Abstract

This project involves the design and implementation of a platform to investigate the activation state of platelets. The final aim is to develop an assay to be used for diagnostic purposes. Blood platelets play a key role in the cardiovascular system. When the en- dothelium of the vessel wall is damaged platelets adhere and become activated, leading to platelet aggregation to maintain physiological haemostasis. Any abnormality in platelet functionality induces thrombus or atherosclerotic plaques leading to life-threatening consequences such as stroke. Recent reports suggest that the detection of activated platelets in the blood may be a useful indicator of the on-set of stroke and other cardiovascular abnormalities. Hence, the detection of the functional state of platelets is of huge clinical importance. A luminescence-based platelet bio-assay which uses high brightness silica nanoparticles (NPs) as super-bright labels is an innovative and promising tool for diagnostics. Compared to assays carried out using conventional single molecule dye labels, the usage of NPs makes it possible to enhance the detection efficiency of the assay and to achieve a reduced limit-of-detection. In this work NPs doped with a near infra-red dye are synthesized and their interactions with the platelets are characterized. Protocols of conjugation of antibodies towards platelet surface antigens to NPs are tested. The second part of the work studies the activation state and the morphology of platelets in different environments by using single molecule dye label assays and with AFM. The confinement of platelets in micrometer sized area using a protein patterning technique makes the analysis innovative and potentially very useful.