Microsystem with Alternating Thrombogenic and Non-Thrombogenic Regions: In Vivo Support System for Brain Imaging in Live Mice

Platelet activation and adhesion involve both positive and negative feedback control. The interaction between these two control mechanisms is not well understood, however, platelets are known to release both activators (e.g. ADP, thromboxane) and inhibitors (e.g. nitric oxide and protein S). To investigate the interaction between positive and negative feedback, we require patterned surfaces in which regions of activating proteins are next to regions with negligible activating properties. The creation of this type of surface has proven to be difficult. The main problems to be overcome are non-specific absorption of proteins onto the non-thrombotic region and rinsing away of the thrombotic proteins, particularly during the step in which platelets are stained with fluorescent dye. The thrombotic regions become diffuse and diluted, with indistinct edges. A 25 μm thick PDMS is spin coated over the silane glass slide. The slide is then masked with double-sided Kapton tape and exposed to a photo-initiator solution. The surface of the slide is then exposed to ultraviolet light and washed to remove retained benzophenone. FITC-labeled fibrinogen was dropped directly on to the masked slide, allowed to incubate for 20 minutes, rinsed and dried. Slides were imaged with a florescence microscope. Recalcified blood was dropped onto the slides. The result was fixed, permeablized, stained, and imaged. A clear region of fibrinogen was present on the region masked (from photo-initiator and ultraviolet) with the tape. Platelets adhered more strongly to areas of fibrinogen adhesion. Thus the slides provide a clear differentiation between a thrombogenic and non-thrombogenic region.