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Electrogenerated chemiluminescence detection method of phospholipid and application of detection method

A technology of electrochemistry and luminescence detection, which is applied in the direction of chemiluminescence/bioluminescence, and analysis by making materials undergo chemical reactions, can solve the problems that cannot be applied to the detection of single-cell phospholipids, and achieve good specificity and detection sensitivity High, easy-to-operate effect

Active Publication Date: 2015-06-24
南京广全环保技术服务有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many methods for studying phospholipids above, none of these techniques can be applied to detect phospholipids in single cells

Method used

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  • Electrogenerated chemiluminescence detection method of phospholipid and application of detection method
  • Electrogenerated chemiluminescence detection method of phospholipid and application of detection method
  • Electrogenerated chemiluminescence detection method of phospholipid and application of detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Electrochemiluminescent detection of pure phospholipids.

[0032] First disperse the pure lecithin with chloroform, dry it with nitrogen in a fume hood, prepare a high-concentration lecithin solution, the solvent is pH=7.4PBS, and sonicate for 15 minutes to completely dissolve the sample. Using ITO as the working electrode, Ag / AgCl and Pt as the reference electrode and counter electrode respectively, an O-ring was attached to the surface of the ITO, and 200 μL of the solution to be tested (PBS solution containing 200 μM luminol and 1 mM lecithin) was added to it, The scanning speed is 1V / S, the voltage of -1.0V ~ 1.0V is applied to the ITO, the voltage of the photomultiplier tube is 500V, and the background luminous intensity I is recorded. 0 ;When recording the signal, try to ensure that there is no strong light shining on the instrument to avoid the error of the luminous intensity measured by the photomultiplier tube. After the baseline is stable, pause and ...

Embodiment 2

[0033] Example 2 Electrochemiluminescence detection of phospholipids on cell membrane surface.

[0034] (1) Cell membrane phospholipid activation: culture about 7000 Hela cells (with an area of ​​1 cm radius) on the surface of ITO, and after the cells grow on the wall overnight, use low ionic strength containing 0.5mM phosphate buffer (PBS, pH 7.4) and 310mM sucrose solution, incubated at 37°C for 1 hour to activate the phospholipids on the cell membrane surface, and then washed and soaked with 100mM PBS (pH 7.4).

[0035] (2) Detection: Take the ITO with cultured cells, suck off the low-ion solution, wash it with PBS for 3 times, add 190 μL of solution (PBS solution containing 200 μM luminol), ITO as the working electrode, Ag / Agcl and Pt As the reference electrode and the counter electrode respectively, the scanning speed is 1V / S during detection, the voltage is -1.0V~1.0V, the voltage of the photomultiplier tube is 600V, and the background luminous intensity is recorded. Aft...

Embodiment 3

[0041] Example 3 Electrochemiluminescent detection of phospholipids on the surface of single cells.

[0042] For the detection of lipid molecules on the surface of single cells, first use pinhole technology to form a 30 μm single hole on the surface of ITO, treat the cultured Hela cells with trypsin, centrifuge, wash, and soak in a solution with low ionic strength, and finally put the single The cells are introduced into the single hole. Before the single cell is introduced, the ITO single hole must be vacuumed to make the single hole wet. Add the diluted cell solution from one side under the microscope, and then use absorbent paper from the other side to ensure that the cells are introduced into the ring-shaped single hole. Put the electrode into the incubator and culture it for about 1 hour to make the cells stick to the wall. After sticking to the wall, suck off the special high-glucose medium for the culture medium and add 190 μL of 200 μM luminol in PBS solution, so as to ...

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Abstract

The invention provides an electrogenerated chemiluminescence detection method of phospholipid. The method comprises the following steps: (1) soaking phospholipid to be detected into a low-ion-strength solution to activate phospholipid, then flushing the activated phospholipid with phosphate buffer, and soaking for later use; and (2) soaking the phospholipid to be detected and treated in step (1) into luminal or L012 containing phosphate buffer, detecting the lighting intensity I0 by the electrogenerated chemiluminescence method and by using ITO (Indium Tin Oxide) as a working electrode and Ag / AgCl and Pt as a reference electrode and a counter electrode, then adding a mixed solution of phospholipase D and choline oxidase to react for 1 to 2 minutes, then detecting the lighting intensity I1, and calculating the change of the lighting intensity I1 / I0 before and after adding the mixed solution of phospholipase D and choline oxidase. With the adoption of the method, the cell surface phospholipid can be detected, and meanwhile, surface phospholipid of single cell can be detected; the method is high in detection sensitivity and high in specifity.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to an electrochemiluminescence detection method of phospholipids and an application thereof. Background technique [0002] Phospholipids are amphiphilic molecules with nitrogen- or phosphorus-containing groups at both ends and long hydrophobic hydrocarbon chains, which play an important role in cell signaling and apoptosis. According to the number of different bases and fatty acids, phospholipids can be divided into lecithin (PC), phosphatidylserine (PS), lysophosphatidylcholine (LPC), and its skeleton is composed of glycerol. Phospholipids can undergo reactions such as hydrolysis, hydroxylation, acetylation, sulfonation, and activation. [0003] The metabolism of phospholipids is related to the methylation cycle. In vivo, phospholipids can be hydrolyzed by various phospholipases into fatty acids, phosphoric acid, glycerol and various amino alcohols. Glycerol can b...

Claims

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Application Information

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IPC IPC(8): G01N21/76
Inventor 田丹碧左焕桢江德臣庄丽红张卫汤燕
Owner 南京广全环保技术服务有限公司
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