Research On Microfluidic System For Molecular Point-of-Care Testing (POCT)

POCT is a rapidly developing laboratory medicine discipline in terms of analytical and clinical applications.POCT is mainly characterized by proximity to the patient,quantitative or semi-quantitative single measurements,short turnaround time,no sample preparation,no pipetting,use of pre-made reagents,user-friendly dedicated analytical instruments and instant,resultdeduced therapeutic action.The function of"sample in-result out" can be realized,and the experimental error caused by operation can be avoided.In this study,we developed a set of methods for processing complex samples on POCT devices.(1)Firstly,based on the principle of DNA splicing,the primers are designed to be in the exon,across the exon-exon junction,or in the intron to control the single amplification of DNA/RNA or the simultaneous but different length amplification.Using this method,a set of gradient primers with an amplification length of 200 bp to 400 bp were designed for DNA and RNA respectively,and a set of FFPE nucleic acid evaluation system was constructed jointly by combining the nucleic acid absorbance determination by ultraviolet spectrophotometry and nucleic acid fragmentation detection by agarose gel electrophoresis.(2)Nucleic acid extraction from rat FFPE tissues was performed by using the kits of three representative companies with different nucleic acid extraction methods,namely Tiangen,Biobase and Covaris.After the evaluation and comparison of the extraction effects of different commercial kits,we found that ultrasound had a great advantage in POCT used to process FFPE samples.The self-made ultrasonic probe was used in combination with the extraction reagent of Covaris,and the extraction of nucleic acid with high quality was successfully achieved.(3)The PC microfluidic chip was made by the process of fine engraving and hot pressing.The channel was closed by high temperature bonding of PC membrane,and PCR reagent was added into the chip.The temperature was controlled by PID,and the reciprocating movement of liquid was controlled by the extrusion cap of stepping motor,so as to realize automatic rapid amplification of 200-500 bp Lambda DNA fragment.(4)Using the principle of three-state equilibrium of water,the PCR reaction solution was lyophilized by adding appropriate proportion of trehalose,PEG and BSA as lyophilized protectant,and the water content in the PCR reaction solution was rapidly reduced through the three stages of pre-freezing,sublimation and desorption,so as to achieve the purpose of getting rid of cold chain transportation.The development of this method for handling complex samples on POCT devices opens a new way of thinking for handling complex samples on POCT devices,and enriches the theoretical basis for the subsequent commercialization of more POCT devices.