A silicon-based array micro-reaction pool digital PCR chip and its preparation method

Abstract

The invention belongs to the technical field of PCR (polymerase chain reaction), and particularly relates to a digital PCR chip with silicon substrate arrays and micro-reaction pools and a method for manufacturing the digital PCR chip. The digital PCR chip mainly comprises an upper cover and a chip. Micro-pores which are arrayed to form honeycombs are formed in the chip, and the upper cover is fixed onto a chip groove. The method includes particular steps of selecting a silicon chip with a single polished surface; cleaning the silicon chip; coating a layer of uniform photoresist on the polished surface of the silicon chip in a spinning manner; forming circular graphic arrays by means of exposure; etching silicon by the aid of a dry process in masks, namely, the circular graphic arrays to form micro-pore structures; removing the photoresist and scribing the chip to completely manufacture the digital PCR chip.

Description

technical field [0001] The invention belongs to the technical field of PCR, and in particular relates to a silicon-based array micro-reaction pool digital PCR chip and a preparation method thereof. Background technique [0002] The application of DNA molecular amplification technology has promoted the development of molecular biology technology, and the precise quantification of DNA copy number is one of the important applications of modern molecular biology and medicine. In 1997, James F. Brown, Jonathan E. Silver and Olga V. Kalinina constructed the prototype of digital PCR (digital polymerase chain reaction, dPCR) technology and obtained the US patent (patent number: US6143496 A). Different from traditional PCR technology, digital PCR fully dilutes nucleic acid samples so that the number of sample templates in each reaction chamber is less than or equal to 1. After PCR amplification, the fluorescent signals of each reaction chamber are statistically analyzed to solve Rea...

AI Technical Summary

Problems solved by technology

Glass is transparent, but poor thermal conductivity

PDMS material has excellent biological inertia and good light transmission, so it has been widely used in digital chips, but it has certain limitations in the application of digital PCR chips, mainly because PDMS has a certain degree of gas permeability. , while the thermal cycle temperature of PCR is relatively high, the high temperature is generally 94-95°C, and at the same time, it needs to be maintained at the high temperature stage for 5-10 minutes during the pre-denaturation process. If the sealing material has a certain degree of air permeability, the water vapor will volatilize, causing the water solvent in the reaction solution to decrease, and the component concentration of the reaction solution will change. The gas will also enter the reaction cell due to the gas permeability of PDMS to form bubbles, which will affect the sealing of the reaction cell

At the same time, the thermal conductivity of PDMS is poor (about 0.18W / mK), and the heat transfer efficiency of the PCR chip prepared by using it is low during the thermal cycle. If the thermal cycle time is not prolonged, the PCR amplification efficiency will inevitably be affected. make an impact

[0005] There are many problems in the existing technology, for example, it is difficult to uniformly enter the liquid into the array microwells on the chip, during the PCR reaction process, the liquid is easy to evaporate, and different areas are heated unevenly

Easy crosstalk between microwells on the chip

The polarity of the reaction micropore cannot match the reaction system, and the catalytic enzyme is easily adsorbed on the micropore wall and cannot participate in the reaction

Microfluidic chips are expensive

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