Research On Key Technologies Of Nucleic Acid Detection System Based On Microfludic For Point-of-care Testing

As people living standard unceasing enhancement,people urgently need to detect diseases quickly and precisely.Nucleic acid detection has become the“gold standard”for the detection of pathogens due to its advantage of good specificity and high sensitivity.However,the existing detection methods have problems of complicated operation,many instruments are required and they are not easy to carry.The on-site nucleic acid detection system based on microfluidics can effectively compensate for the shortcomings of existing nucleic acid detection methods due to its integration,rapid,automation and portable features.The system is mainly composed of a microfluidic chip and a detecting device which involves a number of key technologies.Although,the corresponding products have been commercialized in the abroad,their manufacturing and assemble processes are complicated,the low yield and the cost of single detection is expensive,they are not suitable for the needs of on-site detection of nucleic acid in China,and they cannot be applied in the clinical massively.Therefore,it is urgent to develop an on-site nucleic acid detection system independently and studies the key technologies involved.This paper aims to develop an on-site nucleic acid detection system based on microfluidics.Three key technologies are studied in this paper:the hot pressing bonding of microfluidic chips,ultrasonic lysis method for nucleic acid extraction,temperature control for nucleic acid amplification.The applicability and feasibility of the aforementioned methods were verified by built the corresponding platforms.The main content were as follow:(1)A hot pressing machine for hot pressing bonding of microfluidic chips was developed.A special fixing structure for chip was designed in the hot pressing machine.In the hot pressing machine,temperature can be adjusted from 0 to 250? and pressure can be adjusted from 0 to 150Kg,the temperature error was less than 1?.Taking polycarbonate(PC)as the material,a microfluidic chip was designed independently.Through orthogonal experiment,a set of hot pressing bonding process with film as cover-chip was developed,under the condition of thickness of film less than 0.175mm,used hot pressing temperature:160-17?,hot pressing pressure:80-100kg,hot pressing time:60?90s can achieve good bonding effect.(2)Using ultrasonic lysis as a method of nucleic acid extraction,in order to transmit ultrasonic energy with optimal efficiency,a spherical vibration transmission structure model of chip was established,in which the spherical curvature radius R=16.25mm,spherical width A=4mm,spherical thickness T?1 mm,through calculating the top six modal of the vibration transmission structure,the 40kHz ultrasonic frequency was analyzed which can match inherent frequency of the structure.A nucleic acid extraction experimental platform with ultrasonic lysis based on microfluidics was built,and the ultrasonic power and time were explored.Under the condition of 20W ultrasonic power,the EV71 virus could be lysed in only 10s,the rate was better tharn the mature mechanical oscillation method which verified the practicability of vibration transmission model of the chip.(3)For the nucleic acid amplication process,In order to reduce the the volume of device and the delay of temperature change between the sample in chip and reaction tank,the thermoelectric cooler was used as the temperature source and the copper was used as the material of the reaction tank,the double-sided symmetric heating structure is designed which size was 90mmx80mmx 120mm.The delay of temperature between the sample in chip and the reaction tank was less than 1 s,and the rate of heat and cooling were 2.35 ?/s and 2.72 ?/s,respectively.Futhermore,the nucleic acid amplification experimental platform is built.The tobacco mosaic virus was subjected to nucleic acid amplification experiments,which verified that the system has good heat transfer performance and can achieve stable amplification of nucleic acids.The tobacco mosaic virus was subjected to nucleic acid amplification experiments,which verified that the system has good heat transfer performance and can achieve stable amplification of nucleic acids.