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Digital PCR chip and method based on surfactant-modified PDMS

A surfactant and chip technology, applied in the fields of medicine, environmental science, and biology, can solve the problems of affecting PCR reaction efficiency and poor sealing effect, and achieve the effects of low cost of consumables, easy observation and statistical analysis, and simple operation

Active Publication Date: 2020-11-13
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of pre-filling the BSA-based digital PCR chip based on the micropipe for pre-blocking will affect the injection of the PCR reaction solution due to the impure removal of the pre-blocking solution, and adding BSA and surfactants to the PCR master mix will affect the PCR reaction efficiency, and Poor closure

Method used

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  • Digital PCR chip and method based on surfactant-modified PDMS
  • Digital PCR chip and method based on surfactant-modified PDMS
  • Digital PCR chip and method based on surfactant-modified PDMS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the design and manufacture of mold

[0035] On the basis of studying the effects of various chip cavity shapes, microchannel sizes, bonding and sampling methods on liquid flow resistance and stability, the present invention finally finds that the microchannel connects the cylindrical cavity, and after the chip is thermally bonded, Using its own vacuum negative pressure sample injection method, the sample can be quickly introduced and automatically separated, and the stability of the sample in the micro reaction chamber is good.

[0036] The chip structure is composed of three parts: the sample inlet area, the array area and the sample outlet area (see figure 1 PDMS microarray chip layer). First, use CAD software to design patterns and print the mask plate, and then use negative photoresist SU8 3050 to fabricate micropipes and microreaction chambers in layers on the silicon wafer. The width of the micro-pipes in the sample inlet area and the sample outlet...

Embodiment 2

[0037] Embodiment 2: the preparation of PDMS chip

[0038] After the silicon wafer mold was fabricated, the PDMS chip was cast by soft etching molding. First mix the PDMS prepolymer and the curing agent at a ratio of 10:1 (mass ratio), stir well, and then add 0.1 to 2.0 g of surfactants (such as tween 20, span 80, Triton100 or betaine per 100 g of the mixture) Surfactants commonly used in biological reactions can be mixed again, stirred evenly, degassed by vacuum, poured on the above mold, and thinly coated with the same mixed liquid PDMS on the glass slide, let stand together for 1h , after curing, peel off the PDMS on the mold, punch holes, stick the pipe surface and the coated glass coating surface together, discharge the air in the veneer, and finally put the bonded PDMS into the 85°C Heat on a hot plate for 10 min to fully bond. In the present invention, the surfactant is directly doped into the PDMS monomer, and the surfactant gathers on the surface of the PDMS after c...

Embodiment 3

[0039] Example 3: Automatic sampling of liquid samples and generation of microdroplet arrays

[0040] PCR premix: Here, the PCR reaction solution is compatible with the conventional PCR reaction solution. The PCR reaction system is: 20 μL of the premix solution contains 10ul Roche 480Probe Premix, 250nM EGFR gene exon 21 upstream and downstream primers, 200nMTaqMan probe, 10 ng of genomic DNA.

[0041] First, prepare the PCR premix, that is, the sample, inject the sample into the chip inlet after vacuuming and degassing, and use the negative pressure of the PDMS chip after vacuuming to suck the sample, and the sample gradually fills the microchannel and the holes on both sides along the microchannel. cavity (see figure 2 : sample injection process), after all the reaction chambers are filled with samples, inject the pre-mixed oil phase into the injection port, and paste a small piece of degassed PDMS blank block on the sample port of the chip to guide the introduction of the...

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Abstract

The invention relates to a digital PCR chip based on surfactant modified PDMS, a preparation method and application. The digital PCR chip is characterized in that the digital PCR chip is a PDMS digital PCR array chip prepared by a PDMS material doped by a certain amount of a surfactant; sampling and distributing processes are realized by means of a high air dissolution characteristic of the pre-degassing thin type PDMS chip, and a sandwiched structure of glass-modified PDMS-glass is prepared to inhibit water volatilization. According to a design method of the chip, electrostatic adsorption of PDMS on biological molecules is reduced, and the stability and the volatility resistance of liquid drops are effectively improved, so that the amplification efficiency of PCR is improved. Moreover, compared with a reported digital PCR chip at present, the digital PCR chip is low in cost, simple to operate and quite wide in application prospect.

Description

technical field [0001] The invention relates to a digital PCR chip and method for quantitative detection of nucleic acid, more specifically to a digital PCR chip and method based on surfactant-modified PDMS, which is expected to be applied in the fields of biology, medicine, environmental science and the like. Background technique [0002] The fluorescence quantitative PCR (Fluorescence Quantitative Polymerase Chain Reaction, FQ-PCR, qPCR) proposed in the late 1990s has developed into a key technology and routine technology in the field of molecular biology, which has greatly promoted the development of various fields of life science. However, there are many factors affecting PCR amplification efficiency, and it is difficult to ensure that the amplification efficiency between actual samples and standard samples and different samples is the same, which leads to the basis of its quantitative analysis-cycling threshold value (Cycling Threshold Value, Ct ) is not constant. Ther...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2523/31C12Q2527/125
Inventor 景奉香符亚云李刚
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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