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Detection device and detection method for cell in-situ single vesicle inclusions

A detection device and detection method technology, applied in the field of biotechnology and medical detection, can solve the problems of limited application of coating modification, inability to effectively detect the concentration of free neurotransmitters, and difficulty in realizing the penetrating recording mode. Simple, wide-ranging effects

Pending Publication Date: 2021-04-02
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method uses extremely thin carbon fiber electrodes to directly penetrate cells, and there are still many difficulties in terms of technology and application: 1. The penetration recording mode is mainly used for larger cells in culture, which means that in closer It is difficult to achieve relevant electrochemical recordings at the brain slice level in physiological state or under in vivo conditions
2. The penetrating recording mode requires extremely thin carbon fiber tips, which limits the application of coating modification
Therefore, this method is only suitable for the inspection of individual (higher electrochemically active) transmitters that can directly react with carbon fiber electrodes, while for other neurotransmitters such as glutamic acid, specific enzyme coating of carbon fiber electrodes is required. The composition of the electrochemical detection can only be realized after layer modification, and the penetrating recording mode is difficult to achieve
3. The penetrating recording mode cannot effectively detect the concentration of free neurotransmitters in the cell matrix while detecting the amount of transmitters contained in a single vesicle

Method used

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  • Detection device and detection method for cell in-situ single vesicle inclusions
  • Detection device and detection method for cell in-situ single vesicle inclusions
  • Detection device and detection method for cell in-situ single vesicle inclusions

Examples

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Embodiment 1

[0085] This embodiment provides a method for detecting the contents of a single cell vesicle in situ, and the detection method specifically includes the following steps:

[0086] (1) Preparation of brain slices:

[0087] (a) After the surgical instruments (including surgical scissors, ophthalmic scissors single-sided scalpel, double-sided blade, tweezers, utility spatula, superglue, filter paper, etc.) Dissect the head of the mouse with surgical scissors, ophthalmic scissors, tweezers, etc., and take out the brainstem. This process will be immersed in ice slicing solution every 5s (the slicing solution includes: 125mM sodium chloride, 25mM sodium bicarbonate, 3mM inositol , 2mM sodium pyruvate, 2.5mM potassium chloride, 1.25mM sodium dihydrogen phosphate, 25mM glucose, 0.1mM calcium chloride and 3mM magnesium chloride, the pH of the slicing solution is 7.4, and there are 95% oxygen and 5% carbon dioxide of binary gas);

[0088] (b) Glue the brainstem tissue to the slicing pl...

Embodiment 2

[0102] Sensitivity and immunity testing

[0103] This example provides a method for detecting the contents of a single cell vesicle in situ. The carbon fiber electrode uses a traditional enzyme-based electrode structure and contains a sensitive enzyme (glutamic acid oxidase 33U / mL), a cross-linking agent of 1% ammonium The enzyme coating of dialdehyde and stabilizer 1% bovine serum albumin (concentration ratio is 2:1:1) is modified, and o-phenylenediamine is used as an interfering substance to isolate the film, and platinum nanoparticles are electroplated on the surface of the carbon fiber To improve the response to the reporter molecule produced by the enzymatic reaction. For different transmitters, the effect of specific detection can be achieved by changing the type and combination of enzymes. The linear detection range of the glutamic acid electrochemical electrode to glutamic acid is 1-156 μ M (R 2 =0.998), matching with the change range of glutamate concentration in th...

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Abstract

The invention provides a detection device and a detection method for cell in-situ single vesicle inclusions. The detection device comprises an optical microscope, a micro-manipulator, a computer workstation, an AD / DA converter, a patch clamp amplifier, an infrared camera and an electrochemical electrode system, the electrochemical electrode system comprises a carbon fiber electrode, a glass microelectrode, a reference electrode, an electrolyte and a propeller. The operation method is simple, the application range is wider, and a direct, high-time and high-spatial-resolution detection method isprovided for the qualitative and quantitative research of neurotransmitters at the single vesicle level.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and medical detection, and in particular relates to a detection device and a detection method for the content of a single cell vesicle in situ. Background technique [0002] Flow cytometry is one of the most common methods for multi-parameter and rapid quantitative analysis of single cells and other biological particles today. It has the characteristics of high speed, high precision and good accuracy, but this technology is only applicable to Cells or other particles to be sorted, but nano-scale vesicles (such as synaptic vesicles, exosomes, etc.) cannot be isolated and analyzed. [0003] At present, the method for detecting vesicle content is mainly based on the principle of in vitro separation and lysis detection, and has been applied to the quantitative detection of vesicle content in liposomes, chromaffin cells, PC12 cells, and catecholaminergic neurons. (Omiatek et al., 2009, 2010, 201...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/487G01N15/10G01N15/14
CPCG01N33/48728G01N33/487G01N15/1031G01N15/1434
Inventor 柳孜俐孙坚原申雪峰赵帅男
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI