Fusion method for nanoliter-sized micro droplets in small-scale reaction tube

A technology of reaction tubes and micro-droplets, which is applied in chemical instruments and methods, biochemical equipment and methods, and microbial measurement/inspection, and can solve problems such as inability to perform single-cell reaction screening operations

Inactive Publication Date: 2018-07-13
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The volume of the droplet sorted by flow cytometry is only 2 nanoliters. When flying into the multiwell plate pre-loaded with mineral oil, 30 nanoliters of reagents are added using the high-precision droplet pipetting operation developed by us. After centrifugation, the liquid The efficiency of droplet fusion is only about 40%, and effective single-cell reaction screening cannot be performed

Method used

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  • Fusion method for nanoliter-sized micro droplets in small-scale reaction tube
  • Fusion method for nanoliter-sized micro droplets in small-scale reaction tube
  • Fusion method for nanoliter-sized micro droplets in small-scale reaction tube

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Such as figure 1 As shown, the small reaction tube used in Example 1 is a 96-well PCR plate (1). This example examines the effect of the concentration of Span 80 surfactant in mineral oil (2) on the efficiency of fusion of two added nanoliter volume droplets. The experimental steps are as follows:

[0040] 1) Prepare mineral oil (2) containing different concentrations of Span 80 (0%, 0.01%, 0.02%, 0.03%, 0.05%, 0.06%, 0.1%, 0.2%, 0.5%), and load it into a 96-well plate (1 ), the loading volume is 80 microliters / well;

[0041] 2) According to the micro-droplet generation method described in the published patent (Chinese patent, application number: 201410655191.5), load the 1 mole / liter potassium thiocyanide (KSCN) solution micro-droplet (3) of 5 nanoliter volume to step One said 96-well plate;

[0042] 3) According to the micro-droplet generation method described in the published patent (Chinese patent, application number: 201410655191.5), load the second 5 nanoliter...

Embodiment 2

[0054] According to the present invention, the operation flow of genome amplification of microorganism single cell sorted by flow cytometer is carried out.

[0055] Such as Figure 4 Shown, according to the method provided by the invention, adopt the Moflo XDP flow cytometry sorter (6) of MDA kit (Qiagen REPLI-g SingleCell Kit) and Beckman Company to carry out the step and The conditions are as follows:

[0056] 1) The experiment was carried out in a clean room with a grade of 1000 or above. Use a humidifier to increase the humidity of the experimental environment to 50%, and use alcohol to disinfect and remove static electricity from the experimental table;

[0057] 2) Prepare mineral oil containing 0.02% Span 80, filter and sterilize with a 0.22 micron filter membrane, and load it into each well of a 96-well plate with a loading volume of 80 microliters;

[0058] 3) In each well of the 96-well plate, according to the micro-droplet generation method described in the publish...

Embodiment 3

[0066] Such as Figure 5 As shown, mineral oil containing 0.02% (v / v) Span 80 was added to the PCR eight-tube tube, and according to the micro-droplet generation method described in the published patent (Chinese patent, application number: 201410655191.5), continuously in the The tube was filled with a 5 nanoliter droplet that slowly sank vertically to the bottom of the tube. This experiment shows that through the addition of surfactant, the hydrophobic group of the active agent effectively shields the electrostatic charge of the droplet, avoids the electrostatic adsorption and repulsion of the droplet, and ensures the effectiveness of subsequent centrifugal mixing.

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Abstract

The invention provides a fusion method for nanoliter-sized micro droplets in a small-scale reaction tube. The method is carried out in an environment where static electricity is sufficiently eliminated, the small-scale reaction tube in which an oil phase immiscible with the micro droplets is added in advance is provided, and an appropriate amount of a surfactant is added into the oil phase; the small-scale reaction tube can receive two or more nanoliter volume micro droplets prepared by technologies such as an ink-jet technology, a flow-type sorting technology and a droplet microfluidics; andthe two or more droplets deposited on the bottom of the small-scale reaction tube are contacted and fused by utilizing acting force including centrifugal force, vibration force, electromagnetic fieldacting force and the like for promoting droplet fusion. The fusion method for the micro droplets provided by the invention is characterized in that through addition of the appropriate amount of the surfactant in the oil phase, resistance of the micro droplets to enter the oil phase is reduced, surface charges of the droplets are shielded, and sedimentation and fusion of the micro droplets are promoted. The method provided by the invention has broad application prospects in application fields such as micro-volume biochemical reaction screening and single-cell analysis.

Description

technical field [0001] The invention relates to the technical field of high-throughput analysis screening and single cell analysis, in particular to a method for realizing controllable nanoliter volume mixing reaction and screening in conventional small reaction tubes and its application. Background technique [0002] In the fields of chemistry, biology, pharmacology, and clinical diagnostics, the miniaturization and portability of solution analysis is an important trend (Hong, J.W. and S.R. Quake, Integrated nanoliter systems. Nature Biotechnology, 2003.21:1179-1183). Through miniaturization and integration, the consumption of precious samples and reagents can be saved, the reaction and mixing time can be shortened, the sensitivity of detection can be improved, portability and bedside detection can be achieved, and real-time rapid detection and analysis can be realized. [0003] Among many miniaturization strategies, microfluidic chip technology uses a micron-scale channel ...

Claims

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Application Information

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IPC IPC(8): B01J19/00B01L3/00B01F13/00C12Q1/686C12Q1/6806
CPCB01J19/0093C12Q1/6806C12Q1/686B01L3/50273B01L2400/0409B01F33/3021C12Q2527/125C12Q2531/113
Inventor 杜文斌贠娟莉郑小伟徐鹏戴欣黄力
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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