Choi Jonghoon, Wang Nam Sun, Reipa Vytas
Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, USA.
Bioconjug Chem. 2008 Mar;19(3):680-5. doi: 10.1021/bc700373y. Epub 2008 Feb 22.
We have covalently attached multiple photoluminescent silicon nanoparticles (SNs) to streptavidin molecules. Conjugation of SNs to a target protein is achieved using the multistage photoassisted procedure. In a first step, the terminal hydrogen in the freshly prepared SNs is substituted with an alkane monolayer that serves as a platform for chemical linkage to a heterobifunctional cross-linker: 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester. A resulting surface coating stabilizes nanoparticles against oxidation and aggregation. Next, an open end of bifunctional cross-linker-diazirine succinimidyl ester is reacted with carboxyl moieties of streptavidin and forms an amide bond. Gel and capillary electrophoresis of the SN-streptavidin complex demonstrated separate elution of the conjugation product and unreacted protein. Then, the number of SNs per protein molecule was determined by measuring complex charge variation by capillary electrophoresis. Conjugate functionality was tested by allowing it to interact with biotinylated polystyrene microbeads. Intense photoluminescence at carefully washed microbeads demonstrated selective binding of silicon nanoparticle bearing streptavidin to biotinylated microbeads. The high quantum yield of streptavidin-SN conjugate in combination with the small size and biocompatibility of silicon nanoparticles presents an attractive platform for the fluorescence labeling in diverse bioassays.
我们已将多个光致发光硅纳米颗粒（SNs）共价连接到链霉亲和素分子上。通过多阶段光辅助程序实现了SNs与目标蛋白的共轭。第一步，将新制备的SNs中的末端氢用烷烃单分子层取代，该单分子层作为与异双功能交联剂：4-叠氮基-2,3,5,6-四氟苯甲酸琥珀酰亚胺酯进行化学连接的平台。由此产生的表面涂层可稳定纳米颗粒，防止其氧化和聚集。接下来，双功能交联剂-重氮烷基琥珀酰亚胺酯的开口端与链霉亲和素的羧基部分反应，形成酰胺键。SN-链霉亲和素复合物的凝胶电泳和毛细管电泳显示共轭产物和未反应蛋白的洗脱分离。然后，通过毛细管电泳测量复合物电荷变化来确定每个蛋白质分子的SNs数量。通过使其与生物素化的聚苯乙烯微珠相互作用来测试共轭物的功能。在仔细洗涤的微珠上的强烈光致发光表明携带链霉亲和素的硅纳米颗粒与生物素化微珠的选择性结合。链霉亲和素-SN共轭物的高量子产率，结合硅纳米颗粒的小尺寸和生物相容性，为各种生物测定中的荧光标记提供了一个有吸引力的平台。
