane solution dissolved in 1,6-diaminohexane (33g, 0.28mol), and reacted at 0°C for 4 hours , and then reacted at room temperature for 20 hours; after the reaction was complete, wash the reaction solution with 4×100mL saturated brine, separate the liquids, dry the organic phase with anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain 14.3g colorless oily liquid, namely N-Boc-1,6-diaminohexane (N-Boc-1,6-diaminohexane, intermediate a) was directly carried out to the next reaction without purification.

[0038] (2) Dissolve [Pt(en)Cl2] (500mg, 1.53mmol) in 50ml DMF, then add AgNO 3 (248mg,...

Embodiment 2

[0041] In this experiment, platinum was labeled with rhodamine, DEAC, gloxalic acid, FITC, TRITC, Alexa fluor 594 and other fluoresceins, and the fluorescence intensity was measured. The results indicated that the cisplatin-mediated complex could combine well with the above fluorescein and emit fluorescence. (n=3, 6, 9 for the cisplatin fluorescent complexes used in this experiment). The marker-Pt coordination compound was dissolved in a 0.1 M tris buffer solution, pH = 7.1, and the fluorescence intensity was measured at room temperature. The relationship between the fluorescence intensity and the complex concentration of organic cisplatin fluorescent compounds (carbon chain lengths are respectively n=3, n=6, n=9) is as follows Figure 3-5 shown.

[0042] We also used organic cisplatin fluorescent complexes to bind biotin, and used avidin and Cy5-labeled fluorescent antibodies to label to form biotin-avidin-fluorescent antibody complexes, and the fluorescent signal was signi...

Embodiment 3

[0044] Experiments of preparing nucleic acid and protein probes using cisplatin fluorescent complexes of the present invention

[0045] 1. Nucleotide labeling method

[0046] Mix 100ug of purified target nucleotide clones with 2ug of purified platinum-biotin-fluorescein (FITC, Cy5) markers (n=6 in the cisplatin fluorescent complex used in this experiment), and react at 80°C for about 40min (the reaction time can be appropriately adjusted according to the type of target molecule, and the RNA reaction time can be shortened to about 20min), suck out the mixture of labeled samples, and pass through the above-mentioned cellulose acetate liquid phase separation column to obtain the purified fluoresceinized nucleotide sequence .

[0047] 2. Protein Molecular Probe Labeling Method

[0048] Mix 100ug of protein (antibody IgG used in this experiment) with 1ug of purified platinum-biotin-fluorescein (FITC, Cy5) label (n=6 in this experiment using cisplatin fluorescent complex), 37°C A...

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Abstract

The invention provides a cis-platinum complex, and also relates to a synthetic method of the cis-platinum complex and an application of the cis-platinum complex in preparing nucleotide and protein probes. The cis-platinum complex can be connected with fluorescein, and can also be firmly connected with DNA, RNA and proteins, so that the cis-platinum complex can be used as a connection main body of the biological probe and is suitable for marking and detecting the target DNA, RNA and protein; the cis-platinum complex is simple in marking operation procedures, and is not limited to the size of a marking molecule and not limited to a pH value of a marking mixed system; the cis-platinum complex can be widely applied to the subdivisions such as nucleotide, protein (antibody) and protein micro arrays, hybridization of comparative genome and the like; in addition, the marking efficiency is significantly improved, the marking time is shortened, the fluorescent intensity stability is improved, and the marking effect is excellent; and therefore, the application prospect in the fields such as preparation of nucleotide and protein probes, research and development of biological medicine, diagnosis of diseases and the like is good.

Description

technical field [0001] The invention belongs to the field of biochemistry, relates to a cisplatin fluorescent complex, and also relates to the application of the cisplatin fluorescent complex in preparing nucleotide and protein probes. Background technique [0002] Fluorescein is a compound with a conjugated double bond system chemical structure. When irradiated by light of a specific wavelength, it is excited into an excited state, and can emit fluorescence during the process of returning from the excited state to the ground state. This characteristic of fluorescein is widely used in the detection of biology and medicine. The use of fluorescein in the fields of biology and medicine began in the late 1980s. At present, in the fields of biology and medicine, fluorescein is mainly used to label nucleotides and proteins. The details are as follows: [0003] Nucleotide (DNA, RNA) fluorescein labeling technology mainly includes: (1) enzymatic biotin labeling nucleic acid method...

Claims

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Application Information

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IPC IPC(8): C07F15/00C12Q1/68G01N33/533G01N33/68
CPCC07F15/0093C12Q1/6841G01N33/533G01N33/68C12Q2563/107
Inventor 章劲夫乐威崔杨张文萍
Owner SHANGHAI TONGREN HOSPITAL
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