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
• • 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
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/548—Carbohydrates, e.g. dextran

Definitions

• This invention relates to sensors, in particular to chemical or biochemical sensors having a dielectric surface layer, such as those based on the principle of frustrated total internal reflection.
• 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
• FTR frustrated total reflection
• 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.
• the sensitised coating layer comprise a layer of immobilised chemical or biochemical species.
• a biocompatible porous matrix such as a hydrogel be employed to achieve this immobilisation [see PCT Patent Application WO 90/05303].
• the matrix typically comprises a dextran which is coupled to the sensor surface (which in SPR is a metal layer) via a linking group, preferably a thiol or a disulphide. Thiol or disulphide linking groups are, however, unsuitable for directly linking a porous matrix to the oxide surface of an FTR-based sensor.
• a biocompatible porous matrix may be used to immobilise the sensitive coating on the surface of a biosensor with a dielectric surface layer, and that this approach offers unexpected advantages over previously-known sensors.
• a biosensor comprising a layer of dielectric material, at least a part of which is coupled to a biocompatible porous matrix containing immobilised biochemicals.
• the sensor according to the invention is advantageous in that a much higher density of immobilised biochemicals can be obtained compared with immobilisation of the biochemical species directly on the sensor surface. This results in an enhanced measuring signal and a greater dynamic range. Also, binding activity of the immobilised species is increased and undesirable desorption of the immobilised species, eg caused by treatment with detergents, is reduced.
• the porous matrix provides a three-dimensional matrix for binding of analyte molecules in a sample under investigation, increasing the effect of such binding on the refractive index of the surface region.
• the porous matrix also renders the sensor surface chemically resistant to the sample media with which it is brought into contact during use, and is compatible with proteins and other biomolecules.
• a great variety of biomolecules may be immobilised in the porous matrix by covalent binding, which enhances the versatility of the sensor, in terms of the range of analytes which may be detected.
• greater sensitivity may be achieved than is possible using the SPR technique, even where the porous matrix is employed as described in WO 90/05303.
• the enhanced sensitivity may be due, for example, to the fact that the propagation distance of the light coupled into the cavity layer may be optimised, eg by appropriate choice of materials and layer thicknesses.
• Coupling of the porous matrix may also be achieved more easily on the inorganic oxide surface of the sensor according to the invention than on the metal surface of an SPR sensor. Also, the fabrication of the dielectric structure of the sensor of the present invention may be simpler and less costly than that of SPR-based sensors involving thin layers of metal. In addition, the inorganic oxide surface of the sensor is not susceptible to tarnishing, as are silver or gold surfaces, or to oxidation, as is the case with aluminium surfaces.
• the sensor according to the invention may be based on the principle of FTR, in which case the dielectric layer forms the cavity layer of the sensor.
• the porous matrix may have a thickness in the range of from a few nm, eg about 5nm, to several hundred nm.
• the porous matrix is most preferably a hydrogel, eg one of those described by Merrill et al [Hydrogels in Medicine and Pharmacy, Vol III, (1986), Ed. Pappas NA, Chapter 1, CRC Press] .
• hydrogels examples include polysaccharides, eg agarose, dextran, carrageenan, alginic acid, starch, cellulose, or derivatives thereof, eg carboxymethyl derivatives, xanthan gum, pectin, or a water-swellable organic polymer such as poly inyl alcohol, polyacrylic acid, polyacrylamide, and polyethylene glycol.
• dextran is a particularly preferred hydrogel.
• the surface of the dielectric layer may be derivatised or activated so as to provide coupling sites for the porous matrix.
• the layer may be reacted with a silane-based coupling compound in a known manner.
• a suitable such reagent is, for example, a terminal amino-alkyl trimethoxysilane, eg the 3-aminopropyl compound, used at a concentration of about 2% w/v in acetone. Details of immobilisation techniques using this reagent have been described by Weetall [see, for example, US Patent 3,652,761 and "Immobilised Biochemicals and Affinity Chromatography", R B Dunlop (Ed), Plenum Press, New York (1974), ppl91-212].
• the amino terminals immobilised on the dielectric layer may in turn be reacted with succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- carboxylate (SMCC), then 2-mercaptoethanol, followed by epichlorhydrin for cross-linking to the porous matrix, excess reagents then being removed and the activated surface with immobilised epoxide groups then being treated with a solution of the species constituting the porous matrix.
• SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1- carboxylate
• An alternative method for coupling the porous matrix to the layer surface involves treatment with epoxy-silane reagents, especially glycidyloxypropyltrimethoxysilane, eg at a concentration of about 2% v/v in toluene for about 2 hours at 70°C as described by Herman et al [J Chromatogr Sci (1981), U_(9), 470-476].
• epoxy-silane reagents especially glycidyloxypropyltrimethoxysilane, eg at a concentration of about 2% v/v in toluene for about 2 hours at 70°C as described by Herman et al [J Chromatogr Sci (1981), U_(9), 470-476].
• aldehyde reagents is unnecessary, since the epoxysilylated dielectric layer can react directly with the porous matrix molecules.
• the porous matrix is coupled directly to the dielectric layer surface ie without derivatisation or activation of the surface.
• Dextran in particular may be coupled directly to the dielectric surface and it is found that devices prepared in this way have improved properties. In particular it is found that there may be an increase in the density of immobilised biomolecules. This may be a result of a higher dextran concentration at the surface. Devices with dextran coupled directly to the surface may nonetheless be as stable as devices in which the dextran is coupled to the surface indirectly. In particular, in both cases various treatments, eg washing at 70°C in 6M urea or washing in high concentrations of detergents, do not remove the dextran.
• Coupling of the porous matrix to the dielectric surface may involve derivatisation of the porous matrix molecules, eg carboxymethylation. Since such derivatisation may involve treatment with reagents which may have a detrimental effect on the surface, it is frequently preferred to perform the derivatisation prior to contact with the surface.
• biochemicals such as antibodies for a particular analyte are immobilised in the matrix. It may be necessary to first activate the matrix.
• a hydrazide function may be created in a dextran matrix for binding biomolecules containing aldehyde groups, eg antibodies in which the carbohydrate chain has been oxidised.
• the dextran matrix is initially modified with carboxymethyl groups which are partly reacted to form hydrazide groups.
• reactive ester functions may be formed by modification of some of the carboxyl groups in carboxymethyl- modified dextran, eg by treatment with an aqueous solution of N-hydroxysuccinimide and N-(3-dimethyl-aminopropyl)-N'- ethylcarbodiimide hydrochloride.
• Biomolecules such as proteins, peptides and amino-modified oligonucleotides which contain amino groups may be coupled to the matrix via activated carboxyl groups.
• the reactive ester function is reacted with a disulphide-containing species such as 2-(2- pyridinyldithio)etha ⁇ amine.
• a disulphide-containing species such as 2-(2- pyridinyldithio)etha ⁇ amine.
• the disulphide containing matrix so obtained can be used to couple thiol-containing biomolecules such as reduced F(ab) fragments of immunoglobulins.
• disulfide-containing ligands eg N-succinimidyl 3-(2-pyridinyldithio)propionate (SPDP) modified proteins, may be immobilised after cleavage of the matrix disulphide bonds, eg by reduction or thiol-disulphide exchange.
• immobilisation may also occur by complex formation.
• the biomolecule may be an antibody to dextran which is conjugated with a specific binding partner for the analyte.
• the hydrogel may incorporate absorbing species, eg dye molecules, which may be linked to the hydrogel molecules. Changes occuring when a sample is contacted with the biosensor surface may be detected as a reduction in the intensity of the reflected radiation, radiation coupled into the dielectric layer being absorbed by the absorbing molecules. This provides a further advantage for the system of the present invention over SPR based sensors.
• absorbing species eg dye molecules
• an FTR-based biosensor will comprise 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 indices 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 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 invention.
• 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 200 ⁇ m, more preferably 30 to 150nm, eg lOOnm.
• 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.
• 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 reflected light will undergo a phase change and it may be the angular position at which this phase change occurs which is detected.
• Changes on the surface of the cavity layer eg binding of antigen to antibody immobilised in the porous matrix, cause changes in the thickness of the layer of immobilised biochemicals and hence shift the angular position of the resonance.
• there may also be a reduction in the intensity of the reflected light eg if the immobilised species are absorbing at the wavelength of the incident radiation. In this case, this reduction in intensity may be used to monitor the binding processes.
• FTR biosensor devices with a dielectric cavity layer are washed with acetone for 5 minutes and allowed to air-dry.
• the surfaces of the devices are silanised with glycidoxypropyltrimethoxy-silane under vacuum at 140°C for 1 hour.
• the devices are coated with a solution of dextran
• the devices are washed with water.
• the immobilised dextran is carboxymethylated by incubating the devices with bromoacetic acid (1.3g) in 2M NaOH overnight at room temperature. vi) The devices are washed with water.
• Carboxymethylated dextran immobilised on the surface by method al), a2) or a3) is treated with 0.IM N- hydroxysuccinimide (NHS) and 0.4M N-ethyl-N'- (dimethylaminopropyl)carbodiimide (EDC) for 10 minutes to activate the carboxyl groups of the dextran to reactive N- hydroxysuccinimide esters.
• NMS N- hydroxysuccinimide
• EDC dimethylaminopropylcarbodiimide
• step i) The product of step i) is treated with a 20-100 ⁇ g/ml solution of antibody/protein/peptide/amino-modified oligonucleotide for 10 minutes in lOmM acetate buffer pH 4.0-5.5.

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• 1992
  • 1992-06-02 WO PCT/GB1992/000992 patent/WO1992021976A1/en not_active Application Discontinuation
  • 1992-06-02 JP JP4509875A patent/JPH06507709A/ja active Pending
  • 1992-06-02 EP EP92910780A patent/EP0643833A1/de not_active Withdrawn

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