Simultaneous Detection Of Multiple Nucleic Acids Using Fluorescence Resonance Energy Transfer Based On Microfluidic Droplets

MicroRNA(miRNA),a kind of endogenous non-encoding RNA,will directly cause cancer and other diseases because of its unusual expression in the body.Therefore,the detection of miRNA has important significance for the prediction,diagnosis and treatment of some diseases.Compared with other detection methods,fluorescence resonance energy transfer technology combined with quantum dots as fluorescent chromophores has a unique advantage in the detection of miRNA.It not only can be used as nano scale to measure the length of short chain nucleic acid,but also the change of its fluorescence intensity can be used as a quantitative basis for the detection of nucleic acids.The microfluidic droplets,as the analysis carriers,provide a multiplexed detection strategy.The microfluidic droplet systems ensure the analytical sensitivity and reproducibility because the droplets feature controllable size,adjustable generating speed and isolated system.In this paper,we built a microfluidic droplet system for multicolor fluorescence detection.By simultaneously detecting four kinds of quantum dots with different fluorescence emission wavelengths,the performance of the system was optimized.Based on the fluorescence resonance energy transfer technique,the detection system was used to respectively detect two kinds of short chain DNA and two kinds of miRNA associated with colon cancer.In chapter one,we firstly introduced the significance of miRNA assayand its conventional detection methods.Secondly,we reviewed application of the fluorescence resonance energy transfer technique in the detection of miRNA and other types of nucleic acids.And then,we introduced the application of microfluidic droplets in the detection of nucleic acids.Finally,we put forward the purpose and the design of this work.In the second chapter,a laser-induced fluorescence-based microfluidic droplet multicolor detection system was built.Four kinds of fluorescence quantum dots were detected simultaneously by optimizing the chip configuration and the optical path.The detection limits of 1.8×10-9-1.2x10-10 mol/L were achieved.In the third chapter,the quenching systems based on the fluorescence resonance energy transfer were developed by using short chain DNA or miRNA as a bridge to quench the fluorescence of the quantum dots.Thereafter,we used the above device to realize the quantitative detections of DNA and miRNA were realized by using the microfluidic droplet-based multicolor detection system.When the concentration of miRNA was in the range of 2×10-6-9×10-6 mol/L,the relationship between the quenching efficiency and the concentration fit with the quadratic function well.In the fourth chapter,we summarized the simultaneous detection of nucleic acids by using the microfluidic droplet-based multi-color fluorescence detection system,and forecasted its applications in biological analysis.