A bio-functionalized microsystem was developed for the capture and release of cancer cells from whole blood. Effective isolation and purification of circulating tumor cells from whole blood provides important capability for clinical application and biological research. Here, we demonstrate a single step surface modification procedure for a microfluidic device based on self-assembly of recombinant spider silk harbouring an affinity domain for antibody binding. The surfaces of microfluidic devices were conjugated/equipped with anti-EpCAM antibody for selective isolation of pancreatic cancer cells from spiked whole blood and finally circulating tumor cells from pancreatic cancer patients. Moreover, a protease-cleavage site in the recombinant spider silk proteins provides the unique option to release the captured cancer cells on command from the device without compromising the cell’s viability. Our approach offers a simple, easy and robust surface modification process with a 85% cancer cell capture efficiency. Subsequent addition of a site-specific protease results in the release of 95% of captured cells from the bio functionalized microfluidic systems.
Detection of circulating tumor cells (CTC) in peripheral blood is indicative of early recognition of tumor progression and such an important biomarker for early diagnosis, staging, monitoring and prognosis of cancer. However, CTC are found in very low concentrations and reliable isolation of these rare cells is challenging. Microfluidics enables precise manipulation of fluids and cells and is ideal for cell sorting methods for clinical diagnostics. The thesis contributes towards the development of microfluidic based CTC isolation methods from peripheral blood. The methods are based on size and immunoaffinity. The first part of the thesis describes the phenomenon of inertial focusing for size based cell separation at high throughputs. In paper 1, we demonstrate continuous filtration of leukocytes from diluted blood, with an efficiency of 78% at a flow rate of 2.2ml/min. In the paper 2, separation of total and subpopulation of leukocytes with a purity of 86% for granulocytes and 91% for lymphocytes is demonstrated. Furthermore, cancer cells spiked into whole blood could be separated at a yield of 88%. Finally, in paper 3 and 4 we unravel parts of the unexplored elasto-inertial microfluidics and was utilized to precisely focus the cells, as part of an integrated optofluidic micro flow cytometer device, capable to simultaneously measure fluorescence and scattering of cells and particles at a rate of 2500 particles/sec (paper 4). Second part of the thesis focuses on acoustophoresis. In (paper 5), a multifunctional acoustic microdevice was developed for isolation of cancer cells from red blood cells with a separation efficiency of 92.4% and trapping efficiency of 93%. In (paper 6), microbubbles activated acoustic cell sorter was developed for affinity based cell separation. As a proof of principle, cancer cells in a suspension were separated at an efficiency of 75%. In the third part, using cellulose nano fibrils (paper 7), we demonstrate efficiently capture and release of cancer cells at a release efficiency of 95%. Finally, a novel, single step self-assembly of spider silk proteins is introduced inside microfluidic channels for effective capture of cancer cells with 85% capture efficiency and subsequent release of captured cells with 95% release efficiency (paper 8). The novel recombinant silk modified microfluidic device was validated using pancreatic cancer patients. In summary, we have developed different microfluidic based isolation technologies for the capture and characterization of CTC.