CA1269546A - Dielectric waveguide sensors and their use in immunoassays - Google Patents
Dielectric waveguide sensors and their use in immunoassaysInfo
- Publication number
- CA1269546A CA1269546A CA000610474A CA610474A CA1269546A CA 1269546 A CA1269546 A CA 1269546A CA 000610474 A CA000610474 A CA 000610474A CA 610474 A CA610474 A CA 610474A CA 1269546 A CA1269546 A CA 1269546A
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- analyte
- fiber
- waveguide
- reactant
- refraction
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
Abstract The present invention relates to novel dielectric waveguide (i.e., fiber optic) sensors for use in spectrophotometric assays of analytes in fluids. More particularly, the use of these sensors in immunoassays is disclosed.
Description
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r11~IIY5LIYY~ 5~ L _AND ~HEIR
USE IN IM~NOASSAYS
Technical Field The present invention relates to novel dielectric waveguide (i.e., fib~r optic) sensors for use in spectrophotometric assays of analytes in fluicls. More partlcularly, the use of these sansors in immLmoassays is disclosed.
.
Back~round Art ~;' ' : 10 Optical waveguides have been used in various analytical test. For example, in an article entitled :~ "Optical ~iber Fluoroprobes in Clinical Analysis", Clin.
: Chem, 29/9, pp 1678-1682 (1983), Michael J. Sepaniak et : al. describe the use of quartz optical fluoroprobes.
~`~ 15 By incorporating a single fiber within a hypodermic ne~dle, the authors hav~ been able to obtain in ViYo measurement of the fluore~cence of various therapeutic drug analytes in interstitial body fluids. 5epaniak et al state that their probe must use a laser radiation sourca as a fluorescence exciter~
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r11~IIY5LIYY~ 5~ L _AND ~HEIR
USE IN IM~NOASSAYS
Technical Field The present invention relates to novel dielectric waveguide (i.e., fib~r optic) sensors for use in spectrophotometric assays of analytes in fluicls. More partlcularly, the use of these sansors in immLmoassays is disclosed.
.
Back~round Art ~;' ' : 10 Optical waveguides have been used in various analytical test. For example, in an article entitled :~ "Optical ~iber Fluoroprobes in Clinical Analysis", Clin.
: Chem, 29/9, pp 1678-1682 (1983), Michael J. Sepaniak et : al. describe the use of quartz optical fluoroprobes.
~`~ 15 By incorporating a single fiber within a hypodermic ne~dle, the authors hav~ been able to obtain in ViYo measurement of the fluore~cence of various therapeutic drug analytes in interstitial body fluids. 5epaniak et al state that their probe must use a laser radiation sourca as a fluorescence exciter~
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-2/3-One of the fluoroprobe designs uses a capillary action design for sampling. A length of optical fiber is stripped of its protective coating and slid inside a standard glass capillary tube, touching the walls of the capillary tube at random but not extending the whole length of the tube. This assen~ly is placed within a : hypodermic neadle.
An immunoassay apparatus developed by T.
Hirschfeld is disclosed in U.S. Patent No. 4,447,546 issued May 8, 1984, which employs total internal reflection at an interface between a solid phase and a fluid phase of lower index of refraction to produce an evanescent wave in the fluid phase. Fluorescence excited by the wave is obsexved at angles greater th~n the critical angle, by total reflection within the solid medium. The solid phase is arrangQd and illuminated to provide multiple total internal reflections at the interface. Typically, the solid phase is in the form of : am optical fiber to which i5 immobilized a component o~
a oomplex formed in an i~munochemical reaction. A
~luorophore is attached to another component of the ~:~ complex. The fluorescent labeled component may be ~:~ either the complement to or the analog of the immobilized component, depending upon whether competitive or sandwich assays are to be performed.
: In the case of competitive assays, the labelled ~ component is typically preloaded to the .
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immobilized component in a controlled concentrationO
The fiber and the attached constituent of the assay are immersed in a fluid phase sample and the exciting illumination is injected into an input end of the fiber. The evanescent wave is used to excite fluorescence in the fluid phase, and that fluorescence which tunnels back into the solid phase (propagating in direction greater than the critical angle) is detected at the input end of the fiber.
The observed volume of sample is restricted not only by the rapid decay of the evanescent wave ;15 as a function of distance from the interface, but by an equally fast decrease with distance of the efficiency of tunneling, the more distant fluorophores not only being less intensely excited and thus fluorescing less, but their radiation is less efficiently coupled into the fiber.
~-Consequently the effective depth of the sensed layer is much reduced compared to the ~one observed by total reflection fluorescence alone, the coupling efficiency effectively scaling down the zone.
Multiple total internal reflections in the solid phase allow the illuminating beam to excite repeatedly an evanescent wave, thereby more efficiently coupling the small excitation source to the sample volume. This also increases the amount of sample sensed. The latter is also enhanced by diffusive circulation of the sample past the fiber surface and to which the material being assayed adheres by reaction as it passes. Diffusion makes ~ :
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~269SA6 the actually sampled layer thickness much larger than the thin surface layer.
All of the radiation that tunnels back into the fibers is within the total reflection angle, and is thus trapped within the fiber. The power available from the fluorescence increases with the length of fiber within the fluorPscing material. However, the optical throughput of tha system (determined by the aperture and the numerical aperture of the fiber) remains constant.
The total fluorescent signal coming from the entire surface of the fiber, multiplied by tha increase in sample volume due to diffusion, thus becomes available in a very bright spot (that is the cross-section of the fiber in diameter) exiting the fiber at its input end through a restricted angle determined by the critical angle of reflection within the fiber. Such signal is easily collected at high efficiency and throughput when matched to a small detector.
Various aspects of this invention are as follows:
A multi-element dielectric waveguide for use in a spectrophotometric assay of an analyte in a fluid comprising:
a) a support fiber having an index of refraction (NA) and an opening therethrough;
b) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and c) a means for maintaining the axial position of the core fiber within the support fiber.
A ~ethod of spectrophotometrically assaying an analyte in a fluid comprising:
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5a a) coatiny a multi-element dielectric waveguide comprisingf i) a support fiber having an index of refraction (NA) and an opening therethrough;
ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and iii) a means for maintaîning the axial position of the core fiber within the hollow fiber;
with an immobilized reactant wh.ich interacts ~ with the analyte to form a signal radiation;
;~ 15 b) contacting the waveguide array with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation down the core fiber so as to irradiate the combination of analyte : 20 and reactant;
d) detecting the signal radiation from the ~
irradiated analyte and reactant interaction by monitoring the waveguide.
~: A method o~ spectrophotometrically assaying an an~lyte in a fluid comprising:
~` ~ a) coating a dielectric waveguide : comprising:
i) a support fiber having an index of : refraction ~NA) and an opening therethrough; and ii) a second core fiber axially ~: positioned within the support fiber : opening and having an index of ~ refraction (NB); and ., ~
5b iii) a means for maintaining the axial position o~ the core fiber within the hollow fiber;
with an immobilized reactant which interacts with the analyte to form a signal radiation;
b) contacting the waveguide with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation down the support fiber so as to irradiate the combination of analyte and reactant;
d) detecting the signal radiation from the ~ irradiated analyte and reactant interaction by :~ monitoring the waveguide.
, : 15 A method of spectrophotometrically assaying an analyte in a fluid comprising:
a) coating a multi-element dielectric wavegulde comprising:
: i) a support fiber having an index of refraction (NA) and an opening therethrough;
ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB~; and iiij a means for maintaining the axial position of the core fiber within ~: the hollow fiber;
: with an immobilized reactant which interacts ~: 30 with the analyte to ~orm a signal radiation;
: b) contacting the w~veguide with the fluid for a time sufficient for the analyte and : reactant to be able to interact;
c) propagating radiation within the fluid so ~, ~
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5c as to irradiate the combination of analyte and reactant;
; d) detecting the signal radiation from the irradiated analyta and reactant interaction by monitoring the waveguide.
Disclosure of the Invention The present invention co~prises three novel dielectric waveguide structures that are useful in spectrophotometric assays of analytes in ~luid. Also, it comprises novel methods of spectrophotometrically assaying analytes using these novel waveguides.
~; The first dielectric waveguide has a core, a cladding, and a reactant coating on the core. Of particular interest is that the core has at least an opening in the core material which is exposed to the analyte-containing fluid, and may be hollow t;hroughout. For descriptive purposes, the :
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S~6 waveguide comprises a core transmissive to electromagnetic radiation, preferably visible light, having an index of refraction (N1) and an opening in the core. The core thickness is sufficient to propagate the exciting radiation substantially down the core. A cladding with an index of refraction (N2) (which is less than N1) is about the outside of the core. The cladding is thick enough to contain substantially all of the exciting radiation launched below the critical angle of the waveguides, but to permit penetration of the evanescent wave into a reactant coating.
Finally, a reactant coating is placed abou-t the core opening which, in the presence of electromagnetic radiation, interacts with the analyte to form a signal radiation.
The light propagation in this and the other waveguide structures to be discussed consists of modes with propagation constant, ~, such that E X e ie, where E is the lightwave electric fleld amplitude and z the distance along the waveguide.
; Oscillatory solutions for E, i.e., bound modes, are obtained for N2k <~ < ~lk where k = 21r and is the free space wavelength of the 1 ~ . Leaky modes for which N3k <~ < N2k are also obtained hut these generally decay with length z (where N3 is the index of refraction of the fluid surrounding the waveguide). With a suitable combination of spot size and launch angle, the penetration of light into the analyte can be controlled~
For example, if N2 = N3 for simplicity, then the extension of the electric field into the analyte is given by:
... , I .
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., .. ;
E _ K~ (r r) for r>a where 2a denotes the thickness of the core region,~
is the mode number, ~ = (N12k2 _~ 2)1/2 and K is the modified Hankel function. This applies strictly to the case of a concentric circular fiber but may be used approximately here. The mathematical matching of this evanescent electric field to the core mode electric field gives the value of~. For the lowest order mode, vis.,~ = O, ~; E ~ e -l~r for r>a r Thus, the penetration distance of the light into ; the analyte depends on which in turn depends on the mode(s) selected by the launch ~initial) conditions ( ~), the indices of refractions of the waveguide (Nl and N2) and analyte (N3~, and the wavelength of the light (~).
The above hollow waveguide can be used in the ~; following manner. The coated waveguide is placed in the analyte-containing fluid for a time sufficient for the analyte to interact with the reactant coating and to form an electromagnetically detectable complex or moeity. Then either while the fiber is still in the fluid or after it has been removed, electromagnetic radiation is propagated down the waveguide core so as to irradiate the interacting moeity, which then produces a signal radiation. The last step is to detect the resulting signal radiation by monitoring the core of the waveguide. Typically, the waveguide is a fiber having two ends, either one of which can be monitored.
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Another novel dielectric waveguide has two concentric fibers. A support fiber with an index of refraction (NA) has an opening therethrough, i.e., is hollow. A second core fiber with an index _ of refraction (NB) is axially positioned concentric with the support fiber opening. A means for maintaining this axial position is incorporated to form a multi-element dielectric waveguide. The relationships of NA/NB depends upon how one intends on using the waveguide in an assay. NB can be ~10 either greater than, equal to, or less than NA.
;The selection of materials and waveguide design parameters such as thickness~ follow principles either ]cnown to the art or described above.
There are three general methods of using the multi-element dielectric waveguide. In the first, the exciting radiation is propagated down the core ~~ fiber. The evanescent wave from this propagation -~ interacts with either the analyte itself or the combination of a,nalyte and reactant coating on ~` either of the fibers to produce a signal radiation.
Either the core or the support fiber can be monitored to detect the signal radiation, however, the detecting waveguide should have an index of refraction, equal to or greater than the excitating waveguide.
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Another method uses the hollow support waveguide to propagate the excitation radiation.
Again, either waveguide can be used for detection, but the de~ecting waveguide should have an index of refraction equal to or greater than the exciting waveguide.
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g The third method does not use the waveguide as an exciter. Rather, the analyte-containing fluid is used as the propagating medium for the excitation radiation. Either waveguide is used for detection. of course, the fluid must be able to propagate the excitation radiation.
; The third dielectric wavegu:ide is an elongated member having a series of claddings about a hollow core. Specifically, the core with an index of refraction (Nx) has an opening therethrough, i.e., is hollow. A series of claddings with alternating indices of refraction, (N2) followed by (Nl) (where N2 ~5 less than Nl and only one of either can equal Nx), is positioned about the core. The number and thic]cness of the claddings is sufficient to enable electromagnetic radiation to propagate within the holl~w core. Such configurations are known to the art as Bragg waveguides. The selection materials and design parameters such as thickness, follow principles either known to the art or described above.
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For assay purposes, one coats the interior core surface of a Bragg waveguide with a reactant which, in the presence of electromagnetic radiation, interacts with the analyte to form a detectable signal radiation.
The coated Bragg waveguide can be used in an assay in a method simiIar to the first hollow waveguide; however, the excitation and signal radiation are both launched and carried down the ; opening of the core fiber rather than the fiber itself.
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Of course, an apparatus useful for practicing the above method would include the following elements: an electromagnetic radiation source; a means for guiding the radiation from the source to the interior of the waveguide, where it is propagated; a signal radiation cletection means; and a means for guiding the signal radiation from the waveguide, to the detection means. All of these means are conventional and well known to the ~ skilled artisan.
:~ 10 Description of the Drawings FIGURE 1 is a cross-sectional view of a hollow waveguide.
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; FIGURE 2 is a cross-sectional ~iew of a ~ multi-element waveguide.
: ' FIGURE 3 is a cross-sectional view of a Bragg 2~ waveguide.
FIGURE 4 is a diagrammatic view of an apparatus for use with the above waveguides.
Modes of Carrying Out_the Invention A preferred embodiment of a hollow waveguide is shown in Figure 1. The waveguide 10 comprises a hollow glass cylindrical core 12 having an index of refraction Nl, an internal core diameter of about 100 microns, and a thickness ~ of about 250 microns. The core is covered on the ; outside by a glass cladding 14 having an index of xefraction N2, where N1, and a thickness of about 250 microns. Those skilled in the art of optical :
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~l~i~jL6 fibers know how to select suitable optically transmissive materials, such as glass or plastic, and how to make such a structure, therefore a detailed description of the various processes is superfluous. However, the following disclosures are given as exemplary references on both multi-mode and single mode waveguide construction:
U.S. 3,595,915 to Maurer et al; U.S. 3,711,262 to Keck et al; U.S. 3,775,075 to Keck et al; and U.S.
An immunoassay apparatus developed by T.
Hirschfeld is disclosed in U.S. Patent No. 4,447,546 issued May 8, 1984, which employs total internal reflection at an interface between a solid phase and a fluid phase of lower index of refraction to produce an evanescent wave in the fluid phase. Fluorescence excited by the wave is obsexved at angles greater th~n the critical angle, by total reflection within the solid medium. The solid phase is arrangQd and illuminated to provide multiple total internal reflections at the interface. Typically, the solid phase is in the form of : am optical fiber to which i5 immobilized a component o~
a oomplex formed in an i~munochemical reaction. A
~luorophore is attached to another component of the ~:~ complex. The fluorescent labeled component may be ~:~ either the complement to or the analog of the immobilized component, depending upon whether competitive or sandwich assays are to be performed.
: In the case of competitive assays, the labelled ~ component is typically preloaded to the .
: .
. ' ' .. ..
~ .
5~
immobilized component in a controlled concentrationO
The fiber and the attached constituent of the assay are immersed in a fluid phase sample and the exciting illumination is injected into an input end of the fiber. The evanescent wave is used to excite fluorescence in the fluid phase, and that fluorescence which tunnels back into the solid phase (propagating in direction greater than the critical angle) is detected at the input end of the fiber.
The observed volume of sample is restricted not only by the rapid decay of the evanescent wave ;15 as a function of distance from the interface, but by an equally fast decrease with distance of the efficiency of tunneling, the more distant fluorophores not only being less intensely excited and thus fluorescing less, but their radiation is less efficiently coupled into the fiber.
~-Consequently the effective depth of the sensed layer is much reduced compared to the ~one observed by total reflection fluorescence alone, the coupling efficiency effectively scaling down the zone.
Multiple total internal reflections in the solid phase allow the illuminating beam to excite repeatedly an evanescent wave, thereby more efficiently coupling the small excitation source to the sample volume. This also increases the amount of sample sensed. The latter is also enhanced by diffusive circulation of the sample past the fiber surface and to which the material being assayed adheres by reaction as it passes. Diffusion makes ~ :
:
;~ ' : ,,~, .
: . :
:, .
~269SA6 the actually sampled layer thickness much larger than the thin surface layer.
All of the radiation that tunnels back into the fibers is within the total reflection angle, and is thus trapped within the fiber. The power available from the fluorescence increases with the length of fiber within the fluorPscing material. However, the optical throughput of tha system (determined by the aperture and the numerical aperture of the fiber) remains constant.
The total fluorescent signal coming from the entire surface of the fiber, multiplied by tha increase in sample volume due to diffusion, thus becomes available in a very bright spot (that is the cross-section of the fiber in diameter) exiting the fiber at its input end through a restricted angle determined by the critical angle of reflection within the fiber. Such signal is easily collected at high efficiency and throughput when matched to a small detector.
Various aspects of this invention are as follows:
A multi-element dielectric waveguide for use in a spectrophotometric assay of an analyte in a fluid comprising:
a) a support fiber having an index of refraction (NA) and an opening therethrough;
b) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and c) a means for maintaining the axial position of the core fiber within the support fiber.
A ~ethod of spectrophotometrically assaying an analyte in a fluid comprising:
,~
-:
.. .. .
5a a) coatiny a multi-element dielectric waveguide comprisingf i) a support fiber having an index of refraction (NA) and an opening therethrough;
ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and iii) a means for maintaîning the axial position of the core fiber within the hollow fiber;
with an immobilized reactant wh.ich interacts ~ with the analyte to form a signal radiation;
;~ 15 b) contacting the waveguide array with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation down the core fiber so as to irradiate the combination of analyte : 20 and reactant;
d) detecting the signal radiation from the ~
irradiated analyte and reactant interaction by monitoring the waveguide.
~: A method o~ spectrophotometrically assaying an an~lyte in a fluid comprising:
~` ~ a) coating a dielectric waveguide : comprising:
i) a support fiber having an index of : refraction ~NA) and an opening therethrough; and ii) a second core fiber axially ~: positioned within the support fiber : opening and having an index of ~ refraction (NB); and ., ~
5b iii) a means for maintaining the axial position o~ the core fiber within the hollow fiber;
with an immobilized reactant which interacts with the analyte to form a signal radiation;
b) contacting the waveguide with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation down the support fiber so as to irradiate the combination of analyte and reactant;
d) detecting the signal radiation from the ~ irradiated analyte and reactant interaction by :~ monitoring the waveguide.
, : 15 A method of spectrophotometrically assaying an analyte in a fluid comprising:
a) coating a multi-element dielectric wavegulde comprising:
: i) a support fiber having an index of refraction (NA) and an opening therethrough;
ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB~; and iiij a means for maintaining the axial position of the core fiber within ~: the hollow fiber;
: with an immobilized reactant which interacts ~: 30 with the analyte to ~orm a signal radiation;
: b) contacting the w~veguide with the fluid for a time sufficient for the analyte and : reactant to be able to interact;
c) propagating radiation within the fluid so ~, ~
.
. . .
. .
'" ;"
, ~:
~6g~
5c as to irradiate the combination of analyte and reactant;
; d) detecting the signal radiation from the irradiated analyta and reactant interaction by monitoring the waveguide.
Disclosure of the Invention The present invention co~prises three novel dielectric waveguide structures that are useful in spectrophotometric assays of analytes in ~luid. Also, it comprises novel methods of spectrophotometrically assaying analytes using these novel waveguides.
~; The first dielectric waveguide has a core, a cladding, and a reactant coating on the core. Of particular interest is that the core has at least an opening in the core material which is exposed to the analyte-containing fluid, and may be hollow t;hroughout. For descriptive purposes, the :
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S~6 waveguide comprises a core transmissive to electromagnetic radiation, preferably visible light, having an index of refraction (N1) and an opening in the core. The core thickness is sufficient to propagate the exciting radiation substantially down the core. A cladding with an index of refraction (N2) (which is less than N1) is about the outside of the core. The cladding is thick enough to contain substantially all of the exciting radiation launched below the critical angle of the waveguides, but to permit penetration of the evanescent wave into a reactant coating.
Finally, a reactant coating is placed abou-t the core opening which, in the presence of electromagnetic radiation, interacts with the analyte to form a signal radiation.
The light propagation in this and the other waveguide structures to be discussed consists of modes with propagation constant, ~, such that E X e ie, where E is the lightwave electric fleld amplitude and z the distance along the waveguide.
; Oscillatory solutions for E, i.e., bound modes, are obtained for N2k <~ < ~lk where k = 21r and is the free space wavelength of the 1 ~ . Leaky modes for which N3k <~ < N2k are also obtained hut these generally decay with length z (where N3 is the index of refraction of the fluid surrounding the waveguide). With a suitable combination of spot size and launch angle, the penetration of light into the analyte can be controlled~
For example, if N2 = N3 for simplicity, then the extension of the electric field into the analyte is given by:
... , I .
. .
.
., .. ;
E _ K~ (r r) for r>a where 2a denotes the thickness of the core region,~
is the mode number, ~ = (N12k2 _~ 2)1/2 and K is the modified Hankel function. This applies strictly to the case of a concentric circular fiber but may be used approximately here. The mathematical matching of this evanescent electric field to the core mode electric field gives the value of~. For the lowest order mode, vis.,~ = O, ~; E ~ e -l~r for r>a r Thus, the penetration distance of the light into ; the analyte depends on which in turn depends on the mode(s) selected by the launch ~initial) conditions ( ~), the indices of refractions of the waveguide (Nl and N2) and analyte (N3~, and the wavelength of the light (~).
The above hollow waveguide can be used in the ~; following manner. The coated waveguide is placed in the analyte-containing fluid for a time sufficient for the analyte to interact with the reactant coating and to form an electromagnetically detectable complex or moeity. Then either while the fiber is still in the fluid or after it has been removed, electromagnetic radiation is propagated down the waveguide core so as to irradiate the interacting moeity, which then produces a signal radiation. The last step is to detect the resulting signal radiation by monitoring the core of the waveguide. Typically, the waveguide is a fiber having two ends, either one of which can be monitored.
~ .
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. .
- ~.
t5~;
Another novel dielectric waveguide has two concentric fibers. A support fiber with an index of refraction (NA) has an opening therethrough, i.e., is hollow. A second core fiber with an index _ of refraction (NB) is axially positioned concentric with the support fiber opening. A means for maintaining this axial position is incorporated to form a multi-element dielectric waveguide. The relationships of NA/NB depends upon how one intends on using the waveguide in an assay. NB can be ~10 either greater than, equal to, or less than NA.
;The selection of materials and waveguide design parameters such as thickness~ follow principles either ]cnown to the art or described above.
There are three general methods of using the multi-element dielectric waveguide. In the first, the exciting radiation is propagated down the core ~~ fiber. The evanescent wave from this propagation -~ interacts with either the analyte itself or the combination of a,nalyte and reactant coating on ~` either of the fibers to produce a signal radiation.
Either the core or the support fiber can be monitored to detect the signal radiation, however, the detecting waveguide should have an index of refraction, equal to or greater than the excitating waveguide.
:: ~
Another method uses the hollow support waveguide to propagate the excitation radiation.
Again, either waveguide can be used for detection, but the de~ecting waveguide should have an index of refraction equal to or greater than the exciting waveguide.
.
'' ."~
.
.
~2~
g The third method does not use the waveguide as an exciter. Rather, the analyte-containing fluid is used as the propagating medium for the excitation radiation. Either waveguide is used for detection. of course, the fluid must be able to propagate the excitation radiation.
; The third dielectric wavegu:ide is an elongated member having a series of claddings about a hollow core. Specifically, the core with an index of refraction (Nx) has an opening therethrough, i.e., is hollow. A series of claddings with alternating indices of refraction, (N2) followed by (Nl) (where N2 ~5 less than Nl and only one of either can equal Nx), is positioned about the core. The number and thic]cness of the claddings is sufficient to enable electromagnetic radiation to propagate within the holl~w core. Such configurations are known to the art as Bragg waveguides. The selection materials and design parameters such as thickness, follow principles either known to the art or described above.
;'~,.
For assay purposes, one coats the interior core surface of a Bragg waveguide with a reactant which, in the presence of electromagnetic radiation, interacts with the analyte to form a detectable signal radiation.
The coated Bragg waveguide can be used in an assay in a method simiIar to the first hollow waveguide; however, the excitation and signal radiation are both launched and carried down the ; opening of the core fiber rather than the fiber itself.
~:
;, ", , ~. ".
: - , , - , . ;, ~ : ::- ,-,, - . . .: . . . ,:: . , ,: . . .
~S~L~
Of course, an apparatus useful for practicing the above method would include the following elements: an electromagnetic radiation source; a means for guiding the radiation from the source to the interior of the waveguide, where it is propagated; a signal radiation cletection means; and a means for guiding the signal radiation from the waveguide, to the detection means. All of these means are conventional and well known to the ~ skilled artisan.
:~ 10 Description of the Drawings FIGURE 1 is a cross-sectional view of a hollow waveguide.
lS
; FIGURE 2 is a cross-sectional ~iew of a ~ multi-element waveguide.
: ' FIGURE 3 is a cross-sectional view of a Bragg 2~ waveguide.
FIGURE 4 is a diagrammatic view of an apparatus for use with the above waveguides.
Modes of Carrying Out_the Invention A preferred embodiment of a hollow waveguide is shown in Figure 1. The waveguide 10 comprises a hollow glass cylindrical core 12 having an index of refraction Nl, an internal core diameter of about 100 microns, and a thickness ~ of about 250 microns. The core is covered on the ; outside by a glass cladding 14 having an index of xefraction N2, where N1, and a thickness of about 250 microns. Those skilled in the art of optical :
.~ .
.~ ':~ ' , .
.. .
' . : .
~l~i~jL6 fibers know how to select suitable optically transmissive materials, such as glass or plastic, and how to make such a structure, therefore a detailed description of the various processes is superfluous. However, the following disclosures are given as exemplary references on both multi-mode and single mode waveguide construction:
U.S. 3,595,915 to Maurer et al; U.S. 3,711,262 to Keck et al; U.S. 3,775,075 to Keck et al; and U.S.
3,823,995 to Carpenter.
The interior surface of the waveguide core is covered with an immobilized reactant coating 16.
The chemical composition of this coating varies according to the type of analyte being detected and the type of signal radiation one is trying to generate. As for analytes suitable for detection ; with the present waveguides, the main requirement is for the reactant coating to be able to bind the~
analyte directly. For example, if the analyte is an immunological substance (i.e., antibody, antigen, or hapten), then the reactant coating comprises a complementary immunological substance ; which is secured to the core yet able to bind to the analyte. Thus, an antigen ~Ag) analyte would requixe a complementary antibody (Ab) component to be immobilized to the core as the reactant coating.
Those of skill in the immunoassay art have applied the selective binding property of antibodies to create different types of immunoassays known as "sandwich", I'direct", "limited reagent" and "saturation" assays. See U.SO 4,380,580. The skilled artisan would know how to design an immunoassay by selecting the proper ; immunological substances for a reactant coating ,. ..
:: , . ~ ... ... .
.,.
, ,~, .. ,.. ,,.,. ,.. ~ ~ . , ., . :
:, .. . .
. .~
that would be suitable for use on the present coated, hollow waveguides.
Signal radiation selection can affect the selection of the reactant coating as well. For example, if chemiluminescent production of a particulax signal is desired in an im~unoassay, then the reactant coating can comprise an immobilized chemiluminescent precursor or reactant which, in the presence of the analyte, results in the production of this signal. Alternatively, the precursor can be used according to the methods disclosed in U.S. 4,380,580, where the chemiluminescent precursor is attached to either an antibody or an antigen which would react with the coating~ These configurations are opposed to immunoassays where, if fluorescence is the signal to be monitored, then the art knows how to apply fluorescent "tags" either to the analyte or to a competitive analyte (or analogue thereof) without affecting the makeup of the reactant coating.
. ~ ~
If desired, a mirror coating (not shown~ can be applied to the outside of the cladding. The effect would be to reflect the isotropic signal ra~iation so as to permit more of the signal to be propagated back down the waveguide.
The multi-element waveguide is illustrated in Figure 2. Preferably, the waveguide comprises two spaced fibers. A hollow, cylindrical support fiber 22 having and index of refraction NA, an interior diameter of 1000 microns, and a thickness of 250 microns is coated with a reflective, mirror layer 24. Positioned within the interior of the support iber is a core fiber 25 having an index of :
:. ;~ . , , :. ., . ` - :
.
' ` ''' ', ' ' ` : ~
~6~
refraction NB and a thickness of 250 microns, which may have a cladding 26 about the core having an index of refraction NC and a thickness of 50 microns. NB is greater than NA and less than NC if ~he core fiber is used for detection. Spacer means 27 comprising at least an annular ring keeps the core fiber axially and concentrically positioned within the length of the hollow support fiber.
Finally, a reactant coating 28 covers the cladding surface of the core fiber. Again, as discussed above, this coating can have variable compositions.
~' The third Bragg waveguide 30 has a glass hollow cylindrical core 32 with an interior diameter of 1000 microns and an index of refraction ~; Nx, surrounded on the outside by a multicomponent cladding 34 and on the inside with a reactant coating 36 similar to the ones described above.
The cladding comprises a series of alternating materials having indices of refraction Nl and N2, where N2 < N1 and only one of either N2 or N1 can equal Nx. Th~ cladding thicknesses vary according to the indices of refraction, as mentioned herein.
In general, an apparatus for using these waveguides in spectrophotometric assays 40 has the five elements diagramatically presented in Figure
The interior surface of the waveguide core is covered with an immobilized reactant coating 16.
The chemical composition of this coating varies according to the type of analyte being detected and the type of signal radiation one is trying to generate. As for analytes suitable for detection ; with the present waveguides, the main requirement is for the reactant coating to be able to bind the~
analyte directly. For example, if the analyte is an immunological substance (i.e., antibody, antigen, or hapten), then the reactant coating comprises a complementary immunological substance ; which is secured to the core yet able to bind to the analyte. Thus, an antigen ~Ag) analyte would requixe a complementary antibody (Ab) component to be immobilized to the core as the reactant coating.
Those of skill in the immunoassay art have applied the selective binding property of antibodies to create different types of immunoassays known as "sandwich", I'direct", "limited reagent" and "saturation" assays. See U.SO 4,380,580. The skilled artisan would know how to design an immunoassay by selecting the proper ; immunological substances for a reactant coating ,. ..
:: , . ~ ... ... .
.,.
, ,~, .. ,.. ,,.,. ,.. ~ ~ . , ., . :
:, .. . .
. .~
that would be suitable for use on the present coated, hollow waveguides.
Signal radiation selection can affect the selection of the reactant coating as well. For example, if chemiluminescent production of a particulax signal is desired in an im~unoassay, then the reactant coating can comprise an immobilized chemiluminescent precursor or reactant which, in the presence of the analyte, results in the production of this signal. Alternatively, the precursor can be used according to the methods disclosed in U.S. 4,380,580, where the chemiluminescent precursor is attached to either an antibody or an antigen which would react with the coating~ These configurations are opposed to immunoassays where, if fluorescence is the signal to be monitored, then the art knows how to apply fluorescent "tags" either to the analyte or to a competitive analyte (or analogue thereof) without affecting the makeup of the reactant coating.
. ~ ~
If desired, a mirror coating (not shown~ can be applied to the outside of the cladding. The effect would be to reflect the isotropic signal ra~iation so as to permit more of the signal to be propagated back down the waveguide.
The multi-element waveguide is illustrated in Figure 2. Preferably, the waveguide comprises two spaced fibers. A hollow, cylindrical support fiber 22 having and index of refraction NA, an interior diameter of 1000 microns, and a thickness of 250 microns is coated with a reflective, mirror layer 24. Positioned within the interior of the support iber is a core fiber 25 having an index of :
:. ;~ . , , :. ., . ` - :
.
' ` ''' ', ' ' ` : ~
~6~
refraction NB and a thickness of 250 microns, which may have a cladding 26 about the core having an index of refraction NC and a thickness of 50 microns. NB is greater than NA and less than NC if ~he core fiber is used for detection. Spacer means 27 comprising at least an annular ring keeps the core fiber axially and concentrically positioned within the length of the hollow support fiber.
Finally, a reactant coating 28 covers the cladding surface of the core fiber. Again, as discussed above, this coating can have variable compositions.
~' The third Bragg waveguide 30 has a glass hollow cylindrical core 32 with an interior diameter of 1000 microns and an index of refraction ~; Nx, surrounded on the outside by a multicomponent cladding 34 and on the inside with a reactant coating 36 similar to the ones described above.
The cladding comprises a series of alternating materials having indices of refraction Nl and N2, where N2 < N1 and only one of either N2 or N1 can equal Nx. Th~ cladding thicknesses vary according to the indices of refraction, as mentioned herein.
In general, an apparatus for using these waveguides in spectrophotometric assays 40 has the five elements diagramatically presented in Figure
4. They are: an excitation radiation source 42; a means for guiding the excitation radiation 44 to - the waveguide 46, either at the core, the cladding, or the hollow interior, where it is propagated; a signal radiation detection means 48; a means for guiding the signal radiation, also 44 from the waveguide to the signal detection means and, preferably, a recordation and proce~sing means 49 ,:
.. . . .
'' ' - ' ~s~
which can collect detection data in a more permanent form.
Most of these elements are standard features on spectrophotometers. For example, the exciter can be either a dye-tunable laser or a tungsten bulb. The guide means can comprise focusing lenses, monochromator gratings, mirrors, and - wavelength selective beam splitters. Finally, the detector and recorder can be either a 1 photomultiplier tube or a photo-diode and a ;~ microprocessor with storage and display abilities.
The design of such an apparatus would be within the skill of an optics artisan.
An important aspect of any apparatus using the present waveguides is the waveguide alignment ~ means. That is, part of the function of the - guiding means is to ensure that the excitation radiation is propagated within the waveguide.
Thus, according to known optical principles the ~; waveguide must be properly aligned with this radiation, otherwise bound analyte will not be excited by an evanescent wave of the proper wavelength. More than one gripping arrangement can ' be used, from as simple as a matching cylindrical guide sheath to as complicated as movable opposing ; jaws with precision molded grips.
~ aving described the invention with particular reference to preferred embodiments, it will be - 30 obvious to those skilled in the art to which the invention pertain, that, after understanding the ::~
invention, various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.
.
. . ~ .
, : . ,.: . - ,..... ,, . . , , ;
: :
~ .
.. . . .
'' ' - ' ~s~
which can collect detection data in a more permanent form.
Most of these elements are standard features on spectrophotometers. For example, the exciter can be either a dye-tunable laser or a tungsten bulb. The guide means can comprise focusing lenses, monochromator gratings, mirrors, and - wavelength selective beam splitters. Finally, the detector and recorder can be either a 1 photomultiplier tube or a photo-diode and a ;~ microprocessor with storage and display abilities.
The design of such an apparatus would be within the skill of an optics artisan.
An important aspect of any apparatus using the present waveguides is the waveguide alignment ~ means. That is, part of the function of the - guiding means is to ensure that the excitation radiation is propagated within the waveguide.
Thus, according to known optical principles the ~; waveguide must be properly aligned with this radiation, otherwise bound analyte will not be excited by an evanescent wave of the proper wavelength. More than one gripping arrangement can ' be used, from as simple as a matching cylindrical guide sheath to as complicated as movable opposing ; jaws with precision molded grips.
~ aving described the invention with particular reference to preferred embodiments, it will be - 30 obvious to those skilled in the art to which the invention pertain, that, after understanding the ::~
invention, various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.
.
. . ~ .
, : . ,.: . - ,..... ,, . . , , ;
: :
~ .
Claims (12)
1. A multi-element dielectric waveguide for use in a spectrophotometric assay of an analyte in a fluid comprising:
a) a support fiber having an index of refraction (NA) and an opening therethrough;
b) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and c) a means for maintaining the axial position of the core fiber within the support fiber.
a) a support fiber having an index of refraction (NA) and an opening therethrough;
b) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and c) a means for maintaining the axial position of the core fiber within the support fiber.
2. The dielectric waveguide of claim 1 wherein NB is greater than NA.
3. The dielectric waveguide of claim 1 wherein NB
is equal to NA.
is equal to NA.
4. The dielectric waveguide of claim 1 wherein NB
is less than NA.
is less than NA.
5. The dielectric waveguide of claim 1 wherein a mirror coating is placed about the inside of support fiber.
6. The dielectric waveguide of claim 1 wherein a mirror coating is placed about the outside of support fiber.
7. The dielectric waveguide of claim 1 wherein the support fiber has a cladding on the inside with an index of refraction NC which is greater than NB.
8. The dielectric waveguide of claim 1 wherein the core fiber has a reactant coating which, in the presence of electromagnetic radiation, interacts with the analyte to form a signal radiation.
9. The dielectric waveguide of claim 1 wherein the support fiber has a reactant coating which, in the presence of electromagnetic radiation, interacts with the analyte to form a signal radiation.
10. A method of spectrophotometrically assaying an analyte in a fluid comprising:
a) coating a multi-element dielectric waveguide comprising:
i) a support fiber having an index of refraction (NA) and an opening therethrough;
ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and iii) a means for maintaining the axial position of the core fiber within the hollow fiber;
with an immobilized reactant which interacts with the analyte to form a signal radiation;
b) contacting the waveguide array with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation down the core fiber so as to irradiate the combination of analyte and reactant;
d) detecting the signal radiation from the irradiated analyte and reactant interaction by monitoring the waveguide.
a) coating a multi-element dielectric waveguide comprising:
i) a support fiber having an index of refraction (NA) and an opening therethrough;
ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and iii) a means for maintaining the axial position of the core fiber within the hollow fiber;
with an immobilized reactant which interacts with the analyte to form a signal radiation;
b) contacting the waveguide array with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation down the core fiber so as to irradiate the combination of analyte and reactant;
d) detecting the signal radiation from the irradiated analyte and reactant interaction by monitoring the waveguide.
11. A method of spectrophotometrically assaying an analyte in a fluid comprising:
a) coating a dielectric waveguide comprising:
i) a support fiber having an index of refraction (NA) and an opening therethrough; and ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and iii) a means for maintaining the axial position of the core fiber within the hollow fiber;
with an immobilized reactant which interacts with the analyte to form a signal radiation;
b) contacting the waveguide with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation down the support fiber so as to irradiate the combination of analyte and reactant;
d) detecting the signal radiation from the irradiated analyte and reactant interaction by monitoring the waveguide.
a) coating a dielectric waveguide comprising:
i) a support fiber having an index of refraction (NA) and an opening therethrough; and ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and iii) a means for maintaining the axial position of the core fiber within the hollow fiber;
with an immobilized reactant which interacts with the analyte to form a signal radiation;
b) contacting the waveguide with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation down the support fiber so as to irradiate the combination of analyte and reactant;
d) detecting the signal radiation from the irradiated analyte and reactant interaction by monitoring the waveguide.
12. A method of spectrophotometrically assaying an analyte in a fluid comprising:
a) coating a multi-element dielectric waveguide comprising:
i) a support fiber having an index of refraction (NA) and an opening therethrough;
ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and iii) a means for maintaining the axial position of the core fiber within the hollow fiber;
with an immobilized reactant which interacts with the analyte to form a signal radiation;
b) contacting the waveguide with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation within the fluid so as to irradiate the combination of analyte and reactant;
d) detecting the signal radiation from the irradiated analyte and reactant interaction by monitoring the waveguide.
a) coating a multi-element dielectric waveguide comprising:
i) a support fiber having an index of refraction (NA) and an opening therethrough;
ii) a second core fiber axially positioned within the support fiber opening and having an index of refraction (NB); and iii) a means for maintaining the axial position of the core fiber within the hollow fiber;
with an immobilized reactant which interacts with the analyte to form a signal radiation;
b) contacting the waveguide with the fluid for a time sufficient for the analyte and reactant to be able to interact;
c) propagating radiation within the fluid so as to irradiate the combination of analyte and reactant;
d) detecting the signal radiation from the irradiated analyte and reactant interaction by monitoring the waveguide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000610474A CA1269546A (en) | 1984-09-21 | 1989-09-06 | Dielectric waveguide sensors and their use in immunoassays |
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US65271484A | 1984-09-21 | 1984-09-21 | |
| US652,714 | 1984-09-21 | ||
| US77307485A | 1985-09-06 | 1985-09-06 | |
| US773,074 | 1985-09-06 | ||
| CA000491277A CA1266998A (en) | 1984-09-21 | 1985-09-20 | Dielectric waveguide sensors and their use in immunoassays |
| CA000610474A CA1269546A (en) | 1984-09-21 | 1989-09-06 | Dielectric waveguide sensors and their use in immunoassays |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000491277A Division CA1266998A (en) | 1984-09-21 | 1985-09-20 | Dielectric waveguide sensors and their use in immunoassays |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1269546A true CA1269546A (en) | 1990-05-29 |
Family
ID=27167553
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000610474A Expired - Lifetime CA1269546A (en) | 1984-09-21 | 1989-09-06 | Dielectric waveguide sensors and their use in immunoassays |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1269546A (en) |
-
1989
- 1989-09-06 CA CA000610474A patent/CA1269546A/en not_active Expired - Lifetime
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