Note: Descriptions are shown in the official language in which they were submitted.
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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.
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Back~round Art
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: 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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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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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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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
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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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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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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:
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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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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.
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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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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
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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.
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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
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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.
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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
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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
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invention, various changes and modifications may be
made without departing from the spirit and scope of
the invention as defined by the appended claims.
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