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Fluorescence bio-sensing method for detecting glutathione

A glutathione and biosensing technology, applied in the field of fluorescent biosensing, can solve the problems of high cost, limited application, and many organic probe synthesis and purification steps, and achieves low cost, simple operation and high sensitivity. Effect

Inactive Publication Date: 2017-10-10
MENGCHAO HEPATOBILIARY HOSPITAL OF FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although good results have been achieved, most organic probes have many synthesis and purification steps and high cost, which limits their application
There are also some fluorescent nanomaterials such as quantum dots, gold nanoclusters, fluorescent carbon nanomaterials, and upconversion nanoparticles that have been developed for GSH detection, but there are some shortcomings, such as the use of heavy metal cadmium, gold clusters or fluorescent carbon in the synthesis of quantum dots. Nanomaterials use mercury ions to quench fluorescence, and the synthesis of upconversion nanoparticles is complex and costly

Method used

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  • Fluorescence bio-sensing method for detecting glutathione
  • Fluorescence bio-sensing method for detecting glutathione
  • Fluorescence bio-sensing method for detecting glutathione

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Synthesis of manganese dioxide nanomaterials: After mixing 1mL potassium permanganate solution (10mM) with 2.5mL MES buffer solution (0.1M, pH6.0), add 6.5mL secondary water; after that, place in an ultrasonic cleaner Medium ultrasonic reaction for 30 minutes until the formation of brown flocculent products. The brown product after the reaction was centrifuged at 8000 rpm for 10 minutes and washed 3 times with water to remove unreacted ions. Finally, the obtained manganese dioxide nanosheets were ultrasonically dispersed in secondary water at a concentration of 1 mg / mL. figure 2 It proves that the manganese dioxide material has been successfully synthesized. Among them, (A) is the absorption comparison chart of potassium permanganate solution and manganese dioxide after it is produced; (B) is the transmission electron microscope picture after synthesis, which proves that nanometer-sized Manganese dioxide nanosheets; (C) is the Raman spectrum of manganese dioxide nanos...

Embodiment 2

[0027] Fluorescent detection of glutathione: 2.5 μL of manganese dioxide nanosheets (1 mg / mL) and 200 μL of different concentrations of glutathione solutions (0, 10, 20, 30, 40 μM) were reacted at room temperature for 5 minutes; after that, 5 μL of 6 .8mM o-phenylenediamine (OPDA) solution was mixed and reacted in an oven or water bath at 50°C for 10 minutes. After cooling to room temperature, the fluorescence emission spectrum at 568nm was measured under the excitation of 420nm wavelength. When doing selectivity experiments, react different interfering substances with 2.5 μL of manganese dioxide nanosheets (1 mg / mL) at room temperature for 5 minutes; after that, add 5 μL of 6.8 mM o-phenylenediamine (OPDA) solution and mix well at 50 ° C. After reacting in oven or water bath for 10 minutes. After cooling to room temperature, test the fluorescence spectrum. Fluorescence response of glutathione as image 3 shown. The selectivity of the method for glutathione detection, as ...

Embodiment 3

[0029] Detection of glutathione in cell lysis solution: HeLa cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 U / mL) and streptomycin (100 μg / mL) at 37°C in high humidity containing 5% in a carbon dioxide incubator. The grown HeLa cells were centrifuged, washed 3 times with cold PBS, and the cell concentration was calculated using a cell counter. Disperse the cell suspension into cold PBS so that each 100 μL solution contains 500, 5000, 10000, 20000, 30000, 40000 and other different numbers of cells, and place the cell suspension in an ice bath at 4 °C for 5 minutes sonication , after centrifugation at 10,000 rpm for 5 minutes, the supernatant was taken for testing. The supernatant was diluted to 200 μL and mixed with 2.5 μL of manganese dioxide nanosheets (1 mg / mL) for 5 minutes, then added 5 μL of 6.8 mM o-phenylenediamine (OPDA) solution and mixed, and placed in an oven or water bath at 50 ° C After 10 minutes of reaction. After c...

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Abstract

The invention relates to a fluorescence bio-sensing method for detecting glutathione. According to the invention, a manganese dioxide nanometer material is used as an oxidizing agent and a recognition element; in the absence of glutathione (GSH), an oxidized o-diaminobenzene product is generated by catalytically oxidizing the manganese dioxide nanometer material for fluorescence detection; in the presence of the GSH, the manganese dioxide nanometer material is degraded by the GSH, the oxidizing capacity of manganese dioxide to o-diaminobenzene is inhibited and the fluorescence signal is weakened. The GSH detection is realized in the manner of comparing fluorescence values. Compared with the prior art, the method provided by the invention has the advantages of simple and convenient operation, high sensitivity, simplicity, low cost and specific detection for GSH. The detection method provided by the invention can be used for effectively detecting the GSH in a biological liquid sample.

Description

(1) Technical field [0001] The invention relates to a fluorescent biosensing method for detecting glutathione, belonging to the technical field of biological analysis and detection. (2) Background technology [0002] Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine ​​and glycine. It is the most abundant non-protein thiol in living cells and is the first line of defense against toxins. It plays an important role in maintaining cell balance, cell signal transduction, gene regulation, detoxification and so on. Usually, the imbalance of GSH will cause many diseases, such as liver disease, AIDS, diabetes, cancer and so on. Therefore, it is of great significance to develop sensitive and efficient GSH detection methods. [0003] At present, there are electrochemical methods, high performance liquid chromatography, colorimetric methods, and fluorescence methods for the detection of GSH. Among them, the fluorescence method has the advantages of high sensitivit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428
Inventor 刘景丰刘小龙张晓龙郑爱仙曾永毅
Owner MENGCHAO HEPATOBILIARY HOSPITAL OF FUJIAN MEDICAL UNIV
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