Low-cost wide-adaptation centrifugal digital droplet generation method and device

A centrifugal, low-cost technology, applied in the field of analysis and sensing, can solve the problems of mixed contamination of the syringe pump, external contamination of the pump in the microfluidic chip, and complex structure of the droplet generation device, so as to prevent the flow of droplets and reduce pollution. Effect

Pending Publication Date: 2021-03-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among the droplet generating devices studied in recent years, CN109536590A proposes a single-cell gene detection method based on a microporous chip, but the production of this multi-layer chip is cumbersome and the processing procedure is complicated
CN110841734A proposes a single-pump droplet generation system, but the syringe pump introduces the risk of mixed contamination
In 2018 and 2020, Mou Ying's research group proposed a thermal curing and U...

Method used

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  • Low-cost wide-adaptation centrifugal digital droplet generation method and device
  • Low-cost wide-adaptation centrifugal digital droplet generation method and device
  • Low-cost wide-adaptation centrifugal digital droplet generation method and device

Examples

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Embodiment 1

[0036] The invention discloses an embodiment of the assembly of a centrifugal droplet generating device, which includes an O-shaped rubber ring 1, a 200 μL

[0037] Inner pipette tip 2, high molecular polymer (such as PDMS) 3, micron tube 4 (sampling capillary), 1000μL outer pipette tip 5, 1.5mL centrifuge tube 6, aqueous phase sample 7 (can be prepared Amplified nucleic acid mixture, cell suspension, etc.) are stored inside the modified pipette tip encapsulated with PDMS and capillary. The aqueous phase sample communicates with the capillary. The size of the capillary is 100-200μm. Due to the tension, the aqueous phase sample will not leak out. The inner pipette tip 2 and the outer pipette tip 5 are assembled together through an O-shaped rubber ring. The combined device matches the size of the centrifuge tube and is stuck on the 1.5mL centrifuge tube 6. The inside of the centrifuge tube is equipped with an oil phase 8, and the lower layer The components of the oil phase can ...

Embodiment 2

[0040] Micro-droplets can be combined with nucleic acid isothermal amplification to realize digital nucleic acid amplification, such as Figure 5 Shown is a droplet-based application of nucleic acid loop-mediated isothermal amplification of Staphylococcus aureus. In this application, the aqueous phase components include target nucleic acid, enzyme, buffer (containing MgCl 2 and CaCl 2 ), primers, dNTPs, and calcein as a kind of fluorescent indicator, the sample is distributed into the droplet, followed by isothermal amplification. After the isothermal amplification is completed, in the negative droplet, calcein and Mn 2+Binds and emits dim fluorescence; in positive droplets, the reaction by-product pyrophosphate captures the Mn bound to calcein 2+ Combination, resulting in free calcein molecules and Mg 2+ binds and fluoresces strongly as Figure 5 shown. Quantitative detection of sample nucleic acid can be accomplished by counting positive droplets and total droplets and a...

Embodiment 3

[0042] Embodiment 3 is a kind of implementation case of the liquid droplet ultrafast solidification method, and the ultrafast laser can be used to assist the liquid droplet solidification, and the following is combined with the attached Figure 6 An implementation manner of this example is further described in detail.

[0043] Such as Figure 6 As shown, the water-in-oil droplet 9 is tiled on the glass slide 16, and the slide is placed on the three-dimensional stepping displacement platform 15, and the ultrafast laser emitted by the femtosecond laser 10 is collimated through the lens group 11, and passed through the SLM 12 The output diffraction spot or pattern, the SLM module is located under the displacement platform, controlled by the controller 13, the beam is then converged by the inverted microscope and focusing system 14 to reach the threshold energy of the oil phase solidification and narrow the spot, and finally incident into the oil phase for a specific The position...

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Abstract

The invention discloses a low-cost wide-adaptation centrifugal digital droplet generation method and device, an upper-layer water phase centrifugal unit is embedded in a lower-layer oil phase centrifugal unit, a micron tube is arranged at the lower part of the upper-layer water phase centrifugal unit, and under the action of centrifugal force, the upper water phase overcomes the surface tension effect, liquid drops generated through the micro-tube channel enter the lower oil phase to form water-in-oil liquid drops with uniform sizes, and the sizes of the water-in-oil liquid drops are adjustedby adjusting the diameters of the micro-tubes and/or the centrifugal force. A mutually soluble laser-induced curing agent can be added into the oil phase, and the oil phase is quickly crosslinked andcured under the action of an induction source; the invention is applied to nucleic acid isothermal amplification to realize digital nucleic acid amplification. According to the invention, water-in-oildroplets with uniform size and high quality can be generated. The micro-liter aqueous phase solution can quickly generate high-flux water-in-oil droplets within a few minutes, the high-flux water-in-oil droplets are combined with isothermal amplification of nucleic acid, and meanwhile, ultrafast solidification of the droplets can be realized by virtue of a solidification platform.

Description

technical field [0001] The invention belongs to the field of analysis and sensing, and mainly relates to a low-cost and widely applicable centrifugal digital droplet generation method and device, which can be used for single-molecule nucleic acid detection and the like. Background technique [0002] In recent years, digital nucleic acid detection technology has played a huge role in the fields of biological detection, chemical sensing, and medical diagnosis. As a third-generation nucleic acid amplification method, the sample is diluted to the single-molecule level by distributing the sample into tens of millions of units using the technical concept of micro-droplets. Each droplet contains nucleic acid or does not contain nucleic acid. After amplification, the droplet containing nucleic acid has a fluorescent signal, and the number of fluorescent droplets conforms to the Poisson distribution. Absolute quantitative detection of samples such as target nucleic acids can be achi...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12M1/00C12M1/36C12M1/34C12M1/26
CPCC12Q1/6851C12Q2563/159C12Q2531/119
Inventor 何赛灵程潇羽张文耀贺泽玮纪佳丽纪长
Owner ZHEJIANG UNIV
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