Development Of Microfluidic Droplet Technique For Gene Quantification And Screening Of Protein-ligand Binding

In recent years,microfluidic chips with the advantages of low sample/reagent consumption,high throughput,miniaturization,integration and automation,have become the dominating platforms for gene quantification and high throughput screening of protein-ligand binding.Currently,various microfluidic chip-based systems have been rapidly developed and widely used in the fields of biology,medical science and pharmacy.In Chapter 1,the current development and application of microfluidic digital PCR are described.Various microfluidic digital PCR systems are summarized and classified according to the technical properties.We have also reviewed the current methodologies and applications of protein-ligand binding affinity analysis.In Chapter 2,we developed the surface-assisted multifactor fluid segmentation(SAMFS),an automated,fast,and flexible approach for generating a two-dimensional droplet array with tunable droplet volumes under an oil layer,on the basic of the combination of robotic liquid handling and surface-assisted droplet generation techniques.In the SAMFS approach,droplet volume is mainly determined by the pump flow rate and stage moving speed,hence the droplet volume could be flexibly and reliably changed by simply adjusting the pump flow rate and stage moving speed.The SAMFS approach was adopted to form an oil-covered multivolume droplet array required for multivolume digital PCR.And the droplet array system was applied in absolute quantification of plasmid DNA and measurement of HER2 expression levels in different breast cancer cell lines,with a dynamic range spanning 4 orders of magnitude.In Chapter 3,we developed a microfluidic droplet-based thermal shift assay(dTSA)system for label-free and high throughput screening of protein-ligand binding with low sample consumption.With the combination of robotic liquid handling and two-dimensional planar droplet array,the mixing operation of protein droplets with a massive of different compound droplets and formation of high-density droplet reactors on single chip could be realized automatically.The dTSA system could enable high throughput screening of 100 small molecule compounds in a single chip with femotole consumption of protein for each screening condition.