CA2033168C - Method of optical analysis - Google Patents
Method of optical analysisInfo
- Publication number
- CA2033168C CA2033168C CA002033168A CA2033168A CA2033168C CA 2033168 C CA2033168 C CA 2033168C CA 002033168 A CA002033168 A CA 002033168A CA 2033168 A CA2033168 A CA 2033168A CA 2033168 C CA2033168 C CA 2033168C
- Authority
- CA
- Canada
- Prior art keywords
- refractive index
- waveguide
- light
- sample
- layer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N21/03—Cuvette constructions
- G01N2021/0346—Capillary cells; Microcells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
- G01N2021/6465—Angular discrimination
Abstract
The invention relates to a method of optical analysis which can advantageoulsy be used in conjunction with optical assay techniques, particularly those wherein evanescent signals are to be analysed. The method makes use of the properties of an optical structure (described herein) which allows easy angular discrimination of optical signals emerging from a test object. The invention also relates to apparatus for use in said method.
Description
WO 90/i5985 ~ i ~ ~ PCT/GB90/00953 Method of Optical Analysis This invention relates to methods of optical analysis of evanescent signals and to apparatus for use in such methods.
More particularly, the invention relates to methods of, and apparatus for, detecting evanescent signals produced during ligand/specific binding partner assay procedures, particularly immunoassays.
The prior art contains various disclosures of analytical devices for handling and metering small volumes of test samples, and for the use of such devices in methods of optical analysis.
For example, US Patent 3939350 describes optical measurement of fluorescent material bound to the surface of a solid transparent prism by a method involving single total internal reflection and interaction of the evanescent wave at the surface of the prism with the bound material.
EP-A-75353 makes reference to the exponentially decaying (evanescent) external radiation due to light which is propagated longitudinally in a fibre, and its interaction with coatings.
EP-A-170376 describes, inter alia, methods of optical analysis of fluorescence emerging from a waveguide which is part of a chemical test device, whilst EP-A-171148 describes in detail certain chemical test devices, namely fluorescence capillary fill devices, and methods for their manufacture.
As described in EP-A-171148, a fluorescence capillary fill device (hereinafter FCFD) typically consists of two pieces of transparent material, e.g.
glass, separated by a narrow gap. one plate acts as an optical waveguide and carries an immobilised reagent appropriate to the test to be carried out in the device.
WO 90/ 15985 ~ ~~ ~ ~~ ~ ~ ~ PCT/G B90/00953 For use in an immunoassay, the immobilised reagent may for example be an antibody to an antigen desired to be detected and one of the plates may carry a dissoluble reagent comprising antigen labelled with a fluorescent dye. When a sample is presented to one end of the FCFD
it is drawn into the gap by capillary action and dissolves the reagent. In a competition assay, the fluorescently labelled antigen will compete with sample antigen for the limited number of antibody binding sites immobilised on the waveguide. Because the capillary gap is narrow (typically about 100 microns) the reaction -will generally go to completion in a short time, possibly less than 5 minutes. In a sandwich assay, the waveguide will carry a specific binding partner for the ligand desired to be detected and one of the plates will carry a dissoluble reagent comprising a further specific binding partner labelled with a fluorescent dye. In a sandwich immunoassay for an antigen, a sample antigen will form a sandwich complex with a fluorescently labelled antibody and an antibody immobilised on the waveguide. Thus the amount of fluorescently labelled antibody which becomes indirectly bound to the waveguide by virtue of complex formation will be a function of the concentration of antigen in the sample.
The term "antigen" as used herein will be under-stood to include both antigenic species (for example, proteins, bacteria, bacterial fragments, cells, cell fragments and viruses) and haptens which may be rendered antigenic under suitable conditions.
When using an FCFD in an assay, evanescent wave coupling avoids the need for a separation or washing step. When the labelled reagent is optically excited by a light source of suitable wavelength, fluorescence emission occurs both from fluorophores in the liquid phase and from fluorophores bound, directly or indirectly, to the waveguide. The optical properties of the media involved are such that most of the CVO 90/1'5985 P(T/G B90/00953 fluorescence arising from surface-bound fluorophores emerges from the transverse edge of said waveguide at angles to the normal whose magnitude is less than the magnitude of a certain angle a, whilst substantially all of the fluorescence arising from fluorophores in the liquid phase emerges from the edge of said waveguide at angles to the normal whose magnitude is greater than the magnitude of a (a being such that -%=n<a<1-,a). The value of a depends on the refractive indices of the liquid solution in the cavity and of the material of the waveguide anti on the wavelengths of light involved, but is given approximately by a ~ arcsin (n22 - n12)~~, wherein nl represents the refractive index of the liquid phase and n2 represents the refractive index of the solid waveguide. Thus, under ideal conditions, angular discrimination of fluorescence emerging from the edge of the waveguide would be sufficient to separate the different emission signals.
Although the discussion herein is conducted with reference to angles to the normal whose magnitude is compared to the magnitude of a as defined herein, it should be understood that these angles may deviate from said normal in either a positive or negative sense.
In practice, however, the sensitivity of assays employing simple angular discrimination of fluorescence emission is limited by a number of experimental factors including, inter a 'a: inefficient filtering out of the light of excitation wavelength and secondary fluorescence in all directions from the optical filter (typically of colloidal glass); and aberration of the lenses used to focus the fluorescence emission onto the detectors.
Surprisingly, we have now found that assay techniques such as those described above can be WO 90/15985 ~ ~ ~ '3 ' ~ ~ PCf/GB90%00953 ~~r:l-~~~, ~i implemented without the need for excessive filtering or costly and complex systems of lenses, by using instead an additional optical structure placed close to the edge of said waveguide to provide efficient angular -discrimination of fluorescence emission, or by analogy phosphorescence or luminescence emission.
Thus, according to one aspect of the invention we provide a method of optical analysis of a test sample having fluorescent, phosphorescent or luminescent properties, which sample is partly in a liquid phase and partly bound, directly or indirectly, to an adjacent solid surface, comprising the steps of (i) providing as said solid sur~ace a surface of a transparent (at least at the wavelengths of radiation involved in the analysis) solid optical waveguide;
(ii) measuring light from the sample material bound to said solid surface which has propagated through said waveguide and emerged therefrom at an angle deviating from the optical axis of said waveguide by an angle having a magnitude less than the magnitude of a where a ~ arcsin (n22 - n12)', wherein n2 is the refractive index of the material of the waveguide and n1 is the refractive index of the adjacent liquid; and (iii)excluding from said measurement substantially all light emerging from said waveguide at an angle that deviates from said optical axis by ~a~ or more, said method being characterized in that an optical structure comprising a layer of transparent material of refractive index n3 interposed directly between layers of transparent material of refractive index n4, [the materials of refractive indices n3 and n4 being chosen such that n 2 - n 2 ~ n 2 - n 2 3 4 2 1 ' WO 90/ i 5985 ~ ~' ~ ~ -~ L ~ PCT/GB90/00953 and the layers of material of refractive index n4 being adapted such that the major part of the light passing into them from the layer of refractive index n3 does not pass back into said layer of refractive index n3)-is 5 placed close to the edge of said waveguide such that substantially all evanescently coupled light emerging from the edge of said waveguide passes into the said layer of refractive index n3.
In a preferred embodiment of the invention, the external surfaces only of the layers of material of refractive index n4 are coated with a light-absorbing material such that light passing through the layers is significantly attenuated at the interface of the light-absorbing material and the material of refractive index, n4. In general, attenuation at the said interface will be such that at least 800 of the light impinging on the interface is absorbed. Other embodiments will be described hereinafter.
It should be understood from the above discussion that the refractive index n2 is greater than nl, and correspondingly n3 is greater than n4.
Although, for reasons of practical convenience, the external surfaces of the layers of material of refractive index n4 of the optical structure are substantially parallel, this is not essential, and the optical structure may take various geometrical forms, e.g. of trapeziform cross-section.
In another aspect, the invention comprises apparatus suitable for carrying out a method of optical analysis as defined above comprising (i) a test object such as a slide or cell located at a test object location, suitable for containing a sample material with fluorescent, phosphorescent or luminescent properties, said test object comprising a transparent solid body, e.g, a sheet or fibre, to act as a ' waveguide, and to the surface of which the sample material can become bound from a solution or liquid WO 90/15985 ;-, ~ ~ ,~ , ~ _~ PCT/GB90/00953 ci ~.~ i l3 t~
More particularly, the invention relates to methods of, and apparatus for, detecting evanescent signals produced during ligand/specific binding partner assay procedures, particularly immunoassays.
The prior art contains various disclosures of analytical devices for handling and metering small volumes of test samples, and for the use of such devices in methods of optical analysis.
For example, US Patent 3939350 describes optical measurement of fluorescent material bound to the surface of a solid transparent prism by a method involving single total internal reflection and interaction of the evanescent wave at the surface of the prism with the bound material.
EP-A-75353 makes reference to the exponentially decaying (evanescent) external radiation due to light which is propagated longitudinally in a fibre, and its interaction with coatings.
EP-A-170376 describes, inter alia, methods of optical analysis of fluorescence emerging from a waveguide which is part of a chemical test device, whilst EP-A-171148 describes in detail certain chemical test devices, namely fluorescence capillary fill devices, and methods for their manufacture.
As described in EP-A-171148, a fluorescence capillary fill device (hereinafter FCFD) typically consists of two pieces of transparent material, e.g.
glass, separated by a narrow gap. one plate acts as an optical waveguide and carries an immobilised reagent appropriate to the test to be carried out in the device.
WO 90/ 15985 ~ ~~ ~ ~~ ~ ~ ~ PCT/G B90/00953 For use in an immunoassay, the immobilised reagent may for example be an antibody to an antigen desired to be detected and one of the plates may carry a dissoluble reagent comprising antigen labelled with a fluorescent dye. When a sample is presented to one end of the FCFD
it is drawn into the gap by capillary action and dissolves the reagent. In a competition assay, the fluorescently labelled antigen will compete with sample antigen for the limited number of antibody binding sites immobilised on the waveguide. Because the capillary gap is narrow (typically about 100 microns) the reaction -will generally go to completion in a short time, possibly less than 5 minutes. In a sandwich assay, the waveguide will carry a specific binding partner for the ligand desired to be detected and one of the plates will carry a dissoluble reagent comprising a further specific binding partner labelled with a fluorescent dye. In a sandwich immunoassay for an antigen, a sample antigen will form a sandwich complex with a fluorescently labelled antibody and an antibody immobilised on the waveguide. Thus the amount of fluorescently labelled antibody which becomes indirectly bound to the waveguide by virtue of complex formation will be a function of the concentration of antigen in the sample.
The term "antigen" as used herein will be under-stood to include both antigenic species (for example, proteins, bacteria, bacterial fragments, cells, cell fragments and viruses) and haptens which may be rendered antigenic under suitable conditions.
When using an FCFD in an assay, evanescent wave coupling avoids the need for a separation or washing step. When the labelled reagent is optically excited by a light source of suitable wavelength, fluorescence emission occurs both from fluorophores in the liquid phase and from fluorophores bound, directly or indirectly, to the waveguide. The optical properties of the media involved are such that most of the CVO 90/1'5985 P(T/G B90/00953 fluorescence arising from surface-bound fluorophores emerges from the transverse edge of said waveguide at angles to the normal whose magnitude is less than the magnitude of a certain angle a, whilst substantially all of the fluorescence arising from fluorophores in the liquid phase emerges from the edge of said waveguide at angles to the normal whose magnitude is greater than the magnitude of a (a being such that -%=n<a<1-,a). The value of a depends on the refractive indices of the liquid solution in the cavity and of the material of the waveguide anti on the wavelengths of light involved, but is given approximately by a ~ arcsin (n22 - n12)~~, wherein nl represents the refractive index of the liquid phase and n2 represents the refractive index of the solid waveguide. Thus, under ideal conditions, angular discrimination of fluorescence emerging from the edge of the waveguide would be sufficient to separate the different emission signals.
Although the discussion herein is conducted with reference to angles to the normal whose magnitude is compared to the magnitude of a as defined herein, it should be understood that these angles may deviate from said normal in either a positive or negative sense.
In practice, however, the sensitivity of assays employing simple angular discrimination of fluorescence emission is limited by a number of experimental factors including, inter a 'a: inefficient filtering out of the light of excitation wavelength and secondary fluorescence in all directions from the optical filter (typically of colloidal glass); and aberration of the lenses used to focus the fluorescence emission onto the detectors.
Surprisingly, we have now found that assay techniques such as those described above can be WO 90/15985 ~ ~ ~ '3 ' ~ ~ PCf/GB90%00953 ~~r:l-~~~, ~i implemented without the need for excessive filtering or costly and complex systems of lenses, by using instead an additional optical structure placed close to the edge of said waveguide to provide efficient angular -discrimination of fluorescence emission, or by analogy phosphorescence or luminescence emission.
Thus, according to one aspect of the invention we provide a method of optical analysis of a test sample having fluorescent, phosphorescent or luminescent properties, which sample is partly in a liquid phase and partly bound, directly or indirectly, to an adjacent solid surface, comprising the steps of (i) providing as said solid sur~ace a surface of a transparent (at least at the wavelengths of radiation involved in the analysis) solid optical waveguide;
(ii) measuring light from the sample material bound to said solid surface which has propagated through said waveguide and emerged therefrom at an angle deviating from the optical axis of said waveguide by an angle having a magnitude less than the magnitude of a where a ~ arcsin (n22 - n12)', wherein n2 is the refractive index of the material of the waveguide and n1 is the refractive index of the adjacent liquid; and (iii)excluding from said measurement substantially all light emerging from said waveguide at an angle that deviates from said optical axis by ~a~ or more, said method being characterized in that an optical structure comprising a layer of transparent material of refractive index n3 interposed directly between layers of transparent material of refractive index n4, [the materials of refractive indices n3 and n4 being chosen such that n 2 - n 2 ~ n 2 - n 2 3 4 2 1 ' WO 90/ i 5985 ~ ~' ~ ~ -~ L ~ PCT/GB90/00953 and the layers of material of refractive index n4 being adapted such that the major part of the light passing into them from the layer of refractive index n3 does not pass back into said layer of refractive index n3)-is 5 placed close to the edge of said waveguide such that substantially all evanescently coupled light emerging from the edge of said waveguide passes into the said layer of refractive index n3.
In a preferred embodiment of the invention, the external surfaces only of the layers of material of refractive index n4 are coated with a light-absorbing material such that light passing through the layers is significantly attenuated at the interface of the light-absorbing material and the material of refractive index, n4. In general, attenuation at the said interface will be such that at least 800 of the light impinging on the interface is absorbed. Other embodiments will be described hereinafter.
It should be understood from the above discussion that the refractive index n2 is greater than nl, and correspondingly n3 is greater than n4.
Although, for reasons of practical convenience, the external surfaces of the layers of material of refractive index n4 of the optical structure are substantially parallel, this is not essential, and the optical structure may take various geometrical forms, e.g. of trapeziform cross-section.
In another aspect, the invention comprises apparatus suitable for carrying out a method of optical analysis as defined above comprising (i) a test object such as a slide or cell located at a test object location, suitable for containing a sample material with fluorescent, phosphorescent or luminescent properties, said test object comprising a transparent solid body, e.g, a sheet or fibre, to act as a ' waveguide, and to the surface of which the sample material can become bound from a solution or liquid WO 90/15985 ;-, ~ ~ ,~ , ~ _~ PCT/GB90/00953 ci ~.~ i l3 t~
dispersion contacting the solid body; and (ii) an optical structure as defined hereinbefore, which in use can be positioned so that when a test object is at the test object location the optical structure is optically coupled to the transparent solid body of the test object, such that light propagating in the transparent solid body can be received by the optical structure.
In order to minimise degradation of the information contained in the light signal emerging from the transparent solid body as a result of, for example, malformations at the optical edge of said body, it may be desirable for the said body to be optically coupled to the optical structure by means of an index-matching technique.
Thus, in order to reduce scattering of light emerging from an optical edge of an optical waveguide, said optical edge is maintained in intimate contact with an index-matching substance which itself also forms or intimately contacts a further optical component.
By "index-matching substance" is meant a substance having a refractive index similar to that of the material of the waveguide, e.g. having a refractive index differing from that of the waveguide by up to 20%.
Intimate contact between the optical edge and the index-matching substance may be achieved by, for example, selecting an index-matching substance which is a liquid or gel, or by employing precursors for a substantially transparent solid which pliably moulds to the surface of the optical edge before subsequently setting or otherwise solidifying. Examples of suitable index-matching substances include cedar oil, Canada balsam, silicones, ethanol, pentanol, phenylamine, , benzene, glycerol, paraffin oil, turpentine, silicone gels, plastics such as silane elastomers, optical cements or adhesives selected from appropriate epoxy and acrylate systems.
N'() 90/15985 ~ ~ ~' ~ PCT/GB90/00953 The test object may, for example, be a device of the general type described in EP-A-171148.
If desired, the test object (i) and the optical structure (ii) may be coupled together so as to form an S integral structure.
The apparatus may further comprise one or more of the following: a photodetector arranged so that, in use, the optical structure is optically coupled to the photodetector, such that light propagating in the optical structure can be received by the photodetector after its emergence;
a light source arranged so that, in use, it can illuminate the test material with light (e. g. at some transverse angle to the principal direction of propagation of the light in the waveguide, for example, at or about 90°), such that light from the test material passes into and through both the transparent solid body of the test object and the optical structure, via total internal reflections to the photodetector; an optical filter (which may be secured in intimate contact with the optical structure) to allow differential transmission through the optical train of the wavelengths of light involved in the method; and a microprocessor or similar processing unit, adapted to analyse in a desired manner the electrical signal emerging from the photodetector.
The invention will be more particularly described hereinafter with reference to fluorescence but it will be understood that analogous considerations apply to phosphorescence or luminescence.
The invention relies on the angular distribution of the light emerging from the waveguide part of the test object, which is dependent on the amounts of fluorophores which are in solution and those which are adsorbed on the surface of said waveguide. Suitable materials for the waveguide are known in the art (see, for example, EP-A-170376 and EP-A-171148) and include, WO 90/15985 ~.a ~.. - PCT/GIi90/00953 inter alia, glass, silica and plastics materials; in these cases light from material in a liquid adjacent to the waveguide, where said liquid has a refractive index close to that of water, i.e. lower than that of the waveguide, emerges from the waveguide at rather large angles to the axis thereof. Fluorescent, phosphorescent or luminescent molecules bound to the surface of the waveguide can additionally emit light into angles which would be "forbidden" by classical ray optics (i.e.
angles greater than the critical angle for the waveguide/sample interface), by the known evanescent wave phenomenon.
In the case of a glass waveguide and an aqueous solution, and fluorescent light of a wavelength of 510 nm, the angle a given by a = arcsin (n22 - nl2)-is approximately ~47°. Thus light from an adsorbed layer can be distinguished from that arising from the liquid by the emergence of the former light from the waveguide over a wider range of angles, including angles deviating from the axis of the waveguide by less than about 47°, The optical structure of the method of the invention leads to enhanced discrimination in use, by guiding substantially all the evanescently coupled light arising from surface-bound fluorophores while causing radiation emerging from the waveguide of the test object at larger angles to be removed. Said optical structure may be placed adjacent to the end of the waveguide of said test object, thereby minimizing loss of fluorescent radiation. The angular discrimination of fluorescence is achieved by use of an optical structure the layers of which are composed of materials of suitably chosen refractive index.
For a better understanding of the present l/ C~ ti s ~~ -invention, reference is made to the accompanying drawings wherein:-Figure 1 shows diagrammatically a prior art arrangement for a method of analysis, said arrangement being such as is described in EP-A-170376.
Figure 2(a) shows by means of a diagrammatic section how, in use, optical coupling between the test object as hereinbefore described and one embodiment of an optical structure as hereinbefore described leads to angular discrimination of fluorescence. Figure 2(b) shows an alternative embodiment of the optical structure.
Figure 3 shows (a) a transverse cross-section and (b) a longitudinal cross-section through an alternative optical structure which may be employed in accordance with the invention.
Figure 4 shows in detail an optical structure fabricated for use in accordance with the present invention.
Figure 5 shows graphically the results of an assay carried out in accordance with the invention.
Referring to Figure 1, the sample liquid 1 is contained within the test object 2, in this case a fluorescence capillary fill device. During the course of the test being carried out, fluorophores within the sample liquid may become bound to a thin layer of material (not shown) at the surface of the waveguide 3 of the test object. The sample liquid 1 is contained in the volume between the layer of material 4 and the waveguide 3. In the embodiment shown in Figure 1, illumination is provided by a light source 5, whose output may be focused using lenses 6, 7 and filtered by means of a filter stack 8 to provide light of suitable excitation wavelength. The waveguide 3 has an edge 9 which is substantially both smooth and perpendicular to the long dimensions. Light from the liquid 1 and from the surface layer may propagate through the waveguide wO 90/15985 ~ ~ ~ .~ ~ ~ _ PCf /G B90/00953 c_:,~:s with multiple internal reflections, emerging from the edge 9. Light emerging directly out of the device arising from the liquid sample is removed by means of a mask 11 with an aperture. A photodetector 10 is' 5 disposed so that it receives light emerging from the edge 9 which diverges from the long axis of the waveguide by an angle whose magnitude is not greater than a as defined hereinbefore. This is achieved by screening by, for example, an apertured screen 15, with 10 which may be associated one or more convex lenses 12, 13 to collect the light exiting over the desired angular range on to the photodetector. Light emerging from the edge 9 may be filtered by means of a filter 14 so that light of excitation wavelength may be excluded from reaching the detector 10.
It should be emphasised that Figure 1 shows the arrangement of apparatus diagrammatically. As a practical matter, it would in this prior art arrangement be preferable for efficient detection to have a 45' prism (not shown) in contact with the edge 9 as the first element in the optical detection train.
Figure 2 shows how the present invention may lead to enhanced discrimination between light arising from surface-bound fluorophores and light arising from fluorophores in the sample liquid, whilst greatly simplifying the optical train. As in Figure 1, the sample liquid 21 of refractive index nl is contained within the layer 23 of solid transparent material of refractive index n2 and the layer 22, layer 23 being suitable to act as a waveguide. An optical structure 24 is positioned close to the edge 25 of the waveguide such that substantially all the light impinging on the edge 25 of the waveguide passes into the layer 27 of solid material of refractive index n3 contained within the optical structure, via the end aperture 26. In general, the thickness of layer 27 should be greater than or equal to the thickness of layer 23 for optimum optical ;.~ 'v v ~~ - ~ -WO 90/ ~ 5985 PCT/GB90/00953 coupling.
Under certain circumstances it may be desirable to extend the opaque coating 30 over the ends 31 and 32 of the optical structure 24 to define the aperture to be of the same dimensions as, or smaller than, the optical edge 25.
The material of the layer 27 will in general have a refractive index n3 which is greater than n2, but this is not essential. Suitable materials for the layer 27 include, for example, optical glass, silica, phosphate glasses and polymeric materials such as crystal styrene.
In some embodiments it may be desirable that layer 27 be a liquid, confined within the optical structure 24 by suitable means (e. g. thin layers of strong transparent material at the ends). Disposed on either side of layer , 27 are layers 28 and 29 of transparent solid material, both layers usually having refractive index n4, whose external surfaces are in this embodiment coated with a light-absorbing coating 30. Alternatively, layers 28 and/or 29 may be a liquid confined within the optical structure 24 by suitable means, provided always that no total internal reflection occurs at the interface between the liquid and its confining means.
In the embodiment shown, it is essential that the external surfaces of the optical structure which are substantially parallel to the long axis of said structure should be substantially totally coated with the coating 30, for reasons to be discussed hereinbelow.
It is also desirable, but not essential, that the coating 30 extends to cover the ends 31 and 32 of the layers 28 and 29. As an alternative, a physical aperture could be used, to cover ends 31 and 32 of the optical structure when in use. It is by no means essential that the external surfaces of the optical structure are parallel to the long axis thereof. Thus, by way of example, as shown in Fig. 2(b), an optical structure may be produced which has a cross-section the WO 90/15985 ~ ~ 3 1 PCT/G B9tl/00953 shape of a trapezium.
It is also desirable, but not essential,that the coating 30 extends to cover the ends 37 and 39 of the layers 28 and 29. As an alternative, a physical-s aperture could be used, to cover ends 37 and 39 of the optical structure when in use.
Suitable materials for layers 28 and 29 include silica, magnesium fluoride, lithium fluoride, optical glasses (e. g. BK7 or crown glass), phosphate glasses, polymeric materials (e.g. acrylic or silicones) and liquids. For suitable materials (e. g. phosphate glasses), the layers 28 and 29 may, if desired, be applied by spin-coating. The thickness of the layers 28 and 29 should be greater than 2 microns.
The choice of materials for the layers 27, 2o and 29 is important to the success of the method of the invention. In use, the angular discrimination of light 35 arising from fluorophores bound to the surface 33 from light 36 arising from the sample liquid 21 is achieved by careful choice of refractive indices n3 and n4. In general, these will be such that n3 _ n4 ~ n2 _ n1 In some circumstances, however, it may be desirable that the quantity (n32 - n42) is rather less than suggested by the above equation; this will have the effect of causing less of the light emerging from end 25 to be collected by the photodetector from the optical structure 24, particularly at angles close to a as defined hereinbefore.
Suitable materials for the light-absorbing coating 30 include black paint, optionally curable by heat or infrared radiation, silicone paint, dyes, etc., which may be applied by conventional methods such as screen printing or, if appropriate, by spin-coating or spraying. Such materials should attenuate or absorb WO 90/15985 ~ ~ '~ '~ ~' ~ ~ PCT/GB90/00953 light at least at the wavelengths involved in the assay.
The minimum thickness of the coating 30 will typically be 3 microns. The optical structure 24 performs the same role as the components 11-15 of Figure 1 but is much more convenient to use. Furthermore, in contrast to the prior art situation in which a prism was placed in contact with the optical edge of the waveguide, there is in the present invention no necessity for the optical structure as herein defined to be in contact with the said edge: an intervening gap of several millimetres (when using the apparatus described in Example 1 hereinafter) may be present without serious loss of sensitivity.
In a further embodiment, the external surfaces of the layers 28 and 29 of the optical structure 24 may be roughened, so that light refracted into layers 28 and 29 is scattered out of the optical structure 24. In such an embodiment, it is preferred that the external surfaces of layers 28 and 29 be roughened such that substantially all of the light refracted into layers 28 and 29 is scattered out of the optical structure 24. In such circumstances it may be desirable, but not essential, to apply a coating 30 to the optical structure 24 as already described. In an alternative embodiment, light refracted into layers 28 and 29 may be scattered out of the optical structure 24 by providing that the external surfaces of the layers take the form of suitable diffraction gratings.
In a still further embodiment, the thicknesses of the layers 28 and 29 may be sufficiently great that substantially no light propagating in layers 28 and 29 can re-enter layer 27. In such an embodiment it is preferred that layers 28 and 29 be sufficiently thick that substantially no light propagating therein re-enters layer 27. In such a case the thickness h of layer 28 and/or 29 is given by WO 90/15985 ~ ~ ~ ~ ~ ~ ~ PCT/GB90%00953 ;'.':
In order to minimise degradation of the information contained in the light signal emerging from the transparent solid body as a result of, for example, malformations at the optical edge of said body, it may be desirable for the said body to be optically coupled to the optical structure by means of an index-matching technique.
Thus, in order to reduce scattering of light emerging from an optical edge of an optical waveguide, said optical edge is maintained in intimate contact with an index-matching substance which itself also forms or intimately contacts a further optical component.
By "index-matching substance" is meant a substance having a refractive index similar to that of the material of the waveguide, e.g. having a refractive index differing from that of the waveguide by up to 20%.
Intimate contact between the optical edge and the index-matching substance may be achieved by, for example, selecting an index-matching substance which is a liquid or gel, or by employing precursors for a substantially transparent solid which pliably moulds to the surface of the optical edge before subsequently setting or otherwise solidifying. Examples of suitable index-matching substances include cedar oil, Canada balsam, silicones, ethanol, pentanol, phenylamine, , benzene, glycerol, paraffin oil, turpentine, silicone gels, plastics such as silane elastomers, optical cements or adhesives selected from appropriate epoxy and acrylate systems.
N'() 90/15985 ~ ~ ~' ~ PCT/GB90/00953 The test object may, for example, be a device of the general type described in EP-A-171148.
If desired, the test object (i) and the optical structure (ii) may be coupled together so as to form an S integral structure.
The apparatus may further comprise one or more of the following: a photodetector arranged so that, in use, the optical structure is optically coupled to the photodetector, such that light propagating in the optical structure can be received by the photodetector after its emergence;
a light source arranged so that, in use, it can illuminate the test material with light (e. g. at some transverse angle to the principal direction of propagation of the light in the waveguide, for example, at or about 90°), such that light from the test material passes into and through both the transparent solid body of the test object and the optical structure, via total internal reflections to the photodetector; an optical filter (which may be secured in intimate contact with the optical structure) to allow differential transmission through the optical train of the wavelengths of light involved in the method; and a microprocessor or similar processing unit, adapted to analyse in a desired manner the electrical signal emerging from the photodetector.
The invention will be more particularly described hereinafter with reference to fluorescence but it will be understood that analogous considerations apply to phosphorescence or luminescence.
The invention relies on the angular distribution of the light emerging from the waveguide part of the test object, which is dependent on the amounts of fluorophores which are in solution and those which are adsorbed on the surface of said waveguide. Suitable materials for the waveguide are known in the art (see, for example, EP-A-170376 and EP-A-171148) and include, WO 90/15985 ~.a ~.. - PCT/GIi90/00953 inter alia, glass, silica and plastics materials; in these cases light from material in a liquid adjacent to the waveguide, where said liquid has a refractive index close to that of water, i.e. lower than that of the waveguide, emerges from the waveguide at rather large angles to the axis thereof. Fluorescent, phosphorescent or luminescent molecules bound to the surface of the waveguide can additionally emit light into angles which would be "forbidden" by classical ray optics (i.e.
angles greater than the critical angle for the waveguide/sample interface), by the known evanescent wave phenomenon.
In the case of a glass waveguide and an aqueous solution, and fluorescent light of a wavelength of 510 nm, the angle a given by a = arcsin (n22 - nl2)-is approximately ~47°. Thus light from an adsorbed layer can be distinguished from that arising from the liquid by the emergence of the former light from the waveguide over a wider range of angles, including angles deviating from the axis of the waveguide by less than about 47°, The optical structure of the method of the invention leads to enhanced discrimination in use, by guiding substantially all the evanescently coupled light arising from surface-bound fluorophores while causing radiation emerging from the waveguide of the test object at larger angles to be removed. Said optical structure may be placed adjacent to the end of the waveguide of said test object, thereby minimizing loss of fluorescent radiation. The angular discrimination of fluorescence is achieved by use of an optical structure the layers of which are composed of materials of suitably chosen refractive index.
For a better understanding of the present l/ C~ ti s ~~ -invention, reference is made to the accompanying drawings wherein:-Figure 1 shows diagrammatically a prior art arrangement for a method of analysis, said arrangement being such as is described in EP-A-170376.
Figure 2(a) shows by means of a diagrammatic section how, in use, optical coupling between the test object as hereinbefore described and one embodiment of an optical structure as hereinbefore described leads to angular discrimination of fluorescence. Figure 2(b) shows an alternative embodiment of the optical structure.
Figure 3 shows (a) a transverse cross-section and (b) a longitudinal cross-section through an alternative optical structure which may be employed in accordance with the invention.
Figure 4 shows in detail an optical structure fabricated for use in accordance with the present invention.
Figure 5 shows graphically the results of an assay carried out in accordance with the invention.
Referring to Figure 1, the sample liquid 1 is contained within the test object 2, in this case a fluorescence capillary fill device. During the course of the test being carried out, fluorophores within the sample liquid may become bound to a thin layer of material (not shown) at the surface of the waveguide 3 of the test object. The sample liquid 1 is contained in the volume between the layer of material 4 and the waveguide 3. In the embodiment shown in Figure 1, illumination is provided by a light source 5, whose output may be focused using lenses 6, 7 and filtered by means of a filter stack 8 to provide light of suitable excitation wavelength. The waveguide 3 has an edge 9 which is substantially both smooth and perpendicular to the long dimensions. Light from the liquid 1 and from the surface layer may propagate through the waveguide wO 90/15985 ~ ~ ~ .~ ~ ~ _ PCf /G B90/00953 c_:,~:s with multiple internal reflections, emerging from the edge 9. Light emerging directly out of the device arising from the liquid sample is removed by means of a mask 11 with an aperture. A photodetector 10 is' 5 disposed so that it receives light emerging from the edge 9 which diverges from the long axis of the waveguide by an angle whose magnitude is not greater than a as defined hereinbefore. This is achieved by screening by, for example, an apertured screen 15, with 10 which may be associated one or more convex lenses 12, 13 to collect the light exiting over the desired angular range on to the photodetector. Light emerging from the edge 9 may be filtered by means of a filter 14 so that light of excitation wavelength may be excluded from reaching the detector 10.
It should be emphasised that Figure 1 shows the arrangement of apparatus diagrammatically. As a practical matter, it would in this prior art arrangement be preferable for efficient detection to have a 45' prism (not shown) in contact with the edge 9 as the first element in the optical detection train.
Figure 2 shows how the present invention may lead to enhanced discrimination between light arising from surface-bound fluorophores and light arising from fluorophores in the sample liquid, whilst greatly simplifying the optical train. As in Figure 1, the sample liquid 21 of refractive index nl is contained within the layer 23 of solid transparent material of refractive index n2 and the layer 22, layer 23 being suitable to act as a waveguide. An optical structure 24 is positioned close to the edge 25 of the waveguide such that substantially all the light impinging on the edge 25 of the waveguide passes into the layer 27 of solid material of refractive index n3 contained within the optical structure, via the end aperture 26. In general, the thickness of layer 27 should be greater than or equal to the thickness of layer 23 for optimum optical ;.~ 'v v ~~ - ~ -WO 90/ ~ 5985 PCT/GB90/00953 coupling.
Under certain circumstances it may be desirable to extend the opaque coating 30 over the ends 31 and 32 of the optical structure 24 to define the aperture to be of the same dimensions as, or smaller than, the optical edge 25.
The material of the layer 27 will in general have a refractive index n3 which is greater than n2, but this is not essential. Suitable materials for the layer 27 include, for example, optical glass, silica, phosphate glasses and polymeric materials such as crystal styrene.
In some embodiments it may be desirable that layer 27 be a liquid, confined within the optical structure 24 by suitable means (e. g. thin layers of strong transparent material at the ends). Disposed on either side of layer , 27 are layers 28 and 29 of transparent solid material, both layers usually having refractive index n4, whose external surfaces are in this embodiment coated with a light-absorbing coating 30. Alternatively, layers 28 and/or 29 may be a liquid confined within the optical structure 24 by suitable means, provided always that no total internal reflection occurs at the interface between the liquid and its confining means.
In the embodiment shown, it is essential that the external surfaces of the optical structure which are substantially parallel to the long axis of said structure should be substantially totally coated with the coating 30, for reasons to be discussed hereinbelow.
It is also desirable, but not essential, that the coating 30 extends to cover the ends 31 and 32 of the layers 28 and 29. As an alternative, a physical aperture could be used, to cover ends 31 and 32 of the optical structure when in use. It is by no means essential that the external surfaces of the optical structure are parallel to the long axis thereof. Thus, by way of example, as shown in Fig. 2(b), an optical structure may be produced which has a cross-section the WO 90/15985 ~ ~ 3 1 PCT/G B9tl/00953 shape of a trapezium.
It is also desirable, but not essential,that the coating 30 extends to cover the ends 37 and 39 of the layers 28 and 29. As an alternative, a physical-s aperture could be used, to cover ends 37 and 39 of the optical structure when in use.
Suitable materials for layers 28 and 29 include silica, magnesium fluoride, lithium fluoride, optical glasses (e. g. BK7 or crown glass), phosphate glasses, polymeric materials (e.g. acrylic or silicones) and liquids. For suitable materials (e. g. phosphate glasses), the layers 28 and 29 may, if desired, be applied by spin-coating. The thickness of the layers 28 and 29 should be greater than 2 microns.
The choice of materials for the layers 27, 2o and 29 is important to the success of the method of the invention. In use, the angular discrimination of light 35 arising from fluorophores bound to the surface 33 from light 36 arising from the sample liquid 21 is achieved by careful choice of refractive indices n3 and n4. In general, these will be such that n3 _ n4 ~ n2 _ n1 In some circumstances, however, it may be desirable that the quantity (n32 - n42) is rather less than suggested by the above equation; this will have the effect of causing less of the light emerging from end 25 to be collected by the photodetector from the optical structure 24, particularly at angles close to a as defined hereinbefore.
Suitable materials for the light-absorbing coating 30 include black paint, optionally curable by heat or infrared radiation, silicone paint, dyes, etc., which may be applied by conventional methods such as screen printing or, if appropriate, by spin-coating or spraying. Such materials should attenuate or absorb WO 90/15985 ~ ~ '~ '~ ~' ~ ~ PCT/GB90/00953 light at least at the wavelengths involved in the assay.
The minimum thickness of the coating 30 will typically be 3 microns. The optical structure 24 performs the same role as the components 11-15 of Figure 1 but is much more convenient to use. Furthermore, in contrast to the prior art situation in which a prism was placed in contact with the optical edge of the waveguide, there is in the present invention no necessity for the optical structure as herein defined to be in contact with the said edge: an intervening gap of several millimetres (when using the apparatus described in Example 1 hereinafter) may be present without serious loss of sensitivity.
In a further embodiment, the external surfaces of the layers 28 and 29 of the optical structure 24 may be roughened, so that light refracted into layers 28 and 29 is scattered out of the optical structure 24. In such an embodiment, it is preferred that the external surfaces of layers 28 and 29 be roughened such that substantially all of the light refracted into layers 28 and 29 is scattered out of the optical structure 24. In such circumstances it may be desirable, but not essential, to apply a coating 30 to the optical structure 24 as already described. In an alternative embodiment, light refracted into layers 28 and 29 may be scattered out of the optical structure 24 by providing that the external surfaces of the layers take the form of suitable diffraction gratings.
In a still further embodiment, the thicknesses of the layers 28 and 29 may be sufficiently great that substantially no light propagating in layers 28 and 29 can re-enter layer 27. In such an embodiment it is preferred that layers 28 and 29 be sufficiently thick that substantially no light propagating therein re-enters layer 27. In such a case the thickness h of layer 28 and/or 29 is given by WO 90/15985 ~ ~ ~ ~ ~ ~ ~ PCT/GB90%00953 ;'.':
h > =,d(n4-1) ' where d is the length of the optical structure and n4 is defined as hereinbefore. In such circumstances it may still be desirable, but not essential, to apply a coating 30 to the optical structure 24 as already described.
An optical filter (e.g. of colloidal glass) may optionally be incorporated either into or onto the optical structure 24 at the end 26. If the optical filter is incorporated into the optical structure 24 (as in the particular embodiment illustrated in Fig. 4) then it should preferably be made of material having the same, or higher, refractive index as layer 27. When a filter glass of appropriate refractive index cannot be found, the filter 20 may be bound onto the end 26 of layer 27, as is shown in the embodiment of Fig. 2(b).
As a result of the optics of the method of the present invention, any secondary fluorescence arising in this filter and transmitted to the photodetector is minimised. Alternatively, the optical filter can be incorporated either into or onto the optical structure at the end 38.
If the presence of interference filters were desirable in the detection optics, for example to reduce spurious fluorescence and/or phosphorescence arising within the device, suitable multilayer filters could be deposited onto the face 26 and/or the face 38 of the optical structure 24, for example by means of vacuum deposition techniques known ,per se. Alternatively a pre-formed interference filter may be optically coupled onto the face 26 and/or the face 38 of the optical structure.
Figure 3(a) shows a transverse cross-section through a cylindrical variant of the optical structure to be used in accordance with the invention. A
cylindrical layer 40 of material of refractive index n.~
wo 9nms9ss ~ ~ ~ ~ ~ ' is surrounded by a coaxial layer 41 of material of refractive index n4. As previously described, the external surfaces of the layer 41 are coated with a suitable opaque material. -5 Figure 3(b) shows a longitudinal cross-section through this variant, which accords with the structure shown in Figure 2.
Cylindrical structures such as shown in Figure 3(a) ' and 3(b) can advantageously be used in conjunction with 10 cylindrical biosensors such as described, for example, in US 4447546.
In use, the photodetector as hereinbefore described, e.g. a photomultiplier tube, could be placed close to the end of the optical structure from which the 15 fluorescent light emerges, so as to detect directly the light emerging from said optical structure without necessity for additional lenses. Such an arrangement would minimize loss of signal due to reflection.
However, if desired, additional lenses may be used as in conventional methods.
An alternative arrangement may be to optically couple a suitable photodetector (e. g. a silicon photo-detector) to the end face 38 of the optical structure 24.
The discussion above is made with particular reference to immunoassays involving fluorescence capillary fill devices (FCFD's), but it is to be understood that the invention applies also to other methods and devices which involve angular discrimination of optical signals emerging from waveguides, particularly those involving evanescent signals.
The method of the invention is particularly applicable to assays e.g. of antigens or antibodies, i.e. to immunoassays. However, the invention is not to be taken as limited to assays of antibodies or antigens.
Examples of ligands which may be assayed in conjunction with the method of the invention are given in Table 1 PCf/G B90/00953 ~Y::.
An optical filter (e.g. of colloidal glass) may optionally be incorporated either into or onto the optical structure 24 at the end 26. If the optical filter is incorporated into the optical structure 24 (as in the particular embodiment illustrated in Fig. 4) then it should preferably be made of material having the same, or higher, refractive index as layer 27. When a filter glass of appropriate refractive index cannot be found, the filter 20 may be bound onto the end 26 of layer 27, as is shown in the embodiment of Fig. 2(b).
As a result of the optics of the method of the present invention, any secondary fluorescence arising in this filter and transmitted to the photodetector is minimised. Alternatively, the optical filter can be incorporated either into or onto the optical structure at the end 38.
If the presence of interference filters were desirable in the detection optics, for example to reduce spurious fluorescence and/or phosphorescence arising within the device, suitable multilayer filters could be deposited onto the face 26 and/or the face 38 of the optical structure 24, for example by means of vacuum deposition techniques known ,per se. Alternatively a pre-formed interference filter may be optically coupled onto the face 26 and/or the face 38 of the optical structure.
Figure 3(a) shows a transverse cross-section through a cylindrical variant of the optical structure to be used in accordance with the invention. A
cylindrical layer 40 of material of refractive index n.~
wo 9nms9ss ~ ~ ~ ~ ~ ' is surrounded by a coaxial layer 41 of material of refractive index n4. As previously described, the external surfaces of the layer 41 are coated with a suitable opaque material. -5 Figure 3(b) shows a longitudinal cross-section through this variant, which accords with the structure shown in Figure 2.
Cylindrical structures such as shown in Figure 3(a) ' and 3(b) can advantageously be used in conjunction with 10 cylindrical biosensors such as described, for example, in US 4447546.
In use, the photodetector as hereinbefore described, e.g. a photomultiplier tube, could be placed close to the end of the optical structure from which the 15 fluorescent light emerges, so as to detect directly the light emerging from said optical structure without necessity for additional lenses. Such an arrangement would minimize loss of signal due to reflection.
However, if desired, additional lenses may be used as in conventional methods.
An alternative arrangement may be to optically couple a suitable photodetector (e. g. a silicon photo-detector) to the end face 38 of the optical structure 24.
The discussion above is made with particular reference to immunoassays involving fluorescence capillary fill devices (FCFD's), but it is to be understood that the invention applies also to other methods and devices which involve angular discrimination of optical signals emerging from waveguides, particularly those involving evanescent signals.
The method of the invention is particularly applicable to assays e.g. of antigens or antibodies, i.e. to immunoassays. However, the invention is not to be taken as limited to assays of antibodies or antigens.
Examples of ligands which may be assayed in conjunction with the method of the invention are given in Table 1 PCf/G B90/00953 ~Y::.
below, together with an indication of a suitable specific binding partner in each instance.
Table 1 Ligand Specific Binding Partner antigen specific antibody antibody antigen hormone hormone receptor hormone receptor hormone polynucleotide strand complementary polynucleotide strand avidin biotin biotin avidin protein A immunoglobulin immunoglobulin protein A
enzyme enzyme cofactor (substrate) or inhibitor enzyme cofactor enzyme (substrate) or inhibitor lectins specific carbohydrate specific carbohydrate lectins of lectins The method of the invention has very broad applicability but in particular may be used in conjunction with assays of: hormones, including peptide hormones (e. g. thyroid stimulating hormone (TSH), luteinising hormone (LH), human chorionic gonadotrophin (hCG), follicle stimulating hormone (FSH), insulin and prolactin) or non-peptide hormones (e. g. steroid hormones such as cortisol, estradiol, progesterone and testosterone, or thyroid hormones such as thyroxine (T4) and triiodothyronine), proteins (e. g. carcinoembryonic antigen (CEA) and alphafetoprotein (AFP)), drugs (e. g.
digoxin), sugars, toxins, vitamins, proteins, viruses '~'O 90/15985 ~ ~ ~ ~ ~ ~ ~ PCT/GB90/00953 such as influenza, para-influenza, adeno-, hepatitis, respiratory and AIDS viruses, or microorganisms.
The following non-limiting Example is intended to ' illustrate the invention.
Example 1 An Optical immunoassay for human chorionic ~c~onadotrophin Preparation of Startincr Materials (i) Fabrication of an optical structure Figure 4 shows in more detail an optical structure of the type shown in Figs. 2 and 3, fabricated for use in accordance with the invention as follows:
From a plate of Souriel B89/61 optical glass, a piece 46 of dimensions 30.0 mm x 15.0 mm x 3.0 mm was cut using conventional techniques. After polishing all the surfaces of the glass, again using conventional techniques, the glass was glued to two pieces 47,48 of silica (33.0 mm x 15.0 mm x 1.0 mm) which had been cut and polished using standard methods. The adhesive used to bond the layers was Lensbond optical cement (refractive index 1.55). A piece 49 of filter glass (OG
515 colloidal glass), having been cut to the correct dimensions (15.0 mm x 3.0 mm x 3.0 mm) and polished in the normal manner, was glued into the optical structure, using the same adhesive as before. Finally, all the exposed silica surfaces (but not those of either the optical glass or the filter glass) of the optical structure were spray-painted with a black opaque coating (not shown).
(ii) Preparation of anti-hCG antibody coniugated to fluorescein isothiocyanate (FITC) 200 mg of FITC (Sigma Chemical Company Ltd., UK) and 5 mg of a second monoclonal antibody to hCG specific W~ 90/15985 PCT/GB90/00953 ;
Table 1 Ligand Specific Binding Partner antigen specific antibody antibody antigen hormone hormone receptor hormone receptor hormone polynucleotide strand complementary polynucleotide strand avidin biotin biotin avidin protein A immunoglobulin immunoglobulin protein A
enzyme enzyme cofactor (substrate) or inhibitor enzyme cofactor enzyme (substrate) or inhibitor lectins specific carbohydrate specific carbohydrate lectins of lectins The method of the invention has very broad applicability but in particular may be used in conjunction with assays of: hormones, including peptide hormones (e. g. thyroid stimulating hormone (TSH), luteinising hormone (LH), human chorionic gonadotrophin (hCG), follicle stimulating hormone (FSH), insulin and prolactin) or non-peptide hormones (e. g. steroid hormones such as cortisol, estradiol, progesterone and testosterone, or thyroid hormones such as thyroxine (T4) and triiodothyronine), proteins (e. g. carcinoembryonic antigen (CEA) and alphafetoprotein (AFP)), drugs (e. g.
digoxin), sugars, toxins, vitamins, proteins, viruses '~'O 90/15985 ~ ~ ~ ~ ~ ~ ~ PCT/GB90/00953 such as influenza, para-influenza, adeno-, hepatitis, respiratory and AIDS viruses, or microorganisms.
The following non-limiting Example is intended to ' illustrate the invention.
Example 1 An Optical immunoassay for human chorionic ~c~onadotrophin Preparation of Startincr Materials (i) Fabrication of an optical structure Figure 4 shows in more detail an optical structure of the type shown in Figs. 2 and 3, fabricated for use in accordance with the invention as follows:
From a plate of Souriel B89/61 optical glass, a piece 46 of dimensions 30.0 mm x 15.0 mm x 3.0 mm was cut using conventional techniques. After polishing all the surfaces of the glass, again using conventional techniques, the glass was glued to two pieces 47,48 of silica (33.0 mm x 15.0 mm x 1.0 mm) which had been cut and polished using standard methods. The adhesive used to bond the layers was Lensbond optical cement (refractive index 1.55). A piece 49 of filter glass (OG
515 colloidal glass), having been cut to the correct dimensions (15.0 mm x 3.0 mm x 3.0 mm) and polished in the normal manner, was glued into the optical structure, using the same adhesive as before. Finally, all the exposed silica surfaces (but not those of either the optical glass or the filter glass) of the optical structure were spray-painted with a black opaque coating (not shown).
(ii) Preparation of anti-hCG antibody coniugated to fluorescein isothiocyanate (FITC) 200 mg of FITC (Sigma Chemical Company Ltd., UK) and 5 mg of a second monoclonal antibody to hCG specific W~ 90/15985 PCT/GB90/00953 ;
for a different antigenic determinant were mixed together in 1.4 ml of 0.2 M sodium bicarbonate buffer solution (pH 9.0). The mixture was left for 18 hours at room temperature, during which conjugation of FI'I'C to the monoclonal antibody occurred. The mixture was then purified by gel filtration on Sephadex G-50 superfine.
(iii) Preparation of hCG standard solutions A freeze-dried preparation of hCG calibrated against the first international reference preparation (75/537) was obtained from Biodata SpA, Milan, Italy.
This sample Was diluted in horse serum (Serono Diagnostics Ltd., Woking UK) to give the range of hCG
standards required.
(iv) Apparatus used in the measurement of the hcg assay For the assay according to the present invention, components corresponding to labels 1-10 of Fig. 1 were used. The test object 2 was a fluorescence capillary fill device of a type disclosed in EP-A-171148, on the internal surface of the waveguide part 3 of which was immobilised by conventional methods a monoclonal antibody against hCG. The light source 5 was a Heinmann Xenon flash lamp. The filter stack 8 comprised three filters: a BG7 Schott glass filter (Ealing Electro Optics UK Ltd., Watford UK), a 450-480 nm FITC bandpass interference filter (Optometrics Ltd., UK) and a 475 nm shortpass interference filter (Comar Instruments Ltd., Cambridge UK).
The optical structure fabricated as in paragraph (i) above was placed adjacent to the edge 9 of the waveguide (see also Fig. 2). The photodetector 10 was a Hamamatsu R931A photomultiplier tube (Hakuto UK Ltd).
For comparison, a conventional assay using components labelled 11-15 in Fig. 1 was also carried out. In this latter case, the filter 14 used was a Schott OG515 515 nm colloidal glass filter.
'~O 90/15985 ~ ~ ~ r ~ PCT/GB90/00953 Assay Methodology The ahCG-FITC conjugate was added to each of the hCG standards to give about 0.5 ml of test solution. The conjugate concentration was 1 ~g/ml. Test cells were chosen and ~sach was filled with a different test solution. The signal from the photodetector was recorded after a 20-minute incubation period in a humid environment at 20°C.
Figure 5 shows graphically the results of an assay carried out using apparatus including an optical structure as described hereinbefore, in comparison with results from a conventional assay using apparatus shown schematically in Fig. 1. The assay carried out in accordance with the present invention exhibits significantly better precision than the prior art (5.10 compared with 9.0%).
(iii) Preparation of hCG standard solutions A freeze-dried preparation of hCG calibrated against the first international reference preparation (75/537) was obtained from Biodata SpA, Milan, Italy.
This sample Was diluted in horse serum (Serono Diagnostics Ltd., Woking UK) to give the range of hCG
standards required.
(iv) Apparatus used in the measurement of the hcg assay For the assay according to the present invention, components corresponding to labels 1-10 of Fig. 1 were used. The test object 2 was a fluorescence capillary fill device of a type disclosed in EP-A-171148, on the internal surface of the waveguide part 3 of which was immobilised by conventional methods a monoclonal antibody against hCG. The light source 5 was a Heinmann Xenon flash lamp. The filter stack 8 comprised three filters: a BG7 Schott glass filter (Ealing Electro Optics UK Ltd., Watford UK), a 450-480 nm FITC bandpass interference filter (Optometrics Ltd., UK) and a 475 nm shortpass interference filter (Comar Instruments Ltd., Cambridge UK).
The optical structure fabricated as in paragraph (i) above was placed adjacent to the edge 9 of the waveguide (see also Fig. 2). The photodetector 10 was a Hamamatsu R931A photomultiplier tube (Hakuto UK Ltd).
For comparison, a conventional assay using components labelled 11-15 in Fig. 1 was also carried out. In this latter case, the filter 14 used was a Schott OG515 515 nm colloidal glass filter.
'~O 90/15985 ~ ~ ~ r ~ PCT/GB90/00953 Assay Methodology The ahCG-FITC conjugate was added to each of the hCG standards to give about 0.5 ml of test solution. The conjugate concentration was 1 ~g/ml. Test cells were chosen and ~sach was filled with a different test solution. The signal from the photodetector was recorded after a 20-minute incubation period in a humid environment at 20°C.
Figure 5 shows graphically the results of an assay carried out using apparatus including an optical structure as described hereinbefore, in comparison with results from a conventional assay using apparatus shown schematically in Fig. 1. The assay carried out in accordance with the present invention exhibits significantly better precision than the prior art (5.10 compared with 9.0%).
Claims (13)
1. A method of optical analysis of a test sample having fluorescent, phosphorescent or luminescent properties, which sample is partly in a liquid phase and partly bound, directly or indirectly, to an adjacent solid surface, comprising the steps of (i) providing as said solid surface a surface of a transparent (at least at the wavelengths of radiation involved in the analysis) solid optical waveguide;
(ii) measuring light from the sample material bound to said solid surface which has propagated through said waveguide and emerged therefrom at an angle deviating from the optical axis of said waveguide by an angle having a magnitude less than the magnitude of .alpha. where .alpha. ~ arcsin (n2 2 - n1 2)1/2, wherein n2 is the refractive index of the material of the waveguide and n1 is the refractive index of the adjacent liquid; and (iii) excluding from said measurement substantially all light emerging from said waveguide at an angle that deviates from said optical axis by ~.alpha.~ or more, said method being characterized in that an optical structure comprising a layer of transparent material of refractive index n3 interposed directly between layers of transparent material of refractive index n4, [the materials of refractive indices n3 and n4 being chosen such that n3 2 - n4 2 ~ n2 2 - n1 2, and the layers of material of refractive index n4 being adapted such that the major part of the light passing into them from the layer of refractive index n3 does not pass back into said layer of refractive index n3] is placed close to the edge of said waveguide such that substantially all evanescently coupled light emerging from the edge of said waveguide passes into the said layer of refractive index n3.
(ii) measuring light from the sample material bound to said solid surface which has propagated through said waveguide and emerged therefrom at an angle deviating from the optical axis of said waveguide by an angle having a magnitude less than the magnitude of .alpha. where .alpha. ~ arcsin (n2 2 - n1 2)1/2, wherein n2 is the refractive index of the material of the waveguide and n1 is the refractive index of the adjacent liquid; and (iii) excluding from said measurement substantially all light emerging from said waveguide at an angle that deviates from said optical axis by ~.alpha.~ or more, said method being characterized in that an optical structure comprising a layer of transparent material of refractive index n3 interposed directly between layers of transparent material of refractive index n4, [the materials of refractive indices n3 and n4 being chosen such that n3 2 - n4 2 ~ n2 2 - n1 2, and the layers of material of refractive index n4 being adapted such that the major part of the light passing into them from the layer of refractive index n3 does not pass back into said layer of refractive index n3] is placed close to the edge of said waveguide such that substantially all evanescently coupled light emerging from the edge of said waveguide passes into the said layer of refractive index n3.
2. A method as claimed in claim 1 wherein at least one surface of a layer of material of refractive index n4 not in contact with the layer of material of refractive index n3 is coated with a light-absorbing material.
3. A method as claimed in claim 2 wherein at least 80 per cent of the light impinging on the interface between the layer of material of refractive index n4 and the coating of light-absorbing material is absorbed.
4. A method as claimed in claim 1 wherein at least one surface of a layer of material of refractive index n4 not in contact with the layer of material of refractive index n3 is roughened such that light impinging on said surface is scattered out of the optical structure.
5. A method as claimed in claim 4 wherein said surface is coated with a light-absorbing material.
6. A method as claimed in claim 1 wherein the layer of transparent material of refractive index n3 is cylindrical and the layer of transparent material of refractive index n4 is a surrounding coaxial cylinder.
7. A method as claimed in claim 1 wherein the thickness h of the or each layer of material of refractive index n4 is given by h > 1/2d(n2 4-1)1/2 where d is the length of the optical structure.
8. Apparatus for use in a method as claimed in claim 1 comprising:
(i) a test object located at a test object location, adapted to retain a sample material with fluorescent, phosphorescent or luminescent properties, said test object comprising a transparent solid body to act as a waveguide and to the surface of which the sample material can become bound from a solution or liquid dispersion contacting the solid body; and (ii) an optical structure as defined in claim 1, which in use can be positioned so that when a test object is at the test object location the optical structure is optically coupled to the transparent solid body of the test object, such that light propagating in the transparent solid body can be received by the optical structure.
(i) a test object located at a test object location, adapted to retain a sample material with fluorescent, phosphorescent or luminescent properties, said test object comprising a transparent solid body to act as a waveguide and to the surface of which the sample material can become bound from a solution or liquid dispersion contacting the solid body; and (ii) an optical structure as defined in claim 1, which in use can be positioned so that when a test object is at the test object location the optical structure is optically coupled to the transparent solid body of the test object, such that light propagating in the transparent solid body can be received by the optical structure.
9. Apparatus as claimed in claim 8 wherein the test object is a specifically-reactive sample-collecting and testing device possessing a cavity or cavities each having a dimension small enough to enable sample liquid to be drawn into the cavity by capillary action, one surface of the or each cavity carrying an immobilised reagent appropriate to the test to be carried out in the device, said surface being a surface of a transparent solid plate adapted to act as a light-transmissive waveguide, said plate having an edge which is substantially optically smooth and transverse to the plane of the plate.
10. Apparatus as claimed in claim 9 wherein the test object and the optical structure are coupled together so as to form an integral unit.
11. Apparatus as claimed in any one of claims 8 to 10 wherein the optical structure is optically coupled to the transparent solid body of the test object via an index-matching substance in intimate contact with the optical structure and the transparent solid body.
12. A method of assay of a test sample having fluorescent, phosphorescent or luminescent properties, which sample is partly in a liquid phase and partly bound, directly or indirectly, to an adjacent solid surface, wherein said sample is subjected to a method of optical analysis as claimed in any one of claims 1 to 7.
13. A method of assay of a test sample having fluorescent, phosphorescent or luminescent properties, which sample is partly in a liquid phase and partly bound, directly or indirectly, to an adjacent solid surface, wherein said test sample comprises an antigen or antibody and said sample is subjected to a method of optical analysis as claimed in any one of claims 1 to 7.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8914343.2 | 1989-06-22 | ||
| GB898914343A GB8914343D0 (en) | 1989-06-22 | 1989-06-22 | Optical method of analysis |
| GB9008261.1 | 1990-04-11 | ||
| GB909008261A GB9008261D0 (en) | 1990-04-11 | 1990-04-11 | Method of improving assay sensitivity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2033168A1 CA2033168A1 (en) | 1990-12-23 |
| CA2033168C true CA2033168C (en) | 1998-08-18 |
Family
ID=26295521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002033168A Expired - Lifetime CA2033168C (en) | 1989-06-22 | 1990-06-21 | Method of optical analysis |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US5166515A (en) |
| EP (1) | EP0429612B1 (en) |
| JP (1) | JP2612641B2 (en) |
| AT (1) | ATE98773T1 (en) |
| AU (1) | AU619120B2 (en) |
| CA (1) | CA2033168C (en) |
| DE (1) | DE69005270T2 (en) |
| DK (1) | DK0429612T3 (en) |
| ES (1) | ES2047334T3 (en) |
| WO (1) | WO1990015985A1 (en) |
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| GB9008261D0 (en) * | 1990-04-11 | 1990-06-13 | Ares Serono Res & Dev Ltd | Method of improving assay sensitivity |
| WO1992008966A1 (en) * | 1990-11-13 | 1992-05-29 | Block Myron J | Fluorescence assay apparatus |
| US5192510A (en) * | 1991-01-30 | 1993-03-09 | E. I. Du Pont De Nemours And Company | Apparatus for performing fluorescent assays which separates bulk and evanescent fluorescence |
| AU749504B2 (en) * | 1993-05-18 | 2002-06-27 | University Of Utah Research Foundation | Apparatus and methods for multianalyte homogeneous fluoroimmunoassays |
| US5677196A (en) * | 1993-05-18 | 1997-10-14 | University Of Utah Research Foundation | Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays |
| US5512492A (en) | 1993-05-18 | 1996-04-30 | University Of Utah Research Foundation | Waveguide immunosensor with coating chemistry providing enhanced sensitivity |
| US5919712A (en) | 1993-05-18 | 1999-07-06 | University Of Utah Research Foundation | Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays |
| ATE209782T1 (en) * | 1993-05-18 | 2001-12-15 | Univ Utah Res Found | DEVICE AND METHOD FOR HOMOGENEOUS MULTIANALYTE IMMUNO ASSAY |
| JPH08116961A (en) * | 1994-10-25 | 1996-05-14 | Hamamatsu Photonics Kk | Container for observing culture and device for observing the same |
| US5814565A (en) * | 1995-02-23 | 1998-09-29 | University Of Utah Research Foundation | Integrated optic waveguide immunosensor |
| US5745231A (en) * | 1995-06-12 | 1998-04-28 | American Research Corporation Of Virginia | Method of fluorescence analysis comprising evanescent wave excitation and out-of-plane photodetection |
| US5832165A (en) * | 1996-08-28 | 1998-11-03 | University Of Utah Research Foundation | Composite waveguide for solid phase binding assays |
| EP0922214A1 (en) * | 1996-08-29 | 1999-06-16 | Novartis AG | Optical chemical / biochemical sensor |
| US6222619B1 (en) | 1997-09-18 | 2001-04-24 | University Of Utah Research Foundation | Diagnostic device and method |
| JP4662623B2 (en) * | 1998-02-05 | 2011-03-30 | ノバルティス アーゲー | Method and apparatus for measuring luminescence |
| US6300638B1 (en) | 1998-11-12 | 2001-10-09 | Calspan Srl Corporation | Modular probe for total internal reflection fluorescence spectroscopy |
| WO2001025759A1 (en) * | 1999-10-01 | 2001-04-12 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Method and device for determining substances such as e.g., dna sequences, in a sample |
| CA2397943A1 (en) * | 2000-01-20 | 2001-07-26 | Ikonisys, Inc. | Device and method for detecting and localizing cells by means of photosensitive waveguides |
| WO2002001194A1 (en) | 2000-06-25 | 2002-01-03 | Affymetrix, Inc. | Optically active substrates |
| EP1311892B1 (en) * | 2000-08-18 | 2004-11-03 | Koninklijke Philips Electronics N.V. | Optical component and method of manufacturing same |
| US6947651B2 (en) * | 2001-05-10 | 2005-09-20 | Georgia Tech Research Corporation | Optical waveguides formed from nano air-gap inter-layer dielectric materials and methods of fabrication thereof |
| US6890619B2 (en) * | 2001-11-13 | 2005-05-10 | Agilent Technologies, Inc. | Optical systems and refractive index-matching compositions |
| US7029631B2 (en) * | 2002-04-19 | 2006-04-18 | Agilent Technologies, Inc. | Apparatus for improved light collection |
| US7154598B2 (en) * | 2002-07-12 | 2006-12-26 | Decision Biomarkers, Inc. | Excitation and imaging of fluorescent arrays |
| US7196788B2 (en) * | 2002-07-22 | 2007-03-27 | Ikonisys, Inc. | Device and method for detecting and localizing cells by means of photosensitive waveguides |
| US20060127946A1 (en) * | 2002-08-16 | 2006-06-15 | Montagu Jean I | Reading of fluorescent arrays |
| US7384742B2 (en) * | 2002-08-16 | 2008-06-10 | Decision Biomarkers, Inc. | Substrates for isolating reacting and microscopically analyzing materials |
| US20070196863A1 (en) * | 2006-02-17 | 2007-08-23 | Hanson Technologies, Inc. | Prion protein detection |
| US20080026373A1 (en) * | 2006-07-26 | 2008-01-31 | Rodionova Natalia A | Assays Based On Light Emission From Analyte Complexes Within A Cassette |
| US8331751B2 (en) * | 2009-03-02 | 2012-12-11 | mBio Diagnositcs, Inc. | Planar optical waveguide with core of low-index-of-refraction interrogation medium |
| US9658222B2 (en) | 2009-03-02 | 2017-05-23 | Mbio Diagnostics, Inc. | Planar waveguide based cartridges and associated methods for detecting target analyte |
| US9212995B2 (en) | 2009-03-02 | 2015-12-15 | Mbio Diagnostics, Inc. | System and method for detecting multiple molecules in one assay |
| US8300993B2 (en) * | 2009-03-02 | 2012-10-30 | Mbio Diagnostics, Inc. | Waveguide with integrated lens |
| JP5351815B2 (en) * | 2010-03-31 | 2013-11-27 | 富士フイルム株式会社 | Optical member and surface plasmon resonance measuring apparatus |
| US9453791B2 (en) * | 2014-07-01 | 2016-09-27 | Octrolix Bv | Flow cytometry system and method |
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| US3990779A (en) * | 1975-07-24 | 1976-11-09 | International Telephone And Telegraph Corporation | Single optical fiber connector |
| US4575181A (en) * | 1983-04-26 | 1986-03-11 | Tokyo Shibaura Denki Kabushiki Kaisha | Optical fiber assembly with cladding light scattering means |
| CA1231136A (en) * | 1984-06-13 | 1988-01-05 | Ian A. Shanks | Capillary action chemical test device |
| AT383684B (en) * | 1984-09-17 | 1987-08-10 | Avl Verbrennungskraft Messtech | ARRANGEMENT FOR FLUORESCENT OPTICAL MEASUREMENT OF SUBSTANCE CONCENTRATIONS IN A SAMPLE |
| US4668636A (en) * | 1984-11-06 | 1987-05-26 | Battelle Memorial Institute | Analytical assembly usable in apparatuses for optically determining species in solution |
| US4844869A (en) * | 1985-09-09 | 1989-07-04 | Ord, Inc. | Immunoassay apparatus |
| AU604364B2 (en) * | 1987-08-13 | 1990-12-13 | Dow Chemical Company, The | Sulfur dioxide removal from gas streams using hydroxyalkyl substituted piperazinones |
-
1990
- 1990-06-21 WO PCT/GB1990/000953 patent/WO1990015985A1/en active IP Right Grant
- 1990-06-21 JP JP2508670A patent/JP2612641B2/en not_active Expired - Fee Related
- 1990-06-21 ES ES90909063T patent/ES2047334T3/en not_active Expired - Lifetime
- 1990-06-21 DK DK90909063.1T patent/DK0429612T3/en active
- 1990-06-21 US US07/623,817 patent/US5166515A/en not_active Expired - Lifetime
- 1990-06-21 EP EP90909063A patent/EP0429612B1/en not_active Expired - Lifetime
- 1990-06-21 DE DE69005270T patent/DE69005270T2/en not_active Expired - Fee Related
- 1990-06-21 AT AT90909063T patent/ATE98773T1/en not_active IP Right Cessation
- 1990-06-21 CA CA002033168A patent/CA2033168C/en not_active Expired - Lifetime
- 1990-06-21 AU AU58277/90A patent/AU619120B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| EP0429612A1 (en) | 1991-06-05 |
| DK0429612T3 (en) | 1994-03-07 |
| AU619120B2 (en) | 1992-01-16 |
| AU5827790A (en) | 1991-01-08 |
| JPH04501612A (en) | 1992-03-19 |
| ATE98773T1 (en) | 1994-01-15 |
| US5166515A (en) | 1992-11-24 |
| CA2033168A1 (en) | 1990-12-23 |
| ES2047334T3 (en) | 1994-02-16 |
| EP0429612B1 (en) | 1993-12-15 |
| DE69005270T2 (en) | 1994-07-07 |
| DE69005270D1 (en) | 1994-01-27 |
| JP2612641B2 (en) | 1997-05-21 |
| WO1990015985A1 (en) | 1990-12-27 |
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| EEER | Examination request | ||
| MKLA | Lapsed | ||
| MKEC | Expiry (correction) |
Effective date: 20121202 |