Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-12-03
1996-03-05
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
385129, 385130, 385131, 422 8205, 422 8211, 435 791, 435176, 435808, 436164, 436518, 436527, 436805, G01N 33573
Patent
active
054967015
DESCRIPTION:
BRIEF SUMMARY
This invention relates to methods for the qualitative or quantitative determination of biomolecules in solution, in particular to methods for the determination of enzymes and their substrates in samples of biological origin.
Many devices for the automatic determination of biochemical analytes in solution have been proposed in recent years. Typically, such devices (biosensors) include a sensitised coating layer which is located in the evanescent region of a resonant field. Detection of the analyte typically utilizes optical techniques such as, for example, surface plasmon resonance (SPR), and is based on changes in the thickness and/or refractive index of the coating layer resulting from interaction of that layer with the analyte. This causes a change, e.g. in the angular position of the resonance.
Other optical biosensors include a waveguide in which a beam of light is propagated. The optical characteristics of the device are influenced by changes occurring at the surface of the waveguide. One form of optical biosensor is based on frustrated total reflection. The principles of frustrated total reflection (FTR) are well-known; the technique is described, for example, by Bosacchi and Oehrle [Applied Optics (1982), 21, 2167-2173]. An FTR device for use in immunoassay is disclosed in U.S. Pat. No. 4,857,273 and comprises a cavity layer bounded on one side by the sample under investigation and on the other side by a spacer layer which in turn is mounted on a substrate. The substrate-spacer layer interface is irradiated with monochromatic radiation such that total reflection occurs, the associated evanescent field penetrating through the spacer layer. If the thickness of the spacer layer is correct and the incident parallel wave vector matches one of the resonant mode propagation constants, the total reflection is frustrated and radiation is coupled into the cavity layer. The cavity layer must be composed of material which has a higher refractive index than the spacer layer and which is transparent at the wavelength of the incident radiation.
More recently, FTR biosensors have been described [see, for example, PCT Patent Application WO 90/06503] in which the cavity layer is a thin film of relatively high refractive index material, typically an inorganic oxide.
In all biosensors, it is necessary that the sensitised coating layer comprise a layer of immobilised chemical or biochemical species. Methods have been disclosed [see PCT Patent Application No WO 90/11510] for the determination of one member of an enzyme-substrate pair which comprise immobilising the other member of that pair on the surface of an SPR biosensor and monitoring the effect of deposition of the reaction product on the surface of the device. Obviously, to have an effect on the characteristics of the device, the reaction product must be insoluble and this places great constraints on the range of enzymes and substrates which may be determined by this method. Also, the deposition of the insoluble product on the surface means that the device is generally not re-usable.
We have now devised methods of determining enzymes and their substrates using a biosensor which overcome or substantially mitigate these disadvantages.
According to the invention, there is provided a method of determining one member of an enzyme-substrate pair (the analyte), which comprises bringing the members of the pair into contact so as to form, directly or indirectly, an absorbing reaction product at or in the vicinity of the surface of an optical waveguide biosensor.
The method of the invention is advantageous in that enzymes and substrates which produce soluble, absorbing reaction products are more widely available than those which produce insoluble products. This means that the number of enzymes and substrates which can be directly assayed is significantly greater. In many cases appropriate substrates can be easily synthesised de novo, which is not the case for substrates which result in insoluble products. Also, if it is the enzyme which is immobilised on the sensor surface,
REFERENCES:
patent: 4857273 (1989-08-01), Stewart
patent: 4992385 (1991-02-01), Godfrey
Schaap et al., "Chemiluminescent Substrates for Alkaline Phosphatase: Application to Ultrasensitive Enzyme-Linked Immunoassays and DNA Probes", Clinical Chemistry, vol. 35, No. 9, 1989, pp. 1863-1864.
Chin Christopher L.
Fisons plc
Saunders David
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