We have covalently attached multiple fluorescent silicon nanocrystals (SNs) to streptavidin molecule. Selective conjugation of SNs to a target protein is accomplished using sequential silicon surface termination chemistry. In the first step, freshly prepared hydrogen terminated surfaces of SNs are substituted with alkane monolayer that serves as a platform for chemical linkage to hetero bifunctional crosslinker (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester), and provides optical and chemical stabilities against oxidation and aggregation of nanoparticles. Next, an open end of bifunctional cross linker - diazirine succinimidyl ester forms an amide bond with a carboxyl of target protein. Gel electrophoresis of SNs labeled streptavidin clearly show separate elution of conjugation product and neat protein. Conjugate functionality was verified by allowing it to interact with biotinylated micro beads. A bright fluorescence, characteristic to SN's was observed from vigorously washed micro beads showing selective attachment of nanoparticle bearing streptavidin to biotinylated micro beads. High quantum yield of streptavidin-SN conjugate in combination with the biocompatibility of silicon nanoparticles presents an attractive platform for the fluorescent tagging in diverse bioassays.
Biocompatibility of photoluminescent silicon nanocrystals was tested using standard cytotoxicity protocols with murine macrophage cell line RAW 264.7. We investigated the cytotoxicity and inflammatory responses of cells exposed to silicon nanocrystals by several biological endpoints. Cell death ratio, morphological changes, and the levels of nitric oxide production were studied. Spatial position of the nanoparticles relative to cell body was investigated using fluorescent microscopy. No statistically significant cytotoxicity or inflammatory response was detected with autoclaved silicon nanoparticles at concentrations up to 20 μg/ml in RAW 264.7 cells. The present study of murine macrophages, exposed to autoclaved silicon nanocrystals, will help define safety requirements for comparable nanoparticle biomedical applications.
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