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Microelectrode array and application thereof in detection of unicellular surface activation cholesterol

A micro-electrode array and single-cell technology, applied in the field of medicine, can solve problems such as hindering application and low throughput of detection methods, and achieve the effect of rapid detection of content

Active Publication Date: 2014-03-26
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can study single cells in different disease states, but the detection method has low throughput and requires special electrochemical devices, which hinders its application in the field of biochemistry

Method used

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  • Microelectrode array and application thereof in detection of unicellular surface activation cholesterol
  • Microelectrode array and application thereof in detection of unicellular surface activation cholesterol
  • Microelectrode array and application thereof in detection of unicellular surface activation cholesterol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0045] Example 2 Luminescence detection.

[0046] The ITO electrode array with cultured cells was used as the working electrode. A pulsed triangular wave voltage was applied to the microelectrode through an electrochemical workstation (CHI630E, CH Instrument) and a multi-channel converter (CHI684, CH Instrument). The shortest time for channel switching in this electrochemical system was 2s. The Ag / AgCl electrode and a Pt wire were connected as reference and counter electrodes, respectively. The solution on the microelectrode array for luminescence detection was 500 μL PBS (100 mM pH 7.4) containing 200 μM L012. Initially, a periodic voltage of 0.5-1.0V was applied to the ITO microelectrode with a scanning speed of 1V / S to collect background luminescence signals. Then, add 1U / mL cholesterol oxidase to the buffer, and the luminescence can be detected within 2s-120s. The luminescence ratio at 1.0 V before and after the addition of cholesterol oxidase is referred to as the lumi...

Embodiment 3

[0049] Example 3 Finite Element Simulation.

[0050] We used COMSOL3.5 software (Comsol Inc.) to simulate the distribution of hydrogen peroxide in microcavities after adding cholesterol oxidase. The result is Figure 5 shown.

Embodiment 4

[0051] Example 4 Effects of luminol and voltage on intracellular calcium ion concentration.

[0052] If luminol electroluminescence is applied to detect cell membrane activated cholesterol, the effect of luminol and voltage on cell membrane cholesterol transport should be very small. Since cell membrane cholesterol is related to the balance of calcium ions in the cell, we tracked the concentration of intracellular calcium ions to analyze the effects of luminol and voltage on cell membrane cholesterol. First, soak the cells in 100 μM luminol for 5 minutes, which is about the time for each cell analysis. The fluorescence intensity of cells before and after adding luminol was as follows: image 3 shown. The fluorescence intensity remained basically unchanged, indicating that intracellular calcium ions would not be affected by the introduction of low concentrations of luminol. Second, the effect of voltage on cellular calcium ions was determined. By applying -0.5 ~ 0.5V and -1...

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Abstract

The invention discloses a microelectrode array which contains microelectrodes formed by micro holes of a cell size. Single cells are positioned directly on the microelectrodes containing the micro holes of the cell size, activation cholesterol in cell membranes reacts with cholesterol oxidase in a solution to produce hydrogen peroxide on the electrode surface to cause measurable luminol electroluminescence, and the luminous intensity is related to the activation cholesterol amount in the cell membrane. By connecting with a multichannel converter and a voltage generator, pulse triangle wave voltages are successively applied to the microelectrodes in the array, when a voltage is applied to the surface of a microelectrode, the cell or the microelectrode may shine, and a photomultiplier (PMT) is used for detection of luminol chemiluminescence intensity to obtain the activation cholesterol amount in the membranes. In the microelectrode array, the single cells are selected by the voltages to detect, the microelectrode array facilitates detection automation and analysis of flux. The microelectrode array can realize the rapid detection of the activation cholesterol content in the cell membranes.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to a microelectrode array and its application in detecting activated cholesterol on the surface of single cells. Background technique [0002] The balance of cholesterol inside cells has an important impact on cell function. Although cholesterol is predominantly distributed in the cell membrane, it is also concentrated in sphingolipid-rich regions such as lipid rafts or plasma membrane microvesicles. Cholesterol in these regions is more complex than cholesterol in the phospholipid bilayer, resulting in a higher chemical activity (disengagement propensity), so they are often referred to as activated cholesterol. Recent studies have shown that membrane activated cholesterol can induce cellular cholesterol transport, including inhibition of cholesterol synthesis, reduction of cholesterol intake and enhancement of cholesterol efflux. Therefore, a comprehensive study of cell membrane ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/30G01N27/26
Inventor 方丹君江德臣田春秀马光中
Owner NANJING UNIV
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