Self-priming Compartmentalization Digital PCR Chip For Single Nucleic Acid And Protein Molecule Detection

Digital PCR provides unprecedented opportunities for absolute nucleic acid quantification by counting of single molecule, being known as the third generation PCR technology. The key to digital PCR is the partitioning of the diluted sample and reaction components into hundreds or thousands of reaction chambers so that researchers can get the absolute copies of DNA molecules. The measurement is accomplished by counting the number of positive partitions after endpoint PCR amplification. Advances in nanofabrication and microfluidics have now led to the rapid development of digital PCR, including digital PCR on chips and digital PCR in droplets. However, the reported chip for digital PCR, using integrated microvalves or micro-sized holes array, offers simple microchip device, but the chip needs mechanical connection to an external instrument. For example, many tubes for liquid transportation, syringe pumps for pressure-driven flow, and external air pressure for microvalves control. Although droplet digital PCR methods can perform a large number of droplet reactions, it requires a complicated workflow consisting of microdroplet generator, droplet transfer, microplate sealing and droplet readout system. These pieces of apparatus are often bulky and expensive. Therefore, the world-to-chip fluidic/electrical interconnections of digital PCR techniques are notoriously cumbersome. This limitation hinders maximum utilization of the benefits of microfluidic digital PCR platforms. By learning the integrated fluidic circuits (IFC) digital PCR, a novel valve-free and power-free microfluidic digital PCR device developed by making use of a novel self-priming compartmentalization and simple dehydration control to realize’divide and conquer’ for single DNA molecule detection is for the first time described in this thesis. The high gas solubility of PDMS is exploited to provide the built-in power of self-priming so that the sample and oil are sequentially sucked into the device to realize sample self-compartmentalization based on surface tension. The self-priming compartmentalization digital PCR chip includes two kinds of chip. One chip contains four separate panels, and each panel contains 1280 independent 5.625 nL chambers, which can be used to detect four samples simultaneously. Using this platform, digital loop mediated isothermal amplification (digital LAMP) and digital PCR have been performed to detect the human housekeeping genes, human lung cancer related genes to demonstrate the feasibility and flexibility of the microchip for single DNA/RNA molecule detection, and precision and measurement uncertainty of the device is evaluated by the Poisson distribution. In addition to the single DNA/RNA molecule detection, digital proximity ligation assay (digital PLA) has been developed for the first time to detect single EGFR protein molecule. Compared to the digital ELISA, this method is simpler and more accurate. Another megapixel chip has dimensions of 4 x 4 x 10 μm on an 8 μm pitch, corresponding to sub-pL chambers (160 fL) with a density of 1,562,500/cm2, and a scale of approximately 6,400,000 reactions per device have been made to detect the HBV copies, which provides a theoretical dynamic range of 7 logs. Therefore, This device without world-to-chip connections eliminates the bondage of the complex pipeline control, and is a novel, compact, lower cost and all-on-chip platform, which would be a significant improvement to digital PCR automation and more user-friendly. Taking all these advantages of all-on-chip into consideration, it is foreseeable that the SPC digital PCR chip would provide a valuable platform in life science fields including early diagnosis of cancer, non-invasive prenatal diagnosis, single cell analysis, single cell genomics sequencing and single protein molecule detection, etc.