WO1992021768A1 - Analytical methods - Google Patents
Analytical methods Download PDFInfo
- Publication number
- WO1992021768A1 WO1992021768A1 PCT/GB1992/000991 GB9200991W WO9221768A1 WO 1992021768 A1 WO1992021768 A1 WO 1992021768A1 GB 9200991 W GB9200991 W GB 9200991W WO 9221768 A1 WO9221768 A1 WO 9221768A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- substrate
- enzyme
- biosensor
- reaction product
- analyte
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/808—Optical sensing apparatus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/805—Optical property
Definitions
- 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.
- 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, eg in the angular position of the resonance.
- SPR surface plasmon resonance
- 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), 2 , 2167-2173].
- An FTR device for use in immunoassay is disclosed in US Patent 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.
- 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.
- 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.
- 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.
- the deposition of the insoluble product on the surface means that the device is generally not re-usable.
- a method of determining one member of an enzyme-substrate pair 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, and if the enzymes maintain their activity, the sensor can be re-used for several samples. Again, this is not the case for insoluble reaction products.
- any convenient parameter of the emitted radiation may be monitored. Obviously, the absorbing nature of the reaction product will have an effect on the intensity of the radiation coupled out of the waveguide.
- the product may also be fluorescent or luminescent and it may be the fluorescence or luminescence which is monitored.
- One specific embodiment of the method according to the invention comprises the steps of a) contacting a sample, in which the analyte is to be determined, with the surface of an optical waveguide biosensor on which the other member of the enzyme-substrate pair is immobilised, so as to form (if the analyte is present) a soluble, absorbing reaction product, b) irradiating the biosensor with a beam of electromagnetic radiation such that propagation of light of a wavelength absorbed by the reaction product occurs within the waveguide, and c) monitoring the radiation emitted from the biosensor.
- the method of the invention may also be used to determine analytes which influence the rate of the enzyme-substrate reaction, ie enzyme inhibitors and activators.
- the method may comprise a) contacting a sample, in which an inhibitor or activator of an enzyme is to be determined, with the surface of an optical waveguide biosensor on which an enzyme or a substrate therefor is immobilised, the substrate being one which in the presence of the enzyme produces a soluble, absorbing reaction produc , b) adding to the sample the other member of the enzyme- substrate pair, c) irradiating the biosensor with a beam of electromagnetic radiation such that propagation of light of a wavelength absorbed by the reaction product occurs within the waveguide, and d) monitoring the radiation emitted from the biosensor.
- the formation of absorbing reaction product correlates with the concentration of activator in the sample, or correlates inversely with the concentration of inhibitor.
- a substrate analogue which does do so may be added to the sample before contacting it with the sensor surface.
- the substrate analogue then competes with the natural substrate for binding to the immobilised enzyme.
- the invention also provides a method for determining one member of an enzyme-substrate pair (the analyte) , which comprises a) contacting a sample, in which the analyte is to be determined, with the surface of an optical waveguide biosensor on which a second enzyme is immobilised, b) adding to the sample the other member of the enzyme- substrate pair and a substrate for the immobilised enzyme which, in the presence of a reaction product of the enzyme- substrate pair, produces a soluble, absorbing reaction product, c) irradiating the biosensor with a beam of electromagnetic radiation such that propagation of light of a wavelength absorbed by the reaction product occurs within the waveguide, and d) monitoring the radiation emitted from the biosensor.
- both enzymes may be immobilised on the sensor surface.
- One enzyme reacts with the substrate in the sample to produce a product which in the presence of the substrate for the second enzyme reacts to form the absorbing, soluble reaction product.
- the soluble reaction products must not diffuse out of the evanescent field of the optical waveguide sensor during the course of the assay, but must remain localised close to the immobilised enzyme. This is known to occur for some enzymes.
- the luminescent signal produced remains localised close to the enzyme active site.
- the reaction product may be kept within the evanescent field by means of a physical barrier or by capture on the sensor surface by means of an antibody.
- antibodies may be prepared which bind the product of the enzyme reaction but not the substrate. The antibodies may be immobilised on the same chip as the enzymes.
- reaction products are constrained to the vicinity of the surface at which they are formed, it may be possible to detect several analytes within the same sample.
- Assays for some enzymes may be performed by observing a decrease in the absorbance at a particular wavelength.
- angiotensin converting enzyme may be assayed with the substrate FA-Phe-Gly-Gly (FA is furylacryloyl) by observing a decrease in absorbance.
- Enzymes for which there are no suitable substrates for direct measurement may be detected by the use of a coupling enzyme.
- oxalate oxidase may be detected as follows:
- ⁇ reatine kinase or phosphocreatine may be assayed as follows:
- a resonant biosensor based on the principle of frustrated total reflection (FTR) .
- FTR frustrated total reflection
- Such a biosensor typically comprises a) a cavity layer of dielectric material of refractive index n 3 , b) a dielectric substrate of refractive index n lf and c) interposed between the cavity layer and the substrate, a dielectric spacer layer of refractive index n 2 .
- the interface between the substrate and the spacer layer is irradiated with light such that total reflection occurs.
- 'light' may include not only visible light but also wavelengths above and below this range, eg in the ultra-violet and infra-red.
- Resonant propagation of a guided mode in the cavity layer will occur, for a given wavelength, at a particular angle of incidence of the exciting radiation.
- two basic measurement approaches are possible: scanning the angle of incidence at fixed wavelength or scanning the wavelength at a fixed angle of incidence.
- the former approach using monochromatic radiation, is preferred since it allows the use of a laser source, simplifying the problem of optical collimation, and avoids dispersion effects, thereby simplifying the analysis of the results.
- the angular position of the resonant effect depends on various parameters of the biosensor device, such as the refractive .ndices and thicknesses of the various layers. In general, it is a pre-requisite that the refractive index n 3 of the cavity layer and the refractive index n x of the substrate should both exceed the refractive index n 2 of the spacer layer. Also, since at least one mode must exist in the cavity to achieve resonance, the cavity layer must exceed a certain minimum thickness.
- the cavity layer is preferably a thin-film of dielectric material.
- transmissive dielectric materials for the cavity layer include zirconium dioxide, titanium dioxide, aluminium oxide and tantalum oxide.
- the cavity layer may be prepared by known techniques, eg vacuum evaporation, sputtering, chemical vapour deposition or in-diffusion.
- the dielectric spacer layer must also be suitably transmissive to the incident radiation and must have a lower refractive index than both the cavity layer and the substrate.
- the layer may, for example, comprise an evaporated or sputtered layer of magnesium fluoride.
- an infra-red light injection laser may be used as light source.
- the light from such a source typically has a wavelength around 800nm.
- suitable materials include lithium fluoride and silicon dioxide.
- the spacer layer may be deposited on the substrate by a sol-gel process, or be formed by chemical reaction with the substrate.
- the refractive index of the substrate (n x ) must be greater than that (n 2 ) of the spacer layer but the thickness of the substrate is generally not critical to the performance of the inventio .
- the spacer layer will typically have a thickness of the order of several hundred nanometres, say from about 200nm to 2000nm, more preferably 500 to 1500nm, eg lOOOnm.
- the cavity layer typically has a thickness of a few tens of nanometres, say 10 to 200nm, more preferably 30 to 150nm, eg lOOn .
- the cavity layer has a thickness of 30 to 150nm and comprises a material selected from zirconium dioxide, hafnia, silicon nitride, titanium dioxide, tantalum oxide and aluminium oxide
- the spacer layer has a thickness of 500 to 1500nm and comprises a material selected from magnesium fluoride, lithium fluoride and silicon dioxide, the choice of materials being such that the refractive index of the spacer layer is less than that of the cavity layer.
- Preferred materials for the cavity layer and the spacer layer are tantalum oxide and silicon dioxide respectively.
- any convenient source of radiation may be used as the source of the incident light but it is preferable to use monochromatic radiation and the most convenient source of such radiation is a laser.
- the choice of laser will depend inter alia on the materials used for the various layers of which some examples have already been given, and of course on the particular absorbing reaction product.
- the scanning of angle may be performed either sequentially or simultaneously ie by varying the angle of incidence of a parallel beam of light or by simultaneously irradiating over a range of angles using a fan-shaped beam of light as described (in connection with SPR) in European Patent Application No 0305109A.
- a single-channel detector may be used which is mechanically scanned over a range of angles; in the latter case, in which a range of angles is irradiated simultaneously, it will generally be necessary to use a multi-channel detector having angular resolution.
- the incident light is coupled into the cavity layer by FTR, propagates a certain distance along the cavity layer, and couples back out (also by FTR) .
- the propagation distance depends on the various device parameters but is typically of the order of 1 or 2mm.
- the formation of absorbing reaction product in the course of the methods according to the invention may result in a reduction in the intensity of the reflected light. If the reaction product is fluorescent or luminescent, the increase in intensity at the fluorescent or luminescent wavelengths may be used to determine the analyte.
- Figure 1 shows schematically an assay method for the determination of an enzyme El.
- a substrate SI for the enzyme is immobilised on the surface of the cavity layer C of an FTR biosensor.
- the substrate SI may be immobilised directly on the layer C or may be immobilised in a layer of gel.
- a detectable (absorbing) product Pi is produced.
- Figure 2 shows a corresponding method for the determination of a substrate S.
- the enzyme E which is immobilised on the sensor surface C.
- a coupling enzyme E2 is immobilised on the surface C.
- a substrate SI for that enzyme and a substrate S2 for the coupling enzyme E2 which in the presence of the product Pi (produced from Si by the action of El) produces a detectable (absorbing) product P2.
- Figure 4 shows a scheme in which the substrate Si is the analyte and two enzymes El and E2 are immobilised on the sensor C.
- a coupling substrate S2 is added to the sample containing SI.
- the analyte substrate SI is converted by enzyme El to a product PI which reacts, in the presence of enzyme E2, with the coupling substrate S2 to produce a detectable (absorbing product) P2.
- Figure 5 shows a variation on the scheme shown in Figure 2 in which the reaction product P is captured and retained at the surface C of the sensor by immobilised specific antibodies.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/157,079 US5496701A (en) | 1991-06-04 | 1992-06-02 | Optical biosensor method for determining an analyte |
JP4509874A JPH06507708A (en) | 1991-06-04 | 1992-06-02 | Analysis method |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9111912.3 | 1991-06-04 | ||
GB919111912A GB9111912D0 (en) | 1991-06-04 | 1991-06-04 | Analytical methods |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1992021768A1 true WO1992021768A1 (en) | 1992-12-10 |
Family
ID=10696009
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1992/000991 WO1992021768A1 (en) | 1991-06-04 | 1992-06-02 | Analytical methods |
Country Status (5)
Country | Link |
---|---|
US (1) | US5496701A (en) |
EP (1) | EP0588831A1 (en) |
JP (1) | JPH06507708A (en) |
GB (1) | GB9111912D0 (en) |
WO (1) | WO1992021768A1 (en) |
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Also Published As
Publication number | Publication date |
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GB9111912D0 (en) | 1991-07-24 |
US5496701A (en) | 1996-03-05 |
EP0588831A1 (en) | 1994-03-30 |
JPH06507708A (en) | 1994-09-01 |
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