Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Calorimeter
Patent
1992-09-30
1996-06-18
Redding, David A.
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Calorimeter
422 57, 435 10, 435 11, 435 12, 435 14, 435 20, 435 21, 435 25, 435 26, 435 28, 435805, 435810, G01N 2100
Patent
active
055275095
DESCRIPTION:
BRIEF SUMMARY
This invention relates to apparatus for colorimetric quantitative enzymic analysis of analytes, particularly but not exclusively those analytes which may be oxidised to directly or indirectly to yield hydrogen peroxide. Preferred aspects of the invention also relate to colorimetric enzymic analytical test strips which are substantially dosage independent, that is the accuracy of the apparatus does not depend on application of a precise quantity of a liquid in which the analyte is present.
The use of enzyme systems to detect and obtain quantitative measurement of analyte concentrations is well established. Various enzyme systems have been stabilised and incorporated into dry reagent formats with the necessary co-factors, colour reagents, buffers etc, needed to produce a compact, accurate and disposable analytical element. Examples include: the Reflotron system; BCL Catalogue; Boehringer Mannheim Technical Data; the Ektachem System of Kodak-Eastman (U.S. Pat. No. 3992158 and EP 0013156); the simple Clinistix or Dextrostix for glucose estimation (Ames Corporation), and many others, Analytes which have been measured include ethanol (EP 0110173 (1983)); creatinine and triglyceride, (Clin Chem 24, 1343 (1978)); cholesterol (EP-A-02562 (1981); uric acid (U.S. Pat. No. 3983005); alanine aminotransferase (DE-32067231 (1982)) and amylase (Clin Chem 24, 1343 (1978)), Subsequent techniques have allowed incorporation of immunological reactions into dry chemistry systems, expanding the analytical technique to include substrates for which-direct enzyme determination was not possible. Examples include gentamycin, thyroxine and thyophylline (Clin Chem 30, 194 (1984)) and (EP 0066648 (1987)).
A digital threshold colour control system has been disclosed whereby oxidoreductases utilising NAD(P) as co-factor react on the addition of substrate (the analyte being measured) producing the corresponding product and NAD(P)H (EP 0279988 (1987)). The NAD(P)H was detected by reaction with a tetrazolium salt, using the enzyme diaphorase to mediate colour development. The colour control system utilised the capability of diaphorase, to oxidise NAD(P)H. In the presence of a competing substrate, e.g. benzoquinone, no colour was formed until all the benzoquinone had been reduced. In the presence of excess NAD(P)H the enzyme accepted the tetrazolium salt as a substrate. This reaction may be set to produce colour at a range of levels of the analyte by varying the quantity of competing substrate.
This method has disadvantages. The enzymes utilised require the incorporation of an expensive co-factor. Dehydrogenase enzymes are relatively unstable in the dry state. The number of dye systems that may be used are limited and thus the range of detectable colours is small. Also, colour intensity tends to be low, thus lowering the sensitivity of such systems.
According to a first aspect of the present invention colorimetric enzyme analytical apparatus comprises a test element including a support, the support carrying a zone including a dried enzyme composition, a predetermined amount of a thiol mediator and a dyestuff, the enzyme composition being arranged to produce hydrogen peroxide upon contact of the zone with an analyte, the dyestuff providing a colorimetric display upon reaction with hydrogen peroxide, said colorimetric display being reversible in the-presence of the mediator, the mediator being adapted until exhausted to scavenge hydrogen peroxide and prevent said colorimetric display.
A test element may comprise a test strip or alternative configuration known to those skilled in the art. The term "test strip" will be used below for convenience.
A plurality of zones may be provided on a single strip.
The present invention affords advantages which were not previously attainable. A sharp and clearly visible end point dependent on the concentration of an analyte is very beneficial. Although test strips are known which indicate the presence of more than a minimum quantity of an analyte, simple quantitative strips have been elusive. Complex mechanica
REFERENCES:
patent: 4042329 (1977-08-01), Hochstrasser
patent: 4430427 (1984-02-01), Hopkins
patent: 4734360 (1988-03-01), Phillips
patent: 4900666 (1990-02-01), Phillips
patent: 5200325 (1993-04-01), Blatt et al.
Aston William J.
Gibson Timothy D.
Griffiths David A.
Higgins Irving J.
Woodward John R.
Cranfield Biotechnology Ltd.
Redding David A.
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