New Polymerase Chain Reaction Chip Research

Polymerase chain reaction(PCR)is an in vitro amplification technique for gene fragments consisting of three processes: high-temperature denaturation,low-temperature annealing,and intermediate temperature extension.The traditional PCR amplification model passes 25 to 35 temperature intervals.The repeated cycles of DNA molecular weight increase exponentially.According to the performance of the PCR instrument,a complete amplification process requires 120 to 200 minutes.In recent years,along with the common development of microfluidics(Microfluidics)and microelectromechanical technology(MEMS),it has become possible to realize the implementation of PCR microfluidic chip technology,eventually making it a fluid mechanics,analytical chemistry,The comprehensive technologies of biomolecules,microelectronics and other disciplines,compared with the traditional PCR technology,greatly reduce the amplification time and also reduce the consumption of expensive reagents.This project developed a new type of continuous flow PCR microfluidic chip.First,in order to be able to monitor the flow rate of the reaction system in real time during the amplification process and to correct the flow rate in time,a micro-flow sensor based on a constant heating power was designed in the flow path.The sensor uses a glass substrate of indium tin oxide.(Indium Tin Oxide,ITO)as a heating resistor,polydimethylsiloxane(PDMS)as a channel material,processed by digital lithography projection technology,can achieve more accurate measurement in the range of 10-120?L/min.Secondly,using ITO conductive glass as the heating resistor,an integrated self-heating,self-testing temperature control unit was designed.The unit uses PWM pulse width time length to heat the ITO and measure its own resistance;the temperature control unit uses the temperature sensor and PID controller to establish the temperature resistance linear relationship of the ITO heating resistor in the early stage of development,and then in the PWM wave.In the measurement cycle,the resistance value of the ITO heating resistor is detected by a constant current source,and the temperature value of the heating resistor is inversely introduced through the linear relationship of the temperature resistance obtained in the previous stage.By controlling the microcomputer,the pulse width of the PWM wave is adjusted to achieve accurate temperature control of the ITO conductive glass.The overall dimensions of the temperature control system are: length 5 cm,width 5 cm,height 0.2 cm,and temperature error ±1°C.Then,the structure of the flow path of the PCR chip was designed.With the help of the digital lithography projection system(DLPS)made by our laboratory,a serpentine flow path with a width of 200 ?m,a depth of 80 ?m,and a total length of 2.28 m was realized using PDMS material.Using oxygen plasma technology to achieved the irreversible package of the chip and ITO electrode layer,and the overall chip shape length 4cm,width 3cm,thickness 0.3cm.Finally,a test experiment of the amplification effect was performed after the PCR chip,the heating electrode,and the cooling device were integrally packaged.Among them,for the problem of bubble and channel wall adsorption in the flow path,this article carried out the contrast experiment of glycerol on the inhibition of bubbles under different concentration conditions,and the different concentrations of bovine serum albumin(BSA)against the channel wall.Contrast experiments of adsorption inhibition on the inner surface;this PCR microfluidic chip successfully amplified two DNA fragments and achieved good experimental results.