Hydrodynamic Gating And Its Applications For Bioanalysis On Microfluidic Chips

In recent years, lab-on-a-chip plays an important role in revolutionizing the methodologies for biological researches due to its advantages of miniaturization, integration and automation. Sample introduction is an indispensable standard operation for lab-on-a-chip, which has a great impact on the performance of subsequent analytical operations. Although a variety of researches have been made on sample introduction, most of them are lack of universality in sample category and flexibility in injection mode.In this thesis, we proposed hydrodynamic gating, a simple and flexible flow control strategy for lab-on-a-chip based sample introduction and analysis. It contains two flow states, namely gating state and injection state. In the gating state, buffer solution acts as a virtual gate that prevents the sample solution from leaking into the analysis channel. In the injection state, the virtual gate is open to let the sample enter the analysis channel. The spatio-temporal profile of sample plug can be well controlled in the analysis channel by switching virtual gate in a proper mode and timing. This strategy not only possesses the advantages of gated injection such as flexible flow control and controllable injection volume, but also holds the advantages of hydrodynamic driving such as strong driving force and good biocompatibility, which makes it ideal for applications involving molecules, cells and small organisms in microfluidic systems. We developed three types of sample injection modes, namely pinched mode, full-channel mode and adjustable-interface mode. Theoretical models were established for these modes, and were further validated by numerical simulations and flow visualization experiments. We constructed an experimental platform of hydrodynamic gating, and explored the use of this platform for cell sorting, cell stimulation, droplet purification and liquid chromatographic separation.In the cell sorting, virtual gate was open and close based on fluorescence detection for selectively injecting the target cells into the analysis channel. We first established a fluorescence-activated cell sorter by the pinched mode, and separated the mixture of HeLa cells and the mixture of nematode eggs separately. The result indicated a recovery rate and a purity of both more than 90%, as well as a good biological viability. Furthermore, we proposed an improved cell sorter using the adjustable-interface mode, which has potential to improve purity, throughput and cell viability.In the cell stimulation, we established a chemical signal generator by the pinched mode for chemical stimulation of a single cell located in the analysis channel. System evaluation revealed that the rise time and the fall time of the solution exchange were 19.8±0.8 ms and 20±1 ms respectively. As a proof of concept, adenosine-triphosphate stimulation of HeLa cells was demonstrated by investigating P2Y-IP3 signaling pathway mediated calcium release. Furthermore, preliminary studies have been made on the full-channel mode and the adjustable-interface mode for cell stimulation. The full-channel mode has potential for chemical stimulation of multiple individual cells in the analysis channel simultaneously. The adjustable-interface mode enables cell stimulation in a spatio-temporally controllable manner with a temporal resolution of less than 43 ms and a spatial resolution of 3.4±0.2μm, which can be used to investigate sub-cellular cell dynamics or cell communication.In the droplet purification, we integrated droplet generation and sorting on a single chip for a fluorescence-activated droplet sorter. Droplet generation was achieved with a throughput of 18 droplets/s; droplet sorting was realized by the adjustable-interface mode with both purity and recovery rate of more than 99%. Moreover, we proposed hydrodynamic gating based on an aqueous-oil microfluidic system for selective encapsulation microbeads into picoliter-scale droplets. The result indicated a purity of 92% and a recovery rate of 96%.A tentative research on open-tubular liquid chromatographic separation was also conducted by the pinched mode, and three kinds of fluorescent dyes were resolved using native poly(dimethylsiloxane) as a soild phase and acetonitrile as a mobile phase.In summary, hydrodynamic gating were validated as a versatile and universal strategy for microfluidic sample introduction, and it was expected to be a powerful platform technology for bioanalytical applications of lab-on-a-chip.