Agarose-Based Droplet Microfluidics Research And Application

Droplet-based microfluidics utilizes the shearing force between two immiscible phases (aqueous phase and oil phase) to generate uniform-size, picolitre-volume droplets in microchannel with frequency as high as thousands of herz. As the droplets are separated by oil phase, each of them can be treated as an independent reactor, thus remarkably increasing the total reaction throughput. Droplet-based microfluidics is promising in fields such as gene screening, disease detection, directed enzyme evolution, drug screening, material synthesis and micro-structure fabrication.However, deficiencies still exist in droplet-based microfluidics. First of all, common aqueous droplets tend to coalesce, which makes long-time reservation and follow-up treatments difficult to achieve. Second, it is hard to add or remove a specific composition of the droplet reactor. Compared to common aqueous phase, agarose solution exhibits a temperature-sensitive’sol-to-gel’ characteristic. So, on the one hand, large molecules can be encapsulated inside the agarose gel droplets by simply cooling, which brings great convenience for sample reservation and subsequent analyses, on the other hand, high-throughput in-situ micro-structure fabrication can be achieved inside microfluidic channels with the change of temperature. In addition, agarose gel has a porous structure, small molecules can move across the droplets through osmosis, which simplified experimental operation and chip design. According to the above background, basic principles of agarose-based droplet microfluidics are firstly discussed. Then, an in-situ synthesizable one-way agarose ball valve structure and a micro-pump system are innovatively proposed based on these principles, achieving fluids flowing regulation and reagents introduction respectively. At last, the feasibility of PCR amplification inside agarose droplets is proved in this paper. The main contents of this work include the following aspects:1. Develop a rapid in-lab PDMS microfluidic chip fabricating method.’Soft lithography’is used to fabricate SU-8 male die, female die of the chip is acquired through ’molding’, then irreversible bonding is achieved by’ two-step solidification’ method, microelectrodes can be introduced with’soft lithography-magnetron sputtering’. Compared with conventional MEMS processing technology, this method has lower equipment requirement and is easier to handle, which reduces cost and time consume. This chip fabricating method provides physical basis for our agarose-based droplet microfluidics research.2. Basic principles of droplets generation is discussed. High-throughput agarose droplets generation is achieved in chip using flow-focusing structure. In order to quantitatively analyze the uniformity of droplets, a program which can automatically detect the edge and further calculate the diameters of droplets is designed in MATLAB. According to the quantitative analysis, agarose droplets produced in this paper are highly uniform, with polydispersity less than 5%. The generating frequency of the droplets is stable, with frequency uniformity of 84%. Agarose droplets are collected outside the chip and then reserved in 4℃ fridge. After 48h, the droplets keep intact and uniform, which demonstrate the feasibility of long-time reservation of the agarose droplets.3. Taking advantage of the ’sol-gel’ transferring property, an in-situ one-way agarose ball valve fabricating method is proposed innovatively, and fluid flowing is successfully regulated inside the chip. Based on this ball valve structure, an agarose ball micro-pump system is further designed and reagents introduction is achieved. Compared with other micro-pump systems, our system is easier to fabricate and can be synthesized in situ, thus remarkably reducing the complexity and cost of pump manufacture. Furthermore, the actuating module of our pump is fixed outside the chip, which makes it repairable and replaceable, therefore extends its lifespan.4. Agarose-based droplet microfluidic PCR amplification is achieved. Based on the principle of droplets generation, DNA samples and PCR reagents are encapsulated into agarose droplets. These droplets are collected outside the chip, and PCR is subsequently conducted. At last, amplicons are obtained, which demonstrates the feasibility of high-throughput reaction in agarose droplet reactors.