Microfluidics for single cell analysis

Abstract

Isolation and manipulation of single cells have gained an increasing interest from researchers because of the heterogeneity of cells from the same cell culture. Single cell analysis can ensure a better understanding of differences between individual cells and potentially solve a variety of clinical problems. In this thesis lab on a chip systems for rare single cell analysis are investigated. The focus was to develop a commercial, disposable device for circulating tumour cell (CTC) analysis. Such a device must be able to separate rare cells from blood samples and subsequently capture the specific cells, and simultaneously be fabricated and operated at low costs and be user-friendly. These challenges were addressed through development of two microfluidic devices, one for rare cell isolation based on pinched flow fractionation (PFF) and one for single cell capture based on hydrodynamic trapping. Both devices were fabricated by injection moulding with a nickel master. CTC isolation was realised using PFF, which is a passive, size-based microfluidic technique. The focus was mainly on experimental work; however designs were based on flow calculations and analysed with numerical simulations to support experimental results. Devices were extensively characterised and tested with uorescent nano- and microspheres, and with cancer cells and blood cell samples. It was demonstrated that the separation not only relies on size, but that differences in cell deformability are also exploited, which enabled a successful separation with an efficiency of over 90%. Single cell capture was realised using hydrodynamic cell trapping, which is based on flow and cell interactions with microstructures. The criteria for hydrodynamic single cell capture were investigated and clarified through development of several devices with increasingly optimized designs. The final design provides the possibility of parallel single cell DNA extraction for subsequent off-chip investigations. Because the devices are sensitive to small changes of the structures, the injection moulding process was optimized to improve replication of the structures from the nickel master. A novel method based on freeze-fracture was used to investigate and improve the bonding process used for sealing device microchannels. Structures were intentionally altered by bonding at high temperatures, and the resulting channel cross sections were visualized in a scanning electron microscope. It was demonstrated that chips with the altered structures had an increased capture efficiency. Finally low cost mass-production of the devices was realised using injection moulding in thermoplastics from a nickel master. With this process the price per device rapidly decreases for higher numbers of fabricated devices. In addition devices were fabricated on a Luer-platform that ensures easy connection to external equipment. The devices were used by collaborators in a cancer research lab, which demonstrates their commercial potential.