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

A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling
A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling
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, H-3-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).
enzyme immobilisation, bioanode, biofuel cell, nad(+), nadh, radioisotope labelling, quartz-crystal microbalance, lactate-dehydrogenase, bacillus-stearothermophilus, composite films, electrochemistry, oxidation
0956-5663
965-972
Halliwell, Catherine M.
6cc766e4-a027-4380-8604-c91a27301774
Simon, Evelyne
890fd27c-8885-4929-872f-0afc8d076526
Toh, Chee-Seng
271ecaf6-fbed-454e-aa92-c5e645ada23c
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Cass, Anthony E.G.
6696e860-4ee0-42ea-958f-2cc9ca999e91
Halliwell, Catherine M.
6cc766e4-a027-4380-8604-c91a27301774
Simon, Evelyne
890fd27c-8885-4929-872f-0afc8d076526
Toh, Chee-Seng
271ecaf6-fbed-454e-aa92-c5e645ada23c
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Cass, Anthony E.G.
6696e860-4ee0-42ea-958f-2cc9ca999e91

Halliwell, Catherine M., Simon, Evelyne, Toh, Chee-Seng, Bartlett, Philip N. and Cass, Anthony E.G. (2002) A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling. Biosensors & Bioelectronics, 17 (11-12), 965-972. (doi:10.1016/S0956-5663(02)00088-X).

Record type: Article

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, H-3-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).

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More information

Published date: 2002
Keywords: enzyme immobilisation, bioanode, biofuel cell, nad(+), nadh, radioisotope labelling, quartz-crystal microbalance, lactate-dehydrogenase, bacillus-stearothermophilus, composite films, electrochemistry, oxidation

Identifiers

Local EPrints ID: 19751
URI: http://eprints.soton.ac.uk/id/eprint/19751
ISSN: 0956-5663
PURE UUID: 0da6a796-546b-492e-95c9-9a04e23017c0
ORCID for Philip N. Bartlett: ORCID iD orcid.org/0000-0002-7300-6900

Catalogue record

Date deposited: 20 Feb 2006
Last modified: 08 Jan 2022 02:38

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Contributors

Author: Catherine M. Halliwell
Author: Evelyne Simon
Author: Chee-Seng Toh
Author: Anthony E.G. Cass

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