The Research Of Microfluidic Chip For Single Cell Analysis And Its Application In Isolation And Identification Of Cancer Stem Cells

Cell is the basic unit of life. Traditional cytology studies rely on a large number of cells (103-104), and regard the average value of cell group as the results. However, it was found that the cells even in the same state of group also existed heterogeneity in the phenotype and genotype. Heterogeneity exists between cells, and group analysis will cover the differences between individual cells. Therefore, it is imperative to develop an effective technique for analysis of single cell components. Microfluidic technique can integrate of cell sampling, single cell capture, cultivation, separation, lysis and analysis together, and have the advantages of high-throughput, miniaturization, parallel, automation, portability and low cost, provides a technology platform for the research of single cell.This paper developed a microfluidic chip with the independent intellectual property rights. Microfluidic chip was made of the poly dimethyl siloxane s (poly (dimethylsiloxane), PDMS), realized negative pressure self suction automatic sampling using PDMS polymer’s good gas solubility characteristics. Thus, the chip was easy to be used like clinical vacuum blood collection, no need of auxiliary equipment control flow path. Thousands of cells were passed into the chip and uniformly separated into each reaction chamber, no longer interfered with each other and were not easy to leak. Single cell in the reaction chamber directly performed RT-PCR reaction, saving time. Each reaction chamber volume was 5-10 nL, a chip with 1000 reaction chambers only needed slightly ten microliters of reagents, saving reagent.This study has made the following innovative results:(1) successfully developed a microfluidic chip which can integrates single-cell capture, lysis, RT-PCR reaction on one microfluidic chip, and can used for digital PCR study of single cell. Verified the existence of heterogeneity between different cells through the study of gene expression of cancer stem cell surface markers CD 133 and housekeeping gene GAPDH in single A549 cancer stem cell. (2) The developed single cells microfluidic chip realized a rapid method for identification of tumor stem cells with cancer stem cell surface markers CD 133.In this paper, the development of microfluidic chip is easy to operation, sensitive (single cell), accuracy (countable), and saving reagent (a few microliters/chip), suitable for any laboratory study the identification of tumor stem cells or other rare cells. The project provides a new tool of single cell isolation and identification in cancer stem cell research. It can be used for identification the molecular genetic characteristics and specific markers of tumor stem cells. Provide scientific basis for oncology research, and lay the foundation for The clinical diagnosis, disease monitoring and prediction, treatment etc.