title: A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling.
abstract: A direct method has been developed for the quantitation of the amount of immobilised enzymes on biosensor surfaces. This quantity is of key importance in establishing the activity, kinetics and optimal immobilisation conditions in the construction of both amperometric and optical biosensors. Recombinant L-lactate dehydrogenase incorporating both a biosynthetically introduced radiolabel, 3H-leucine, and a hexahistidine peptide tag was immobilised on a poly(aniline) composite film and then quantitated by liquid scintillation counting. It was found that enzyme mass loading was proportional to the concentration of LDH in solution, and also depended on the morphology of the composite film. The LDH mass loading on the composite film doubled when a surface cysteine containing variant was used, possibly due to the covalent attachment of the cysteine to the diiminoquinoid rings of the poly(aniline).
authors.affiliation: Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 2AY, UK.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Halliwell
authors.forename: Evelyne
authors.initials: E
authors.lastname: Simon
authors.forename: Chee-Seng
authors.initials: CS
authors.lastname: Toh
authors.forename: Philip N
authors.initials: PN
authors.lastname: Bartlett
authors.forename: Anthony E G
authors.initials: AE
authors.lastname: Cass
mesh_terms: D000814:Aniline Compounds; D015374:Biosensing Techniques; D002384:Catalysis; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004563:Electrochemistry / Q000379:methods*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000032:analysis* / Q000737:chemistry; D004867:Equipment Design / Q000379:methods*; D007553:Isotope Labeling / Q000295:instrumentation / Q000379:methods*; D007770:L-Lactate Dehydrogenase / Q000032:analysis* / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008855:Microscopy, Electron, Scanning; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D012588:Scintillation Counting / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D000814:Aniline Compounds; D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; C416807:polyaniline; D007770:L-Lactate Dehydrogenase; D007930:Leucine