Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Patent
1996-01-30
1998-07-07
Naff, David M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
435 26, 435176, 435177, 4352871, C12Q 166, C12Q 132, C12N 1102, C12M 140
Patent
active
057767157
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to biosensors and more particularly to biosensors for detecting nitrate or nitrite ions.
Owing to concern over the growing contamination of ground and surface waters by nitrates and nitrites both the field analysis and continuous monitoring of nitrate and nitrite concentrations are of significant interest. Conventionally, these species are detected in solution by wet chemical analysis methods which are time consuming and costly in terms of the personnel effort required.
An amperometric biosensor has in the prior art been proposed for nitrate detection based upon a reductase enzyme capable of accepting electrons from a mediating donor. However, the mediating donors proposed for use in such a biosensor, viz redox dyestuffs, produce a relatively negative redox potential (-400 mV to -700 mV) causing significant interference currents in real samples by the reduction of oxygen and other reducible substances present in the sample.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide an improved biosensor in which this problem is avoided.
According to a first aspect of the present invention, a biosensor comprises a biochemical capable of producing a biochemical reaction in the presence of nitrate or nitrite ions and a transducer arranged to detect a transducible output agent produced directly or indirectly by the reaction produced by the biochemical in the presence of nitrate or nitrite ions, the biochemical comprising a reductase for the nitrate or nitrite ions and a co-enzyme which is oxidised by reduction of the nitrate or nitrite ions and characterised in that the biochemical includes a photoluminescent ingredient which produces as the said output agent photons directly or indirectly by oxidation of the said co-enzyme and that the said transducer comprises a photodetector.
According to the present invention in a second aspect there is provided a biosensor for detecting nitrate or nitrite ions, the biosensor comprising an immobilised enzyme composition comprising a quantity of a nitrate or nitrite reductase enzyme, a quantity of a co-factor having a reduced state and an oxidised state and which is converted from its reduced state to its oxidised state by the reduction by the said reductase enzyme, a quantity of a co-enzyme capable of inducing an oxidation reaction in the presence of the co-factor in its oxidised state, the co-factor thereby being converted to its reduced state to maintain a supply of the co-factor in its reduced state to permit further reduction by the reductase enzyme, and means for detecting the extent of oxidation induced by the co-enzyme, the extent of oxidation by the co-enzyme being a measure of formation of the co-factor in its oxidised state which in turn is a measure of the quantity of nitrate or nitrite ions reduced by the reductase enzyme, the means for detecting comprising a photodetector for detecting photons emitted directly or indirectly as a result of oxidation induced by the co-enzyme.
The biosensor may include an immobilised enzyme composition which incorporates both a nitrate reductase and a nitrite reductase so that nitrite produced by the nitrate reduction is further reduced by the nitrite reductase.
According to the present invention in a third aspect there is provided a method of detecting nitrate and/or nitrite ions, the method-comprising exposing to the nitrate or nitrite ions a biosensor as in the first or second aspect thereby causing the reductase enzyme to induce a reduction of the nitrate or nitrite ions and thereby produce by oxidation of the co-factor an output of photons, and detecting the output photons by the photodetector.
The co-factor may comprise the nicotinamide enzyme NADPH which is oxidised to NADP.sup.+ in conjunction with the reduction provided by the reductase and subsequently re-reduced to NADPH by the oxidation by the co-enzyme thereby maintaining a supply of NADPH.
The co-enzyme may itself produce the photons or these may be produced via one or more other co-enzym
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patent: 5500351 (1996-03-01), Eccles et al.
Strehlitz, et al., Fresenius J. Anal. Chem. 349:676-678 (1994).
Kobayashi, et al., Agricultural & Biological Chemistry, vol. 45, Jun. 1981, pp. 1403-1408.
British Nuclear Fuels PLC
Naff David M.
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