Covalent method of immobilization is now gaining attention even in diagnostics involving ELISA, ,. In recent years, covalent immobilization of a biomolecule on a solid matrix has become the subject of intense research as it is rapid, stable and efficient. Thus the method is potentially useful for rapid ELISA or covalent immobilization of ligands onto an inert surface without prior activation. ELISA carried out in less than 3 h using photoreactive-BSA showed comparable results with that of conventional ELISA carried out in 18 h. The method is further exemplified by performing ELISA by covalent binding of antigen or antibody on a polystyrene microtiter plate in just 30 min using photoreactive-BSA. When an enzyme is placed on an inert polystyrene matrix in presence of photoreactive-BSA and exposed to light the later forms highly reactive nitrenes some of which bind to the matrix and the rest to the ligand resulting simultaneous formation of covalent bonds with the matrix and the enzyme. We have made photoreactive-BSA – a proteinaceous photolinker by the reaction of bovine serum albumin (BSA) with excess of 1-fluoro-2-nitro-4-azidobenzene (FNAB). Prerequisite of the method is a novel proteinaceous photolinker having multiple light-activable functional groups. Hermanson GT (ed) (1995) Bioconjugate Techniques.A simple and versatile method is developed for covalently binding a protein ligand onto a matrix irrespective of functional groups either on the ligand or the matrix. Sabanayagam CR, Smith CL, Cantor CR (2000) Nucleic Acids Res 28:e33, ii–iv Reichmuth P, Sigrist H, Badertscher M, Morf WE, de Rooij NF, Pretsch E (2002) Bioconjug Chem 13:90–96 Sigrist H, Collioud A, Clemence J-F, Gao H, Luginbuehl R, Saenger M, Sundarababu G (1995) Opt Eng (Bellingham, Washington) 34:2339–2348 Haynes CA, Norde W (1995) J Colloid Interface Sci 169:313–328īuijs J, Norde W, Lichtenbelt JWT (1996) Langmuir 12:1605–1613Ĭhevrier D, Rasmussen SR, Guesdon J (1993) Mol Cell Probes 7:187–197 Wessa T, Rapp M, Sigrist H (1999) Colloids Surf B Biointerfaces 15:139–146 For this study DNA oligonucleotides were chosen as a model system of biomolecular probes, and fluorescence detection of DNA microarrays served as method of detection. Of all photolinkers and substrates tested, PTD as photolinker and COC as substrate showed the highest photolinking efficiencies and fastest reaction times. We compared the overall photolinking efficiency of all photolinkers with respect to the polymer substrate they are applied to, and we found considerable differences for certain photolinker/substrate combinations. The influence of substrate material is discussed, and three different polymers served as representative substrates: poly(methyl methacrylate) (PMMA), polystyrene (PS), and a cycloolefin copolymer (COC). The influence of these variables are investigated for four prominent photolinkers: ketyl-reactive benzophenone (BP) and anthraquinone (AQ), nitrene-reactive nitrophenyl azide (NPA), and carbene-reactive phenyl-(trifluoromethyl)diazirine (PTD). This study addresses the selection of photolinker and the adjustment of reaction conditions, such as the concentration of biomolecule applied, and irradiation time. The use of photolinkers (photoactivatable heterobifunctional crosslinkers) is a popular method to attach biomolecules to polymer surfaces.
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