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Method Article
Pradip Nahar
Nahar's Lab
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Introduction of functional group to an inert polymer is a very challenging and time consuming task. This protocol describes a simple and mild procedure for the preparation of an activated polymer surface, used for immobilization of a protein ligand through a covalent linkage (1). Activation of the polymer surface is carried out by attaching an active functional group through 1-fluoro-2-nitro-4-azidobenzene (FNAB). UV irradiation of FNAB transforms its azido group into a highly reactive nitrene, which binds with the inert polymer surface, whereas the active fluoro group of FNAB, now part of the polymer, remains intact. Covalent linkage between the ligand and the inert surface is established through this active fluoro group in a thermochemical reaction without addition of any catalyst or reagent. The method can be used for activation of different inert polymer surfaces having carbon hydrogen bonds. The efficacy of our method is demonstrated by immobilizing horseradish peroxidase on an activated polystyrene surface. The enzyme, immobilized through the photolinker, is found to give a twofold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. The method may have many applications in the preparation of bioreactors, biostrips, and biosensors, and in diagnostic tests involving the ELISA technique.
Keywords
4-fluoro-3-nitrophenyl azide 1-fluoro-2-nitro-4-azidobenzene heterobifunctional photolinker covalent immobilization biomolecules polymer inert surface polystyrene microtiter plate
Figure 1
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Version History
CURRENT STATUS: Posted
Version 1
Posted 26 Nov, 2013
Community comments: 6
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Subject Areas
Chemistry
Method Article
Pradip Nahar
Nahar's Lab
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 26 Nov, 2013
Community comments: 6
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Chemistry
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Introduction of functional group to an inert polymer is a very challenging and time consuming task. This protocol describes a simple and mild procedure for the preparation of an activated polymer surface, used for immobilization of a protein ligand through a covalent linkage (1). Activation of the polymer surface is carried out by attaching an active functional group through 1-fluoro-2-nitro-4-azidobenzene (FNAB). UV irradiation of FNAB transforms its azido group into a highly reactive nitrene, which binds with the inert polymer surface, whereas the active fluoro group of FNAB, now part of the polymer, remains intact. Covalent linkage between the ligand and the inert surface is established through this active fluoro group in a thermochemical reaction without addition of any catalyst or reagent. The method can be used for activation of different inert polymer surfaces having carbon hydrogen bonds. The efficacy of our method is demonstrated by immobilizing horseradish peroxidase on an activated polystyrene surface. The enzyme, immobilized through the photolinker, is found to give a twofold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. The method may have many applications in the preparation of bioreactors, biostrips, and biosensors, and in diagnostic tests involving the ELISA technique.
Figure 1
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