Hybrid microfluidic devices based on polymeric materials functionalized for cell biology applications

The present thesis work deals with the development of a novel manufacturing protocol for the realization of excimer laser micro-patterned freestanding hydrogel layers (50 to 300 μm thickness) based on thermo-responsive poly-(N-isopropyl)acrylamide (PNIPAAm) which can operate as temperature-triggered actuators for cells-on-chip applications. PNIPAAm based thin films were synthesized in house and manufactured by an injection/compression moulding based technique in order to obtain flat hydrogels attached to rigid polyvinyl chloride (PVC) substrates to facilitate laser focusing. Laser machining parameters were empirically optimized to fabricate arrays of through-holes with entrance diameter ranging from 30 μm to 150 μm and having different exit diameter (from 10 to 20 μm) on the PNIPAAm employing a stencil aluminum mask. After laser processing, the microstructured layers were detached from the PVC using a chemical treatment and then left to swoll in pure water. The KrF excimer laser machined through-holes could be reversibly modulated in terms of size as a consequence of the polymer volumetric phase transition induced by a temperature change above the critical value of 32 °C. Thermo-responsiveness characterization was carried out on the detached water swollen freestanding layers using a thermostat bath, by changing the temperature from 18 °C to 39 °C and each sample could undergo multiple cycles. As a result of the polymer water loss, the shrinkage of the layer caused the holes to shrink homogeneously, thus reducing their original size of about the 50% in the polymer collapsed state. To prove the functionality of these stimuli-responsive smart surfaces in the frame of cells-on-chip systems, they were integrated in a multilayer microfluidic device to operate as self-regulating cell sorting actuators for single cell assays applications. Using mechanical fastening as the packaging strategy, the hydrated hydrogel was sealed between two micro-milled poly-methyl methacrylate (PMMA) components, which provided the fluid accesses and ducts to the cell suspension to be flown over the thermoresponsive actuator (top layer) and the well to collect the sorted sample (bottom layer). The device is also equipped with a thin transparent heater to control the microfluidic chip temperature. When the system is assembled, the temperature-triggered actuation mechanism was exploited to trap a cellular sample in the shrunken exit hole on the top of the hydrogel layer by applying a negative pressure across the film via the bottom PMMA component when the system is kept at 37 °C. Subsequently, the sorting of the trapped cell took place through the micro-capillary when the polymer natural relaxation at room temperature towards its initial state occurred; the operational principle of the device was proved using MG63 cells as a model cell line by monitoring the sorting through the size-modulating structures using optical microscopy.