Hengsakul M, Cass A E
Department of Biochemistry, Imperial College of Science, Technology & Medicine, South Kensington, London, U.K.
Bioconjug Chem. 1996 Mar-Apr;7(2):249-54. doi: 10.1021/bc960007z.
A method is described for the covalent immobilization of macromolecules at defined location on polymer surfaces. A thin film of a photoactivatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin, in salt form) was dried onto polystyrene or nitrocellulose surfaces and then exposed to intense white light through a mask to yield patterns of biotin covalently bound to the polymers. The subsequent addition of avidin resulted in the formation of surfaces to which biotinylated molecules could then be bound through a biotin-avidin-biotin bridge. To develop the pattern of avidin on the surfaces, biotinylated enzymes (alkaline phosphatase and horseradish peroxidase) were specifically immobilized on the surface. These enzymes still retained their catalytic activities and were visualized through the formation of colored or fluorescent products. Various factors such as concentration, irradiation time, and light intensity were shown to determine the amount of active enzyme that could be bound and so by implication the degree of photobiotinylation that had occurred.
描述了一种将大分子共价固定在聚合物表面特定位置的方法。将生物素的光可活化类似物N-(4-叠氮基-2-硝基苯基)-N'-(N-d-生物素基-3-氨丙基)-N'-甲基-1,3-丙二胺（光生物素，盐形式）的薄膜干燥在聚苯乙烯或硝酸纤维素表面上，然后通过掩膜暴露于强光下，以产生与聚合物共价结合的生物素图案。随后加入抗生物素蛋白导致形成表面，生物素化分子随后可以通过生物素-抗生物素蛋白-生物素桥与之结合。为了在表面上形成抗生物素蛋白图案，将生物素化酶（碱性磷酸酶和辣根过氧化物酶）特异性固定在表面上。这些酶仍保留其催化活性，并通过形成有色或荧光产物来可视化。结果表明，诸如浓度、照射时间和光强度等各种因素决定了可以结合的活性酶的量，并因此暗示了发生的光生物素化程度。
