Liquid Marble Based Digital Microfluidics

Abstract

Sample handling with liquid marbles is a recent advancement in digital microfluidics. Liquid marbles are liquid droplets coated with fine hydrophobic/oleophobic particles. The external particle coating isolates the liquid droplet from the surrounding ensuring a contamination-free environment for the droplet. Mobility manipulation of the liquid marble is easier and well developed compared to the droplet counterpart. These advantages have promoted the usage of liquid marbles as a microreactor for a range of biological applications. Liquid marbles may help in reducing the non-reusable contaminated plastic waste generated in conventional biochemical reaction chambers such as plastic vials and microfluidic chips. However, the use of liquid marble as a microreactor is limited to room-temperature applications as they are susceptible to the problems of evaporation at elevated temperatures. The development of a liquid marble based digital platform for biochemical applications at elevated temperature would broaden the scope of liquid marbles, reducing the generation of contaminated plastic waste. Polymerase Chain Reaction (PCR), a DNA amplification technique is a high-temperature process, which finds application in medical diagnosis, agriculture and forensics. Millions of PCRs are carried out in plastic vials and conventional microfluidic chips annually, contributing to the increasing problem of plastic waste. So developing a liquid marble based digital microfluidic platform tailored for PCR has an immense significance. This Ph.D. thesis focusses on the development of a liquid marble-based digital microfluidic platform particularly for carrying out PCR. A basic PCR process consists of three different phases namely sample dispersion, thermal cycling, and output monitoring. Sample dispersion process should be precise and contamination-free. Manual intervention in sample dispersion should be minimised to avoid the contamination of the sample and to achieve the precise volume. We designed, developed and tested an automated on-demand liquid marble generator. The instrument demonstrated a high precision over the volume of the marble and was highly repeatable. Evaporation of the sample liquid is a major problem faced by liquid marbles operating at elevated temperature. Detailed knowledge of the evaporation dynamics of the liquid marble is essential for designing and developing a liquid-marble-based platform for PCR. We carried out experiments to study the evaporation dynamics of liquid marbles at elevated temperatures. Studies revealed that the evaporation of liquid marble also obeys the “d-square law” at higher temperature. The lifetime of the liquid marbles is a function of its volume, temperature, the relative humidity of the surrounding environment, the number of liquid marbles and the distribution of liquid marbles. We demonstrated that the lifetime of a liquid marble can be increased by providing a vapour saturated ambient. Next, we designed an experiment to demonstrate the use of a liquid marble as a microreactor for PCR. With the knowledge acquired from the previous study, a humidity-controlled environment was developed. Custom-built thermal cycler was integrated with the humidity-controlled chamber. Thermal cycling of the PCR mixture inside the liquid marble was carried out. Successful DNA amplification was observed, and the results were comparable with those obtained from the PCR in a conventional commercial machine. However, the sample volume used in this experiment was high and the marble was able to undergo only 9 thermal cycles. Furthermore, we tested the synthesis of core-shell beads using composite liquid marble technology. A volume of 2 μL PCR sample was used as a core liquid where a photopolymer was used as the shell material. We successfully achieved the synthesis of core-shell beads. Thirty thermal cycles were carried out without evaporation. Successful DNA amplification was verified. We achieved twenty-five times reduction in sample volume and 86.1% reduction in plastic waste. The present thesis explains in details the various stages of developing a liquid marble based digital microfluidic platform for PCR. A little research on characterisation and optimisation of the methods proposed in this thesis might result in a commercially viable liquid marble-based platform for PCR.