A Centrifugal-Driven Micro-droplets Technique For Nucleic Acid Detection In Single-Molecule And Single-Cell Level

Nucleic acid is the carrier of genetic information and plays an important role in human life.It can reveal the mechanism of chemical reaction in physiological processes by analyzing nucleic acid at the single-molecule level.On the other hand,the rearch of nucleic acid at the single-cell level can distinguish heterogeneity among cells,which has a great influence on cellular function.Digital PCR has realized the absolute quantitative detection of nucleic acid with higher sensitivity,accuracy,specificity and reproducibility,and contributed to the development of nucleic acid detection at the single molecule level.Recently,scientists have accomplished single-cell whole-genome amplification with higher coverage and lower bias,error rates and environmental DNA contamination by a method of emulsion whole-genome amplification(eWGA).It has been successful in accurate detection of gene copy number variations and single nucleotide variations by combining eWGA and next-generation sequencing.The technology of micro-droplets can provide thousands of uniform droplets with small volume for the application of digital PCR and dMDA.It will take long time to generation droplets with traditional methods,at the same time,there is a waste of sample during droplets generation and the operation is complex.In this work,we have developed a centrifugal-driven droplet technology based a microfluidic chip.It can generate thousands of uniformly stable droplets in one minute without waste of sample by centrifugal.The diameter of droplets is dependent on the channel width and depth.We successfully achieved absolute quantitative detection of nucleic acid at single-molecule level on this chip using digital PCR,and the result is consistent with real-time quantitative PCR,which is the gold standard method of nulcleic acid detection.On the other hand,we realized single glioma stem cell whole-genome amplification by the method of eWGA on the chip.We found it can decresing amplification bias and environmental DNA contamination by searching the fluorescence kinetics of droplets.We also proved that eWGA can achive higher genome coverage and lower bias by analyzing sing-cell whole-genome sequencing data.The centrifugal-driven droplet technology is expected to further research and application in physical,chemical and biomedical.