Cell carrier chip and single cell rapid identifying or sorting method employing same

A single-cell, cell-based technology, applied in biochemical equipment and methods, material excitation analysis, Raman scattering, etc., can solve problems such as inability to guarantee cell viability, cell damage, interference with single-cell Raman spectrum and fluorescence measurement, and achieve Improve accuracy and ease of operation, ensure cell viability, and reduce laser intensity

Active Publication Date: 2013-10-16
长春长光辰英生物科学仪器有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has certain limitations: (1) The currently used coating materials have a Raman background or a fluorescent film, which interferes with the Raman spectrum and fluorescence measurement of single cells; (2) Most of them need to use high-intensity ultraviolet pulsed laser Cell transfer usually causes damage to cells, and cell viability cannot be guaranteed; (3) The identification of target cells is still based on a few factors such as morphology and fluorescent markers, and prior knowledge of cells is required
(4) So far, there is no system coupling single-cell Raman-fluorescence detection system and single-cell ejection system

Method used

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  • Cell carrier chip and single cell rapid identifying or sorting method employing same
  • Cell carrier chip and single cell rapid identifying or sorting method employing same
  • Cell carrier chip and single cell rapid identifying or sorting method employing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Material selection and fabrication of a cell carrier chip

[0033] The base material is a material that does not affect light transmission, including but not limited to silicate glass, quartz glass, calcium fluoride glass; special coating materials that can be used include Ti, TiO 2 , SiO 2 , Si, Al, Al 2 o 3 , Au and Ag etc. Use SEM / TEM sputter coater (model Q150T, purchased from Quorum Technologies company) to coat the above-mentioned materials on the surface of the substrate to form a coating; the thickness of the coating is related to the coating time and current, and the voltage is 2.5KV, and the distance between the target and the material is When the coating thickness is 50mm, the specific formula for calculating the coating thickness is Th=7.5It (Th, coating thickness, Angstrom; I, current, mA; t, time, min). According to needs, the thickness of the coating is 10-1000nm.

Embodiment 2

[0034] Example 2: Rapid measurement method of single-cell Raman spectrum

[0035] The Escherichia coli DH5α used for the test (purchased from Promega (Beijing) Biotech Co., Ltd.) was cultured overnight at 37°C and 150 rpm in a basal medium containing 5 g / l glucose until stationary phase. 1 liter of basal medium contains 2.5g Na 2 HPO 4 , 2.5g KH 2 PO 4 , 1.0g NH 4 Cl, 0.1g MgSO 4 ·7H 2 O, 10 μl saturated CaCl 2 solution, 10 μl saturated FeSO 4 solution and 1ml Bauchop & Elsden solution. Among them, 1 liter of Bauchop&Elsden solution contains 10.75g MgSO 4 , 4.5g FeSO 4 ·7H 2 O, 2.0g CaCO 3 , 1.44g ZnSO 4 ·7H 2 O, 1.12g MnSO 4 4H 2 O, 0.25g CuSO 4 ·5H 2 O, 0.28g CoSO 4 ·7H 2 O, 0.06g H 3 BO 3 and 51.3 ml of saturated HCl solution.

[0036] When measuring the Raman spectrum, the cells in the stationary phase were washed three times with high-purity water and then resuspended in high-purity water, and the cells were diluted to 10 with high-purity water. 6 ...

Embodiment 3

[0039] Example 3: Rapid identification of single cells based on Raman spectra

[0040] Five strains of oral microorganisms Enterococcus faecalis ATCC29212, Actinomyces viscosus ATCC27045, Streptococcus mutans UA159 (ATCC700610), Streptococcus sanguinis ATCC49295 and Porphyromonas gingivalis W83 (ATCC: BAA-308) (these five strains of oral microorganisms were purchased from Guangdong Institute of Microbiology The collection center (Microbial Culture Collection Center of Guangdong Institute of Microbiology, GIMCC) was cultured statically in BHI medium at 37°C until the stationary phase (the BHI medium was purchased from Qingdao Hopebio-Technology Co., Ltd, China) , wherein the cultivation of P.gingivalis W83 was carried out under strict anaerobic conditions. When measuring the Raman spectrum, the cells in the stationary phase were washed three times with high-purity water and then resuspended in high-purity water, and the cells were diluted to 10 with high-purity water. 6 -10 8...

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Abstract

The invention relates to a cell carrier chip which comprises a basal layer and a clad layer, wherein the basal layer is made of materials which do not affect light ray transmission, the basal material is covered with the clad layer which has zero damage to cells; a ratio of the lowest Raman signal noise ratio of a to-be-tested sample to the highest Raman signal noise ratio of the clad layer is more than 3; the clad layer can be used for absorbing lasers and be stripped or melted partially. The invention further relates to the application of the cell carrier chip in a rapid measuring method of the single cell Raman spectrum, and a single cell rapid identifying method and a single cell rapid sorting/separating method which are based on the Raman spectrum. The Raman spectral background of the cell carried chip is reduced, the accuracy and handleability of an ejection system are improved, and the activity of an ejected target cell is guaranteed.

Description

technical field [0001] The invention relates to the technical field of cell identification and sorting, in particular to a cell carrier chip and a method for rapidly identifying or sorting single cells using it. Background technique [0002] All life on earth is formed by the combination and differentiation of single-cell individuals, so single-cell is the basic unit and evolutionary unit of life activities. In-depth and systematic research on single cells can not only reveal the essence of life activities in a panoramic manner, but also the specificity and differentiation process of single cells is of great significance for the study of disease mechanisms and diagnosis and prevention of diseases. Traditional microbiology studies on the characterization of microbial cells are based on the population cell level, but recent studies have shown that in a cell population, even between different individual cells with completely consistent genome information, their phenotypes also ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/65C12N5/00
Inventor 黄巍王允宋一之
Owner 长春长光辰英生物科学仪器有限公司
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