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Method for screening microorganisms by using supermolecular fluorescence microfluidic technology

A microfluidic technology, a microfluidic technology, is applied in the field of high-throughput screening of microorganisms by supramolecular fluorescence microfluidic technology, which can solve the problems of inability to detect metabolites, complicated labeling process, and limited application range, etc. High sensitivity, low detection cost, and simple experimental operation

Active Publication Date: 2020-12-01
态创生物科技(广州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional high-throughput screening of micro-droplets mainly uses fluorescent dyes or fluorescent proteins of cells as tracer molecules. The labeling process is complicated, and the metabolites secreted by cells cannot be detected and screened, which limits its application range.

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  • Method for screening microorganisms by using supermolecular fluorescence microfluidic technology
  • Method for screening microorganisms by using supermolecular fluorescence microfluidic technology
  • Method for screening microorganisms by using supermolecular fluorescence microfluidic technology

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Analysis of tryptophan acid concentration in microfluidic droplets by supramolecular fluorescence;

[0026] 1. Add different concentrations of tryptophan to the supramolecular fluorescent supramolecular solution of CB[8] and MDPP with a molar ratio of 1:1, and use a microplate reader to detect the fluorescence intensity of the supramolecular solution. attached by Figure 4 It can be seen that when tryptophan is added to the supramolecular fluorescence system, the fluorescence of the system decreases gradually with the addition of tryptophan. A π-π stacking occurs, resulting in a fluorescence quenching effect.

[0027] 2. Prepare three sets of solutions as aqueous phase (1): CB[8] (5 μM) and MDPP (5 μM); aqueous phase (2): CB[8] (5 μM), MDPP (5 μM) and tryptophan ( 5 μM); aqueous phase (3): CB[8] (5 μM), MDPP (5 μM) and tryptophan (10 μM). The flow rate of the aqueous phase was 1 μL / min, and the flow rate of the oil phase was 3 μL / min. Droplets were generated into a 1...

Embodiment 2

[0029] Using supramolecular fluorescence to analyze the concentration of tryptophan metabolized by Escherichia coli in microfluidic droplets;

[0030] The E. coli suspension was diluted to 10 with liquid medium 6 individual / mL. The basic components of the medium are: trace elements 0.1%, K 2 HPO 4 2.5%, KH 2 PO 4 0.9%, (NH 4 ) 2 SO 4 0.45%, Glucose 0.3%, MgSO 4 0.01% pH7.2. , select the cell-wrapped chip to wrap the cells with droplets, set the flow rate of the oil phase to 3 μL / min, and the flow rate of the E. coli cell diluent to 1 μL / min, and generate droplets into a 1mL syringe. The droplet diameter is 30 μm, according to the Poisson distribution , the proportion of droplets without cells was 0.6065, the proportion of droplets embedded with single cells was 0.3033, and the proportion of droplets embedded with two or more cells was 0.0902. Put the syringe into a 37°C incubator and incubate for 24 hours, inject the cucurbituril / fluorescent dye supramolecular co...

Embodiment 3

[0032] Screening of Escherichia coli with different tryptophan metabolic abilities using supramolecular fluorescent microfluidic technology;

[0033] The droplet detection chip is placed on the stage, and the position of the stage is adjusted to make the droplet detection chip image clearly on the high-speed camera. Set the droplet flow rate to 0.1μL / min, the oil phase flow rate to 2μL / min, inject the droplet into the droplet detection chip, turn on the laser, set the laser wavelength to 442nm, and the detection wavelength to 500-520nm, adjust the droplet detection chip The position aligns the laser light to the channel detection point of the droplet detection chip. Turn on the photomultiplier tube, set the trigger value of the signal analysis and processing software, the gain value of the photomultiplier tube, and the waveform diagram shows the trigger threshold and the droplet fluorescence intensity value. When the detection value of the detected target droplet exceeds the p...

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Abstract

The invention provides a method for detecting the concentration of amino acid generated in the metabolic process of microorganisms based on a supramolecular fluorescence system and realizing high-throughput screening of the microorganisms by combining a droplet microfluidic technology. The method comprises the following steps of: (1) generating water-in-oil droplets on a micro-fluidic chip, and researching the influence of amino acid on the fluorescence intensity of a cucurbituril / fluorescent dye supramolecular system in the micro-fluidic droplets; (2) wrapping and culturing microorganisms byutilizing a droplet microfluidic technology, then injecting cucurbituril / fluorescent dye into microfluidic droplets, and distinguishing the microorganisms with different amino acid metabolic capabilities according to the change of the fluorescence intensity of the droplets; and (3) carrying out high-throughput screening on the microorganisms with different amino acid metabolic capabilities by utilizing a microfluidic droplet screening technology according to the difference of fluorescence intensity of each microfluidic droplet. Fluorescence of the supramolecular system is researched in the microfluidic droplets for the first time and applied to detection of the metabolite concentration of the microorganisms, and high-throughput screening of the microorganisms with different metabolic capabilities is established accordingly. The detection method has the advantages of high sensitivity, high speed and the like, with the screening flux reaching millions of droplets per hour. By adopting the high-throughput screening method disclosed by the invention, microorganisms capable of producing target products at high yield are obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, the present invention relates to a novel supramolecular fluorescent microfluidic technology for high-throughput screening of microorganisms. Background technique [0002] 2. Microbial high-throughput screening refers to the technology that can automatically, quickly and efficiently screen the functions and products of microorganisms, which is one of the bottlenecks in the development and industrialization of industrial microbial resources. Microbial high-throughput screening technology uses advanced modern automation technology and instrument analysis technology to realize the traditional mutagenesis screening process, which has the characteristics of automation, standardization, and high-throughput quantification, and greatly breaks through the limitations of manual screening in terms of speed, efficiency, and standardization. However, all kinds of high-throughput screening equip...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 余子夷文慧琳张静胡驰
Owner 态创生物科技(广州)有限公司
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