Photoreactive cellulose membrane--A novel matrix for covalent immobilization of biomolecules.

We report a simple and mild procedure for the preparation of a photoreactive cellulose membrane capable of forming a covalent bond with a biomolecule in presence of 365 nm UV light. Photoreactive cellulose membrane was prepared by the reaction of fluoro group of 1-fluoro-2-nitro-4-azidobenzene (FNAB) and hydroxyl group of the cellulose in an alkaline medium. X-ray photoelectron spectroscopy (XPS) of the photoreactive cellulose confirmed the incorporation of FNAB moiety. Azido group of the photoreactive membrane on exposure to UV light transforms into highly reactive nitrene which binds with a protein. The efficacy of the activated membrane was checked by immobilizing glucose oxidase (GOD) onto it in presence of light. Immobilized GOD was found to have improved thermal, pH and storage stability. Photoreactive cellulose membrane was successfully used in enzyme-linked immunosorbent assay (ELISA) technique. The antibody immobilized onto such support by UV irradiation in 30 min showed similar ELISA value than the antibody immobilized onto a polystyrene ELISA plate in 12h incubation at 4 degrees C by conventional method.

[1]  C. Collins,et al.  Immobilization of horseradish peroxidase onto polyaniline polymers , 2003 .

[2]  S. Cramer,et al.  Membrane chromatographic systems for high-throughput protein separations , 1992 .

[3]  E. Entcheva,et al.  Analytical application of membranes with covalently bound glucose oxidase , 1994 .

[4]  Kyoichi Saito,et al.  Protein adsorption capacity of a porous phenylalanine-containing membrane based on a polyethylene matrix , 1991 .

[5]  G. Rao,et al.  A fluorescence lifetime-based solid sensor for water. , 1997, Analytica chimica acta.

[6]  K. Neoh,et al.  Covalent immobilization of invertase onto the surface-modified polyaniline from graft copolymerization with acrylic acid , 2000 .

[7]  Kamalesh K. Sirkar,et al.  Membrane in a reactor: A functional perspective , 1999 .

[8]  L Giorno,et al.  Biocatalytic membrane reactors: applications and perspectives. , 2000, Trends in biotechnology.

[9]  P. Nahar,et al.  Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures. , 2002, Journal of immunological methods.

[10]  N. Chaniotakis,et al.  Lactate Biosensor Based on the Adsorption of Polyelectrolyte Stabilized Lactate Oxidase into Porous Conductive Carbon , 2001 .

[11]  K. Kannan,et al.  Heat-mediated enzyme-linked immunosorbent assay procedure on a photoactivated surface. , 2004, Journal of immunological methods.

[12]  K. Burhop,et al.  Biocompatibility of hemodialysis membranes: evaluation in an ovine model. , 1993, The Journal of laboratory and clinical medicine.

[13]  Zhi‐Kang Xu,et al.  Surface modification of microporous polypropylene membranes by the grafting of poly(γ-stearyl-l-glutamate) , 2003 .

[14]  K. Kannan,et al.  A simple method for functionalization of cellulose membrane for covalent immobilization of biomolecules , 2005 .

[15]  Zhi‐Kang Xu,et al.  A comparative study on lipase immobilized polypropylene microfiltration membranes modified by sugar-containing polymer and polypeptide , 2004 .

[16]  D. Rodríguez‐Puyol,et al.  Cellulose acetate membrane improves some aspects of red blood cell function in haemodialysis patients. , 1990, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[17]  Pradip Nahar,et al.  Introduction of functional groups onto polypropylene and polyethylene surfaces for immobilization of enzymes. , 2002, Analytical biochemistry.

[18]  K. Fernandes,et al.  Covalent immobilisation of horseradish peroxidase onto poly(ethylene terephthalate)–poly(aniline) composite , 2004 .

[19]  Stoyan Tzonkov,et al.  Membranes for optical pH sensors , 1993 .

[20]  N. Kubota,et al.  Recovery of serum proteins using cellulosic affinity membrane modified by immobilization of CU2+ ion , 1996 .

[21]  Jiang Yuan,et al.  Improvement of blood compatibility on cellulose membrane surface by grafting betaines , 2003 .

[22]  J. H. Lee,et al.  Immobilization of proteins on poly(methyl methacrylate) films. , 1993, Biomaterials.

[23]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[24]  V. Gekas.  Artificial membranes as carriers for the immobilization of biocatalysts , 1986 .

[25]  R. P. Gandhi,et al.  Light-induced activation of an inert surface for covalent immobilization of a protein ligand. , 2001, Analytical biochemistry.

[26]  W. M. Azevedo,et al.  Increasing glucose determination range by flow injection analysis (FIA) using glucose oxidase immobilised on polyaniline , 1994 .

[27]  K. Tan,et al.  Covalent immobilization of glucose oxidase on the surface of polyaniline films graft copolymerized with acrylic acid. , 1998, Biomaterials.

[28]  W. Deckwer,et al.  Surface modification of microporous polyamide membranes with hydroxyethyl cellulose and their application as affinity membranes , 1995 .