title: A novel method for immobilization of proteins via entrapment of magnetic nanoparticles through epoxy cross-linking.
abstract: A method for immobilization of functional proteins by chemical cross-linking of the protein of interest and uncoated iron oxide nanoparticles in the presence of Epichlorohydrin is described. As a result of the cross-linking, the proteins form a matrix in which the particles get entrapped. The optimum concentration of Epichlorohydrin that facilitates immobilization of protein without affecting the functional properties of the protein was determined. This method was used to immobilize several functional proteins and the development and functional activity of Protein A-magnetic nanoparticles (MNPs) is described here in detail. The Protein A-MNPs possess high binding capacity due to the increased surface area of uncoated nanoparticles and robust magnetic separation due to the absence of polymeric coating materials. Protein A-MNPs were successfully used for purification of antibodies and also for immunoprecipitation. We also immobilized enzymes such as horse radish peroxidase and esterase and found that by providing the optimum incubation time, temperature and protein to nanoparticle ratio, we can retain the activity and improve the stability of the enzyme. This study is the first demonstration that Epichlorohydrin can be used to entrap nanoparticles in a cross-linked matrix of protein without impairing the activity of immobilized protein.
authors.affiliation: MagGenome Technologies Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India. Electronic address: tessy.m@scigenom.com.
authors.forename: Tessy
authors.initials: T
authors.lastname: Iype
authors.affiliation: MagGenome Technologies Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India.
authors.forename: Jaiby
authors.initials: J
authors.lastname: Thomas
authors.affiliation: SciGenom Labs Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India.
authors.forename: Sangeetha
authors.initials: S
authors.lastname: Mohan
authors.affiliation: MagGenome Technologies Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India.
authors.forename: Kochurani K
authors.initials: KK
authors.lastname: Johnson
authors.affiliation: SciGenom Labs Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India.
authors.forename: Ligi E
authors.initials: LE
authors.lastname: George
authors.affiliation: SciGenom Labs Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India.
authors.forename: Lizebona A
authors.initials: LA
authors.lastname: Ambattu
authors.affiliation: MagGenome Technologies Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India.
authors.forename: Aniruddha
authors.initials: A
authors.lastname: Bhati
authors.affiliation: Department of Obstetrics and Gynecology, 1301 Catherine St., 6436 Medical Sciences Building 1, University of Michigan, Ann Arbor, MI, 48103, USA.
authors.forename: Kristen
authors.initials: K
authors.lastname: Ailsworth
authors.affiliation: Department of Obstetrics and Gynecology, 1301 Catherine St., 6436 Medical Sciences Building 1, University of Michigan, Ann Arbor, MI, 48103, USA.
authors.forename: Bindu
authors.initials: B
authors.lastname: Menon
authors.affiliation: CSIR- Centre for Cellular & Molecular Biology, Hyderabad, 500007, Telangana, India.
authors.forename: Sunayana M
authors.initials: SM
authors.lastname: Rayabandla
authors.affiliation: CSIR- Centre for Cellular & Molecular Biology, Hyderabad, 500007, Telangana, India.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Jesudasan
authors.affiliation: MagGenome Technologies Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India; SciGenom Labs Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India.
authors.forename: Sam
authors.initials: S
authors.lastname: Santhosh
authors.affiliation: MagGenome Technologies Pvt. Ltd., CSEZ, Kakkanad, PIN-682037, Kochi, Kerala, India.
authors.forename: Chaniyilparampu N
authors.initials: CN
authors.lastname: Ramchand
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D004811:Epichlorohydrin / Q000737:chemistry* / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay; D007700:Kinetics; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D013696:Temperature
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D058185:Magnetite Nanoparticles; D004811:Epichlorohydrin; D006735:Horseradish Peroxidase; D004950:Esterases
keywords: Entrapment; Epichlorohydrin; Epoxy cross-linking; Immobilization; Magnetic nanoparticles