Protein Patterning with a Photoactivatable Derivative of Biotin

Abstract

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.

Light‐Triggered Multifunctionality at Surfaces Mediated by Photolabile Protecting Groups

“…[79][80][81][82] Masked light exposure and incubation with streptavidin generated streptavidin patterns that were used for assembly of target biotinylated proteins at selected surface concentrations controlled by the exposure dose. Alternatively, biotin was site-selective reacted with chemical patterns obtained by masked irradiation or laser scanning of surfaces modifi ed with caged coupling agents,…”

Alkaline Phosphatase-Strep tag fusion protein binding to streptavidin: resonant mirror studies 1 1Edited by A. R. Fersht

“…Depending upon the nature of the surface coating and the structure of the fusion protein this approach could offer immobilisation methods that are extremely mild, potentially reversible and with the opportunity to control the orientation of the immobilised biomolecules. We have recently described a method for photopatterning surfaces with biotin (Hengsakul & Cass, 1996) and this could be readily used with streptavidin or avidin-binding sequences fused to the protein that is to be immobilised. One such sequence has been described (Schmidt & Skerra, 1993) which contains ten amino acids (SAWRHPQFGG), X-ray crystallographic (Schmidt et al, 1996) studies have revealed that it is the sequence HPQ that is the primary recognition site by streptavidin, and titration calorimetry has been used to investigate the binding of such sequences to streptavidin in solution (Schmidt et al, 1996).…”

Bio-Microarray Fabrication Techniques—A Review

“…The two main methods are photolithography 9,46,55,66 and direct photochemical patterning. 9,26,68 In photolithography, a positive photoresist layer is spin-coated onto the substrate, exposed to UV light through a photomask and then developed to form micrometer sized open regions where adhesionpromoting molecules are bound (Figure 20(a)). The substrate is then immersed in solvent to remove the remaining photoresist, and adhesion-resistant molecules are bound to the exposed glass surfaces.…”