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In-situ rapid detection method for cell biological process

A technology of cell biology and cell biology, which is applied in the field of rapid detection of cell biological processes in situ, and can solve problems such as not yet cultured and unable to reflect the living environment of cells

Active Publication Date: 2018-02-27
上海氘峰医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But until now, by adding deuterium water to mark active cells, there are several problems: 1) only for microbial cells, and there is no suitable method for eukaryotic cells; 2) only for classical microorganisms that have been isolated and cultured Purely cultivated strains, not targeting unknown, uncultivated or real mixed flora; 3) The medium in the process of microbial cells being marked with deuterium water is artificially prepared medium, which cannot reflect the real living environment of the cells

Method used

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  • In-situ rapid detection method for cell biological process
  • In-situ rapid detection method for cell biological process
  • In-situ rapid detection method for cell biological process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Embodiment 1: the impact of deuterated substrate on microbial growth

[0107] This example verifies that cells cannot distinguish undeuterated substrates from fully deuterated substrates during metabolism.

[0108] The specific implementation plan is as follows:

[0109] Escherichia coli DH5a, Pseudomonas putida UWC1, and Pseudomonas putida G7 were selected (these strains are common commercially available strains), the culture condition of Escherichia coli DH5a was 37°C, and Pseudomonas putida UWC1, Pseudomonas putida The culture condition of Monas G7 was 30°C. The bacterial solution cultured overnight in LB medium was inoculated into fresh MM medium at a ratio of 1:50. The only carbon source for the medium was glucose or naphthalene. The proportions of deuterated glucose were 0, 5, 10, 25, 50, 75, 100%, and the proportions of deuterated naphthalene were 0, 5, 10, 25, 50, 75, 100%. Use a pipette gun to pipette 200 μL of bacterial solution to a 96-well plate, and cul...

Embodiment 2

[0112] Example 2: Identification of antibiotic resistance of unknown non-purely cultured microorganisms in river water without altering the in situ environment

[0113] The river water was collected in the Thames River, England in February 2016 (the coordinates of the sample collection point are 51°44'47.0"N, 1°15'21.0"W). Take 200mL surface river water samples and store them in glass sample bottles. Save it.

[0114] Take 2.4mL river water sample, add 1.6mL deuterium water, and add different concentrations of antibiotics (see Table 1 for specific concentrations), and incubate at room temperature for 24 hours at a rotation speed of 500rpm. The negative control is 2.4mL river water sample added with 1.6mL deionized water, cultured under the same conditions.

[0115] Table 1 Concentration of antibiotics in river water samples

[0116]

[0117] Label the 24-hour river water sample, centrifuge at 5000g for 5 minutes, remove the supernatant, add 4 mL of deionized water and cen...

Embodiment 3

[0122] Example 3: Identification of Bacterial Infection Resistance from Sputum of Patients with Upper Respiratory Tract Infection

[0123] The patient, male, 33 years old, had upper respiratory tract infection. Collect 2mL of its sputum and mix it with 2mL deuterium water. The mixed sample was divided into five equally, four of which were added with different antibiotics, including kanamycin (50 μg / mL), carbenicillin (100 μg / mL), tetracycline (12 μg / mL), chloramphenicol (6 μg / mL), and one without antibiotics was used as a control. Samples were incubated at 37°C for 7 hours.

[0124] Then the sample was centrifuged at 5000g for 5 minutes, the supernatant was removed, an equal volume of deionized water was added to mix, centrifuged at 5000g for 5 minutes, the supernatant was removed and resuspended in the same volume of deionized water. After drying 1 μL of cells on a calcium fluoride glass slide, the Raman spectrum of a single cell was measured with a Raman spectrometer. R...

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Abstract

The invention relates to an in-situ rapid detection method for a cell biological process. After absorption, conversion, metabolism or storage for deuterium-containing matters based on active cells, in-situ marking for deuterium in cells can be realized in different stages of the cell biological process, the cells are detected through a Raman spectrum, whether deuterium exists in the cells is identified, and then the biochemical process, cell character or function related information about the cells is obtained. The method can be used for biological and medical fields such as bacterial infection drug susceptibility testing, environmental microbial metabolism and human body cell detection. Compared with the prior art, the method has the potential of detecting the activity and metabolism of the cells for most important biomolecules, and more complex deuteration matters are allowed to be researched and used, including natural products, medicines and a deuterization mixture containing various chemical matters. The method is extremely valuable for function research of the cells under natural conditions of single cell level.

Description

technical field [0001] The invention belongs to the detection technology of biotechnology, and relates to a method for rapid detection of cell biological processes in situ, in particular to selectively labeling cell biological processes with deuterium-containing substrates, and analyzing the state of the labels through single-cell Raman maps , so as to identify the methods of cell metabolism, synthesis and other traits or functions and their applications in the fields of biosynthesis and medical testing. Background technique [0002] The study of cellular and molecular biology advances our understanding of life on Earth. Modification of biomolecules can be used to monitor cellular changes or functions in the natural environment. The discovery represented by the green fluorescent protein (GFP), which won the Nobel Prize in Chemistry in 2008, has brought about a revolution in the field of chemical biology. However, many small biomolecules and metabolites such as nucleic acid...

Claims

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Application Information

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IPC IPC(8): G01N21/65
CPCG01N21/65
Inventor 宋一之徐嘉宝王允
Owner 上海氘峰医疗科技有限公司
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