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Living cell detection method based on chemiluminescence principle

A chemiluminescence and detection method technology, applied in chemical instruments and methods, chemiluminescence/bioluminescence, luminescent materials, etc., can solve the problems of inability to measure ATP, fast detection speed, and high accuracy, so as to shorten detection time and reduce detection. Time, the effect of rapid detection

Inactive Publication Date: 2012-09-19
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ATP bioluminescence method has the problem of interference from non-fungal "free ATP" and other components. How to remove this background signal easily and reliably without affecting the sensitivity of "microbial ATP" detection has not yet been effectively resolved.
In addition, since the cell surface is covered by cell membrane and cell wall, ATP cannot be determined without treatment of the sample, and false positive signals will also be generated if the ATP extraction process is not handled properly.
In summary, the current rapid detection methods for microorganisms have certain limitations, and there is no method that can truly meet the requirements of fast detection speed, high detection accuracy and low detection limit.

Method used

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  • Living cell detection method based on chemiluminescence principle
  • Living cell detection method based on chemiluminescence principle
  • Living cell detection method based on chemiluminescence principle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1) Dissolve 1 mg of the phosphatase substrate (I-1) (R is octyl, X is the 5-position chlorine element, Y is the ortho-position chlorine element) with 1 ml of anhydrous DMSO to prepare a 1 mmol / l phosphatase substrate reserve solution, sealed and frozen at 2-8°C; add 10 μl of phosphatase substrate stock solution to 5 ml of Tris buffer to prepare 2 μmol / l, 0.2% DMSO phosphatase substrate solution.

[0026]

[0027] 2) Centrifuge the artificial Escherichia coli bacterial liquid of known concentration to collect the bacterial colony, remove the supernatant, add Tris buffer solution (pH8.0), and dilute 10 times into a series of bacterial liquid standards (the number of bacteria is adjusted to 10 4 、10 5 、10 6 、10 7 pieces / ml).

[0028] 3) Pipette 0.2ml of the Escherichia coli liquid with different concentrations above, transfer to a 96-well polystyrene microwell plate, add 0.2ml of the above phosphatase substrate solution, stir well and incubate at room temperature for...

Embodiment 2

[0031] 1) Dissolve 1 mg of lipase substrate (I-2) in 1 ml of anhydrous DMSO (R is a methyl group, X is a 5-position chlorine element, Y is an ortho-position chlorine element), and prepare a 1 mmol / l lipase substrate reserve Liquid, sealed and frozen at 2-8°C. Add 10 μl of lipase substrate stock solution to 5 ml of Tris buffer to prepare a 2 μmol / l, 0.2% DMSO lipase substrate solution.

[0032]

[0033] 2) Centrifuge the Pichia pastoris GS115 bacterial liquid of known concentration to collect the bacterial colony, remove the supernatant, add Tris buffer (pH8.5), and dilute 10 times into a series of bacterial liquid standards (adjust the number of yeast to 10 4 、10 5 、10 6 、10 7 pieces / ml).

[0034] 3) Take 0.2ml of the above-mentioned yeast liquids with different concentrations, transfer them to a 96-well polystyrene microwell plate, add 0.2ml of the above-mentioned lipase substrate solution, stir well and incubate at room temperature for 15 minutes to trigger chemilumin...

Embodiment 3

[0037]1) Dissolve 1 mg of lactase substrate (I-3) (R is ethyl, X is 5-position chlorine element, Y is ortho-position chlorine element) with 1 ml of anhydrous DMSO to prepare 1 mmol / l lactase substrate reserve Liquid, sealed and frozen at 2-8°C. Add 10 μl of lactase substrate stock solution to 5 ml of Tris buffer to prepare a 2 μmol / l, 0.2% DMSO lactase substrate solution.

[0038]

[0039] 2) Centrifuge the lactic acid bacteria liquid of known concentration to collect the flora, remove the supernatant, add Tris buffer (pH8.0), and prepare a certain concentration of the bacterial liquid sample (the number of lactic acid bacteria is adjusted to 10 5 and 10 6 pieces / ml).

[0040] 3) Take 0.2ml of the above-mentioned lactic acid bacteria solution of different concentrations, transfer it to a 96-well polystyrene microwell plate, add 0.2ml of the above-mentioned lactase substrate solution, stir well and incubate at room temperature for 15 minutes to trigger chemiluminescence, a...

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Abstract

The invention designs a new culture-independent quick living cell detection method using intracellular enzymes of living cells as detection targets and using chemiluminescence as detection signals of intracellular enzyme activity. A 1,2-dioxocyclohexane chemiluminescence agent capable of penetrating cell membranes is used as an enzyme substrate in the method; the substrate enters the interiors ofthe cells, then is subjected to hydrolysis reaction with the intracellular active hydrolase and generates chemiluminescence; and the intensity of the chemiluminescence depends on the number and the metabolism intensity of the living cells, so the chemiluminescence can be used as an index for detecting cell activity.

Description

(1) Technical field [0001] The invention relates to a living cell detection method based on the principle of chemiluminescence. (2) Background technology [0002] Conventional methods for detecting cell viability include MTT method, fluorescent staining method and ATP bioluminescence method, etc. The MTT method is based on the mitochondrial dehydrogenase of living cells that can crack the tetrathiazolium salt MTT into blue-purple crystal Formazan. The MTT method is easy to operate, has high sensitivity and good repeatability, which brings great convenience to cell culture technology. However, the MTT method has an insurmountable weakness: the formation of Formazan is affected by the action time, and the measured A value changes with time when there are many samples, which will increase the experimental error. For suspension cells, after MTT staining and dissolving with DMSO, the cells in each well must be aspirated, centrifuged to remove the supernatant, and the precipitat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76C09K11/07
Inventor 焦艳华黄志坚陈森林何睿梁媛媛李霞陈灿玉
Owner HANGZHOU NORMAL UNIVERSITY
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