A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling

• Published in: Biosensors & bioelectronics Vol. 17; no. 11; pp. 965 - 972
• Main Authors: Halliwell, Catherine M., Simon, Evelyne, Toh, Chee-Seng, Bartlett, Philip N., Cass, Anthony E.G.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 01-12-2002; Elsevier Science
• Subjects: Aniline Compounds; Bioanode; Biofuel cell; Biological and medical sciences; Biosensing Techniques; Biosensors; Biotechnology; Catalysis; Coated Materials, Biocompatible - chemistry; Electrochemistry - methods; Enzyme Activation; Enzyme immobilisation; Enzymes, Immobilized - analysis; Enzymes, Immobilized - chemistry; Equipment Design - methods; Fundamental and applied biological sciences. Psychology; Isotope Labeling - instrumentation; Isotope Labeling - methods; L-Lactate Dehydrogenase - analysis; L-Lactate Dehydrogenase - chemistry; Leucine - chemistry; Methods. Procedures. Technologies; Microscopy, Electron, Scanning; NAD; NADH; Radioisotope labelling; Recombinant Proteins - analysis; Recombinant Proteins - chemistry; Scintillation Counting - methods; Sensitivity and Specificity; Various methods and equipments; Enzyme immobilisation; IPTG, isopropylthiol-β-galactoside; NAD +, nicotinamide adenine dinucleotide; LDH, l-lactate dehydrogenase; PANi–PAA, poly(aniline)–poly(acrylic acid) composite film; WT, wild-type; PANi–PSS, poly(aniline)–poly(styrene sulfonate) composite film; Bioanode; CHis, C-terminal hexahistidine sequence; NAD; NADH; SCE, saturated calomel electrode; Biofuel cell; PANi–PVS, poly(aniline)–poly(vinyl sulfonic acid) composite film; Radioisotope labelling; NADH, nicotinamide adenine dinucleotide reduced form; LB, Luria–Bertani media; CCys, C-terminal cysteine sequence; Enzyme; Biosensor; Immobilization; Oxidoreductases; Method; Recombinant protein; Enzyme electrode; L-Lactate dehydrogenase; Liquid scintillation; Quantitative analysis
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/S0956-5663(02)00088-X

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).