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Ratio fluorescent probe for detecting hydroxyl radical and synthesis method and application of ratio fluorescent probe

A fluorescent probe and free radical technology, which is applied in the field of biological detection technology and clinical medical detection, can solve the problem that the measurement environment error cannot be effectively avoided, and achieve the effects of good selectivity, low cytotoxicity and good biocompatibility.

Inactive Publication Date: 2014-04-30
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the probes reported to detect intracellular hydroxyl radicals are single-emission, which cannot effectively avoid errors caused by the measurement environment

Method used

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  • Ratio fluorescent probe for detecting hydroxyl radical and synthesis method and application of ratio fluorescent probe
  • Ratio fluorescent probe for detecting hydroxyl radical and synthesis method and application of ratio fluorescent probe
  • Ratio fluorescent probe for detecting hydroxyl radical and synthesis method and application of ratio fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Synthesis of ratiometric fluorescent probes for hydroxyl radicals

[0032] 1. Under the conditions of 37 ℃ and sufficient magnetic stirring, add 5 mL of 10 mM chloroauric acid aqueous solution to 55 mL of bovine serum albumin aqueous solution with a mass concentration of 50 mg / mL, and then add 0.5 mL of 1 M NaOH solution, continue to react for 12 h; use a dialysis membrane (MWCO: 3500) to dialyze the reaction product in ultrapure water to obtain a BSA-AuNCs solution;

[0033] 2. At room temperature, add a volume of 10 μL of dimethylformamide solution of HPF with a concentration of 5 mM to a phosphate buffer solution with a pH of 7.4, and add 0.0040 g of 1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide and 0.0040g N-hydroxysuccinimide activated the carboxyl groups in HPF, and then added 400 μL of the above BSA-AuNCs solution to react for 10 h; After dialysis for 4 h, a ratiometric fluorescent probe solution was obtained.

Embodiment 2

[0034] Example 2: Ratio probe with · Determination of fluorescence properties after OH reaction

[0035] 1. The generation method of hydroxyl radicals:

[0036] Hydroxyl free radical among the present invention utilizes Fenton reaction to produce, and it reacts according to following:

[0037]

[0038] in · OH to Fe 2+ To quantify, to ensure Fe 2+ Fully reacted, Fe 2+ with H 2 o 2 The concentration ratio is 1:10, the reaction medium is PBS buffer solution with pH 7.4, FeCl 2 solution and H 2 o 2 It is required to be equipped and used now.

[0039] 2. Fluorescence test:

[0040] (1) Measurement parameters of fluorescence spectrum:

[0041] Fluorescence detection was performed with a F-2700FL fluorescence instrument, the voltage of the photomultiplier tube was 700 V, the slit width was 2.5 nm, the scanning range was 500-750 nm, the scanning speed was 1200 nm / min, and the excitation wavelength was 488 nm. · In the presence of OH, the positions of the two fluo...

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Abstract

The invention relates to a ratio fluorescent probe for detecting a hydroxyl radical and a synthesis method and an application of the ratio fluorescent probe. The ratio fluorescent probe is formed by putting a protein coated fluorescent BSA-AuNCs (nano-cluster) and a covalent bond of an organic molecule HPF (2-[6-(4'-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoicacid) having a specific response to the hydroxyl radical together. Under the condition that the probe does not have the hydroxyl radical, the BSA-AuNCs emits reference fluorescence, but when the hydroxyl radical exists in the environment, the HPF on the probe and the hydroxyl radical react to produce a florescent product, and the response fluorescence of the hydroxyl radical is emitted. Due to the existence of the reference fluorescence, the measurement errors caused by the stability of a laser light source, the laser strength, the probe concentration, optical focusing, environment change in a cell and the like are effectively reduced, and the concentration of the hydroxyl radical is more accurately measured.

Description

technical field [0001] The invention belongs to the fields of biological detection technology and clinical medical detection, and specifically relates to a ratio-type fluorescent probe for detecting hydroxyl radicals, a synthesis method and application. Background technique [0002] Active oxygen is an inevitable by-product of biological life metabolism, it plays an important role in the body's innate immunity, signal transduction, cell proliferation, differentiation, apoptosis, etc. There is also oxidative damage, which causes destructive modification of the structure of tissue cells and damages the morphology and function of normal tissue cells (Document 1: Chen X., .Probing oxidative stress: Small molecule fluorescent sensors of metal ions, reactive oxygen species, and thiols, Coord. Chem. Rev., 2011, 40:4783-4804). [0003] Hydroxyl free radicals are currently known to be the most toxic and harmful free radicals to organisms among active oxygen species. They can cause e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N21/76G01Q60/20G01Q60/22
Inventor 庄梅田阳
Owner TONGJI UNIV
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