Research On Digital PCR Mcirodroplet Formation And Amplification System

Digital PCR is an absolute quantitative technique for nucleic acid molecules.As the latest generation of PCR technology,the micro samples are diluted and dispensed in large numbers until each sample contains no more than one of nucleic acid molecules to be tested,and after that all the samples will take part in the PCR amplification reaction under the same conditions and are counted one by one,finally the absolute quantification of the initial sample nucleic acid molecules can be achieved.Digital PCR has a smaller reaction volume,faster reaction speed,lower system noise and higher sensitivity,which compare to conventional qPCR.This researched system can be used in medical diagnostics,individualized medical,food testing,genetically modified organisms,pathogen identification,Immunoassay,forensic science and so on.In this paper,we aimed at developing a set of digital PCR,focusing on the droplet generation and the amplification system:1?The research of digital PCR microdroplet generation system included the process simulation of droplets generation,droplets generation chip design and droplets generation experiment.We simulated the numerical process of droplet formation in the flowing micro-channels of the digital PCR and analyzed the effect of two phases flow velocity on the size and frequency of droplets formation.The results showed that(1)when the velocity of the discrete phase was fixed,the size of the droplet decreased and the frequency increased as the continuous phase flow rate increasing and when the continuous phase velocity was fixed,the droplet size increased with the increase of the discrete phase flow rate,the frequency increased.(2)The size and frequency of the droplets could meet the requirements of digital PCR droplets generation in the 100?m micro-channel.According to the simulated results,we designed the microdroplet generation chip,using the production method of PDMS molding.After that we applied the chip into the experiment.The resulting droplets' volume were 0.7nL,the generation frequency was 46 Hz,and the standard deviation of the droplet area was less than 5%,which met the system design requirements.2?Digital PCR amplification system included hardware circuit design,the underlying software development and Android PC software development.The hardware circuit design included the design of temperature sensing circuit and semiconductor drive circuit.The accuracy of the temperature sensing circuit detection was ±0.15?,the semiconductor drive circuit drive capacity was greater than 120 W.The results showed that the maximum heating rate of the sample cell was greater than 4?/s,the temperature overshoot was less than 0.3?,and the static deviation was less than 0.2? when the temperature was stable,which met the related national standards.Android software could receive real-time temperature data through the Bluetooth,which achieved the Internet connection of the equipment.When the temperature deviation occurred,there was alarm.