The application of microfluidic devices and multifunctional fibers in cancer diagnostics

Sammanfattning: Efficient separation and detection of rare cells in a mixed population is important in many biomedical applications. For instance, isolating and detecting circulating tumor cells (CTCs) from whole blood samples could allow for early cancer diagnosis and prognosis during treatment. CTCs are rare cells circulating in blood detached from the primary tumor site, carrying important information such as the origin of cancer and metastatic information. The detection of CTC from blood samples, besides being a minimally invasive procedure, could be vital in case of difficulty to access the tumor site via traditional biopsies, such as colon and pancreatic cancer. Microfluidics is a research field with great promise towards the development of methods to isolate and separate cells for clinical applications. Microfluidic based cell separation has been demonstrated using biological approaches using cell surface markers, and biophysical approaches using cell size, shape, and deformability. This thesis will focus on developing passive strategy using inertial microfluidics (biophysical, paper 1-4) and affinity biomarker (biochemical, paper 5) based strategy to isolate and analyze CTCs. Inertial microfluidics relies on inherent hydrodynamic forces, inertial forces, in flow through the microfluidic channel. Depending on the geometry of the channel, inertial forces drive the particles and cells to a specific streamline position, allowing for focusing and separation. In contrast, affinity-based isolation relies on biomarkers expressed on the surface of the targeted cells, which is highly specific. In paper 1, using the elasto inertial microfluidic technique, high throughput particle focusing and separation was achieved in a curved rectangular channel with a separation efficiency of 89% for 10 μm and 99% for the 15 μm particles at a high volumetric flow rate (1 mL/min). In paper 2, a detailed analysis of particle focusing was studied experimentally and numerically in a circular cross-section. Using the FENE-P model simulating non-Newtonian fluid and an immersed boundary method to account for the particles, it was observed that a combination of inertia and elasticity leads to several intermediate focusing positions. In paper 3, we developed a portable microflow cytometer using fiberoptics capillaries. By combining elasto inertial microfluidics and optical fibers, we focused particles and cells and demonstrated particle counting at a throughput of 2500 particles/second. In paper 4, we built an all-fiber separation and detection component and demonstrated a separation efficiency of 100% for the 10 μm and 97% for the 1 μm particles as a proof of principle. In addition, the separated 10 μm particles could beiiiquantified in the all-fiber component. In paper 5, an affinity-based separation approach was carried out to utilize the surface markers to capture and release viable CTCs for downstream analysis. A novel layer-by-layer nanofilm coating strategy was developed using cellulose nanofibril (CNF) built into multiple layers and functionalized with antibodies to capture the cells. After capture, the CNF were enzymatically degraded to release the CTCs. HCT116 colon cancer cells were captured with an efficiency of more than 97%, and when spiked in whole blood, an approximately 200 fold average enrichment was achieved compared to white blood cells. 80% of the cancer cells spiked in whole blood were recovered with 97% viability in less than 30 minutes.In summary, this thesis presents different microfluidics-based separation of cancer cells based on biophysical and biochemical properties. Using elasto inertial microfluidics, we developed several approaches to separate and detect cells and particles. Using layer-by-layer coating of CNF, we successfully demonstrated capture and release of cancer cells with maintained high viability. While the thesis has focused on different properties of cells for separation and analysis, combining these methods will be important for efficient isolation and characterization of CTCs for improved diagnostics.

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