Studies On Integrated Digital PCR Microfluidic Devices For Nucleic Acids Purification,Anolification And Detection

In recent years, digital PCR (dPCR) methods on microfluidic chip have obtained considerable development. The dPCR technology with its high accuracy, sensitivity and absolute quantification also caused strong concern. The dPCR technology and microfluidic chip promoted each other and complemented each other, such as integrated fluidic circuit, slipchip, megapixel dPCR chip and a variety of droplet dPCR chips. However, the existing dPCR chips are with more lines or with additional complex equipment and time-consuming operation. Moreover, several commercialized dPCR instruments are expensive, and completely dependent on imports, which limits the popularization of digital PCR technology in the field of life science research in china In addition, the integrated microfluidic chip is a trend. The function components of sample processing, enrichment, separation, purification and the reaction are integrated into a chip by some way, so that they effect in synergistic, and strive to achieve the real sample-in/answer-out. In this thesis, the basis of the principle, development and application of digital PCR technology was summarized, and combined with the actual situation of the laboratory, the following innovative results were obtained. By exploiting PDMS permeability and taking advantage of the principle of filter, we design and develop a digital PCR chip based on the PDMS permeability. This chip includes a μfilter structure like filter and a thin PDMS film like filter membrane. The μfilter structure is composed of two parts, a sampling hole and exhaust hole on it, which is to achieve the pumping and loading. Thin film chip is clamped as the filter membrane in the filter, and there are 650 circular chambers with a diameter of 0.2 mm and 0.23 mm deep. When the syringe connected to μfilter was drawn, pressure of film chip side appeared lower than atmospheric pressure, then the sample enclosed air slowly went through the PDMS film into the syringe, and eventually took reagent into each chamber. The thin film chip can run digitalPCR reaction on a PCR equipment. The PDMS permeability is convenient for loading, but also caused the evaporation of water in thermal cycle of PCR, and influenced the reaction. The evaporation was inhibited by increasing rehydrate layer that larger than the thin film chip. The nucleic acid extraction component and PCR component are integrated by step. The reagent segments for nucleic acid extraction were stored in Teflon tube. When we draw the syringe in the other end, the reagent segments in Teflon tube pass the nucleic acid extraction region in single file. Magnetic particles in sample lysate are captured in the nucleic acid extraction region by a magnet; Washing reagent pass the region and wash the DNA on particles; PCR mix elute the purified DNA on partcles. Because of the negative pressure operation, sample lysis reagent and washing reagent can not enter into the reaction zone of PCR, so prevent the occurrence of pollution. We improve the loading method based on the permeability of PDMS, by placing a large area of gas drainage layer to realize negative pressure sampling in the microfluidic chip. When the syringe connected to the gas drainage layer was drawed, the air in the gas drainage layer was negative pressure, so the air in the chammbers go through the PDMS into the gas drainage layer, and took the reagent into each chammber. And then, the gas drainage layer were filled with water to prevent evaporation. At the same time, nucleic acid extraction components are integrated to digital PCR chip, and it realizes the nucleic acid extraction and digital PCR reaction on one chip.In summary, based on the permeability of PDMS, we have developed a kind of microdevice that can use the negative pressure by syringe to achieve sample loading and digital PCR reaction. The digital PCR microdevice is more convenient, easy operation and good practicality than the existing digital PCR platform. We also integrated the components of nucleic acid extraction and digital PCR, and realized DNA purification from beef meat and digital PCRdetection on the microdevice. Its characteristics are simple and rapid operation, less reagent consumption, good accuracy and suitable for general laboratory. And the microdevice can also be used to extract RNA and capture cells based on the magnetic bead, and it also provides a new method and instrument for more critical issues in the field of life science such as tumor diagnosis, single cell detection, capture and detection of rare cells.