Simultaneous Detection Of Dual Tumor-related MiRNAs By Pneumatic-assisted Microfluidic Droplet System

Cancers have a high mortality rate and threat human’s health seriously.Because the early symptoms of many kinds of cancers are not obvious,the treatments for many patients were usually delayed.However,if early diagnosis could be achieved,it would be easier to save patients’ lives.MiRNAs could be used as promising biomarkers for early diagnosis of cancers.In this paper,a pneumatic-assisted microfluidic droplet system was built.For proof of concept,two kinds of tumor biomarkers,miRNA-21(miR-21)and miRNA-20a(miR-20a)were selected as model targets to develop a method for simultaneous online detection of miRNAs.In chapter one,the methods for droplets generation was summarized,and the ways for passive and active generation of droplets were introduced and compared.Next,the application of droplet-based microfluidic systems,the QD-FRET system and its application were described.Then,the origin of miRNAs,its significance in disease diagnosis and its research progress was depicted.Finally,the purpose and design of this paper was proposed.In chapter two,the pneumatic-assisted microfluidic droplet system was built up.Top layer and middle layer chips were fabricated using soft lithography technique at first.By designing proper channel structures and optimizing spinning speeds of photoresist and PDMS,channels with appropriate structures and depth were fabricated.Thus pneumatic control of disperse phase cold be achieved after bonding of three layers.Then the pneumatic-assisted microfluidic droplet chip was integrated with the lab-made fluorescence detection platform,and the pneumatic-assisted microfluidic droplet system was built finally.In chapter three,miR-21 and miR-20a were detected one by one using the detection platform firstly.And then the simultaneous detection of miR-21 and miR-20a was executed.The linear range and quenching rate in simultaneous detection were similar with the ones in single detection.Thereafter,the selectivity of the detection method was investigated by introducing disturbing miRNAs,and the experimental results showed that the detection system has good selectivity to the target miRNA under the same concentration of target and disturbing miRNA.The linear range for miR-21 and miR-20a detection is 1.00×10-1-1.00×102 pmol/L and 1.00×10-1-1.00×103 pmol/L.The LOD for miR-21 and miR-20a are 40 fmol/L and 50 fmol/L.In chapter four,the working process and experimental results of the simultaneous detection platform for miR-21 and miR-20a were concluded.And the possibility of the proposed platform for more other targets detection at the same time in the future was prospected.