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Claims  |
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What is claimed is:
1. In apparatus for assaying a fluid sample and including a totally
internally reflecting, elongated, substrate transmissive to excitation
radiation directed at a proximal end of said substrate by an optical
system; which radiation, when propagated through said substrate, will
provide an evanescent wave for exciting fluorescence in fluorescent
material disposed at least on a portion of the surface of said substrate,
said substrate also being transmissive to said fluorescence; and a hollow
elongated enclosure spaced from and surrounding the surface of said
substrate, the improvement comprising:
mounting means couplable to said optical system and to said substrate and
to said enclosure for mounting said substrate within said enclosure, said
mounting means comprising:
seat means spaced a fixed predetermined distance from said optical system
when said mounting means is coupled to said optical system, for so
releasably seating said proximal end in a preselected position fixed at
least axially with respect to the long axis of said substrate and with
respect to said optical system, that when said proximal end is seated in
said seat means, substantially all of said excitation radiation entering
said proximal end from said optical system at a solid angle less than or
equal to a maximum acceptance angle determined in part by the refractive
index of said sample, undergoes total internal reflection within said
substrate.
2. In apparatus as defined in claim 1, wherein said mounting means imposed
substantially no restriction on said maximum acceptance angle.
3. In apparatus as defined in claim 1, wherein said mounting means
comprises a base member having a frusto-conically tapering aperture formed
therein, and bore and counterbore means for holding said proximal end of
said substrate so that said substrate (a) is substantially prevented from
moving transversely of its axial dimension with respect to said base
member and (b) is prevented from moving in a first direction along its
axial dimension with respect to said base member, said bore means extends
through said base member in coaxial alignment with said frusto-conically
tapering aperture so as to intersect an apex end of said aperture, and
said counterbore means is coupled with and coaxially aligned with said
bore means and is coupled with the apex end of said frusto-conically
tapering aperture.
4. In apparatus as defined in claim 3, wherein said seat means is provided
in said counterbore means for supporting a peripheral portion of said
proximal end of said substrate so as to prevent said substrate from moving
in said first axial direction with respect to said base member and so that
a fixed spatial relationship is maintainable between said proximal end and
said radiation source.
5. In apparatus as defined in claim 3, said base member comprising
enclosure aperture means for receiving said elongated enclosure so as to
substantially prevent said elongated enclosure from moving transversely of
its axial dimension.
6. In apparatus as defined in claim 5, wherein said base member comprises
quick-release clamp means for releasably securing said elongated enclosure
in said enclosure aperture means so that said elongated enclosure can be
easily secured to and removed from said base member and so that said
elongated enclosure is prevented from moving along its axial dimension
when secured to said base.
7. In apparatus as defined in claim 3, said mounting means comprising
biasing means for urging said elongated substrate in said first axial
direction.
8. In apparatus as defined in claim 1, wherein said mounting means is
coupled to a distal end of said elongated means and comprises an opening
adjacent said distal end through which a fluid sample may be introduced
into said enclosure.
9. In apparatus as defined in claim 1, wherein said mounting means
comprises a hollow body secured to a distal end of said substrate, said
hollow body comprising aperture means for slidably receiving a distal end
of said elongated enclosure so that said distal end of said substrate and
said hollow body are substantially prevented from moving transversely
relative to said distal end of said elongated enclosure, said hollow body
comprising an opening through which a fluid sample may be introduced into
said elongated enclosure.
10. In apparatus as defined in claim 9, said mounting means comprising
biasing means surrounding said elongated enclosure having a first end
attached to said hollow body and a second end attached to said elongated
enclosure for urging said substrate in a first axial direction with
respect to said elongated enclosure.
11. In apparatus as defined in claim 1, said mounting means comprising
spacer means secured to said substrate adjacent a distal end of said
substrate and extending into said elongated enclosure adjacent a distal
end of said enclosure for substantially preventing said distal end of said
substrate from moving transversely relative to said enclosure, said spacer
means being sized so that a gap exists between said spacer means and said
enclosure through which a fluid sample may be introduced into said
enclosure.
12. In apparatus as defined in claim 11, said mounting means comprising
biasing means surrounding said elongated enclosure and having a first end
attached to said spacer means and a second end attached to said elongated
enclosure for urging said substrate in a first axial direction with
respect to said elongated enclosure.
13. In apparatus for assaying a fluid sample and including a totally
internally reflecting elongated substrate transmissive to radiation
capable of providing an evanescent wave for exciting fluorescence in
fluorescent material disposed at leas on a portion of the surface of said
substrate, said substrate also being transmissive to said fluorescence,
and elongated means spaced from said surface of said substrate so as to
define a hollow elongated enclosure surrounding said surface, and optical
means couplable to said mounting means for transmitting a beam of optical
radiation to an end face of said substrate, the improvement comprising:
means coupled to said substrate and said elongated means for mounting said
substrate within said enclosure and for releasably supporting both ends of
said elongated substrate so that said end face is axially positionable at
a fixed location with respect to said optical means and so that said
elongated substrate is (a) substantially prevented from moving
transversely relative to its longitudinal axis and (b) is prevented from
moving in a first axial direction relative to said optical means.
14. In apparatus as defined in claim 13, said optical means further
comprising light source means for transmitting said radiation so as to
enter said substrate at said end face at a solid angle less than or equal
to the maximum acceptance angle determined in part by the refractive index
of said sample, and photo- detector means for detecting said fluorescence
transmitted through said end face.
15. In apparatus as defined in claim 14, said mounting means comprising
seat means for supporting a peripheral portion of said end face so as to
achieve substantially total internal reflection of said radiation which is
transmitted into and is emitted from said elongated substrate.
16. In apparatus as defined in claim 15, wherein said seat means is
positioned at a location that is spaced a discrete distance from said
optical means.
17. In apparatus as defined in claim 15, said mounting means comprising
biasing means coupled to said substrate and said elongated means for
biasing said end face in said first direction against said seat means.
18. In apparatus as defined in claim 17, wherein said biasing means
comprises a coil spring surrounding said elongated substrate and said
elongated means, said coil spring having a first end secured to said
elongated substrate and a second end secured to said elongated means.
19. In apparatus as defined in claim 15, said supporting means comprising
centering means for cooperating with said end face so as to cause said end
face to move radially and axially toward said seat upon insertion of said
substrate into said supporting means.
20. In apparatus as defined in claim 13 wherein said optical means
comprises an objective lens system.
21. In apparatus as defined in claim 13 wherein said supporting means
cooperates with said elongated substrate to hold the latter in coaxial
alignment in said enclosure.
22. In apparatus as defined in claim 13 wherein said substrate is an
optical rod having a circular cross-section and a substantially uniform
diameter.
23. In apparatus for assaying a fluid sample and including a totally
internally reflecting, elongated, substrate transmissive to excitation
radiation from a radiation source, which radiation is capable of providing
an evanescent wave for exciting fluorescence in fluorescent material
disposed at least on a portion of the surface of said substrate, said
substrate also being transmissive to said fluorescence, said substrate
comprising a proximal end through which said excitation radiation and
excited fluorescence may be transmitted, a hollow elongated enclosure
spaced from and surrounding the surface of said substrate, the improvement
comprising:
mounting means couplable to said substrate and to said enclosure for
mounting said substrate within said enclosure, said mounting means
comprising:
means defining a fixed interface between said mounting means and said
radiation source;
seat means for supporting said proximal end in a preselected position with
respect to said interface, so that substantially all of said excitation
radiation entering said proximal end at a solid angle less than or equal
to a maximum acceptance angle determined in part by the refractive index
of said sample undergoes total internal reflection within said substrate;
and
seal means for creating a barrier past which a fluid sample disposed within
said enclosure cannot escape.
24. In apparatus according to claim 23, wherein said seal means is disposed
adjacent both a distal and said proximal end of said enclosure so as to
prevent said fluid sample from escaping from either said distal or
proximal ends of said enclosure past said barrier.
25. In apparatus according to claim 23, wherein said seal means is disposed
adjacent said proximal end of said enclosure so as to prevent said fluid
sample from escaping from said proximal end of said enclosure past said
barrier.
26. In apparatus according to claim 23, wherein said mounting means
comprises a base member for receiving a proximal end of said enclosure and
said proximal end of said substrate, further wherein said seal means
comprises lip means provided in said base member for engaging said
proximal end of said enclosure and said proximal end of said substrate so
as to define a barrier past which a fluid sample disposed within said
enclosure cannot escape.
27. In apparatus according to claim 26, said seal means further comprising
O-ring means disposed adjacent said lip means for ensuring a fluid-tight
seal is achieved between (1) said proximal end of said enclosure and said
proximal end of said substrate and (2) said base member.
28. In apparatus as defined in claim 1 including means associated with the
distal end of said substrate for absorbing said excitation radiation.
29. In apparatus as defined in claim 28 wherein said means for absorbing
has an index of refraction substantially matched to the index of
refraction of the medium immediately surrounding said distal end.
30. In apparatus for assaying a fluid sample and including a totally
internally reflecting elongated substrate transmissive to radiation
capable of providing an evanescent wave for exciting fluorescence in
fluorescent material disposed at least on a portion of the surface of said
substrate, said substrate also being transmissive to said fluorescence,
and elongated means spaced from said surface of said substrate so as to
define a hollow elongated enclosure surrounding said surface, and optical
means couplable to said mounting means for transmitting a beam of optical
radiation to an end face of said substrate, the improvement comprising:
means coupled to said substrate and said elongated means for mounting said
substrate within said enclosure and for releasably supporting both ends of
said elongated substrate so that said end face is axially positionable at
a fixed location with respect to said optical means and so that said
elongated substrate is (a) substantially prevented from moving
transversely relative to its longitudinal axis and (b) is prevented from
moving in a first axial direction relative to said optical means; and
wherein said optical means comprises a tapered fiber disposed in fixed
spatial relationship relative to said fiber end face, said tapered fiber
having an input face and an output face, said tapered fiber being
transmissive to said optical radiation and being tapered smoothly so as to
reduce the diameter of said tapered fiber from said input face to said
output face.
31. In apparatus according to claim 30, wherein said optical means
comprises an optical element and wherein said input face is disposed
adjacent said optical element and said output face is disposed adjacent
said fiber end face.
32. Apparatus as defined in claim 1 wherein said seat means substantially
prevents movement of said proximal end radially with respect to the long
axis of said substrate when said proximal end is seated in said seat
means. |
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Claims |
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Description |
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This invention relates to optical apparatus for carrying out chemical and
biochemical assays, and more particularly to an improved fiber optics
apparatus for such assays.
Among the large variety of chemical and biochemical equipment used for
analysis or assay, is an optical system employing the principles of
attenuated total internal reflection (ATR) spectroscopy. Particularly
useful for immunoassays, such an optical system employs an optical fiber
or rod upon a portion of the outer surface of which an antibody is
covalently immobilized, adsorbed or the like. The antibody is selected to
be reactive with an antigen in a solution to be assayed or tested. A light
beam introduced into one end of the optical rod will be totally internally
reflected in the dense medium of the rod, and will generate in the rarer
medium or test solution an electromagnetic waveform, known as the
evanescent wave component. The latter, for practical purposes,
characteristically effectively extends only a fraction of a wavelength
across the interface between the rod and test solution. This penetration,
however, is sufficient to permit substantial optical interaction between
the evanescent wave component and the immobilized antibody with which the
antigen in the test solution will complex, and only minimally with any
bulk solution in which the antigen was present. Such optical interaction
then permits one to assay the antigen. A number of such systems using
internal total reflection spectroscopy for an assay are known and have
been described, for example, in U.S. Pat. Nos. 4,133,639 in which is
disclosed a system based on absorption of the evanescent wave by the
analyte; and 4,321,057 and 4,399,099 both of which disclose systems that
detect changes in the radiation transmitted through the fiber; 4,447,546
which describes a fluorescence immunoassay system; and others.
An immunoassay apparatus developed by T. Hirschfeld (U.S. Pat. No.
4,447,546 issued May 8, 1984) 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 observed at angles greater than the critical angle,
by total reflection within the solid medium. The solid phase is arranged
and illuminated to provide multiple total internal reflections at the
interface.
Typically, the solid phase is in the form of an optical fiber or rod to
which is immobilized a component of a complex formed in an immunochemical
reaction. A fluorophore 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 labeled component is typically preloaded to the immobilized component
in a controlled concentration.
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 directions 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 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 are less intensely excited and thus
fluoresce 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 zone 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 the actually sampled layer thickness much larger than the
thin surface layer that is all that contributes to the background.
All of the radiation that tunnels back into the fiber within the total
reflection angle is thus trapped within the fiber. The power available
from the fluorescence increases with the length of fiber within the
fluorescing material. However, the optical throughput of the system
(determined by the diameter and the numerical aperture of the fiber)
remains constant. The total fluorescent signal coming from the entire
surface of the fiber, multiplied by the 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 matched to a small detector.
For excitation radiation initially propagating through an optical fiber of
refractive index n.sub.0, otherwise surrounded by a material of refractive
index n.sub.1, the maximum acceptance angle B of input radiation into the
fiber can be found from the equation:
NA = n.sub.2 sinb = sinb = (n.sub.0.sup.2 -n.sub.1.sup.2
where n.sub.2 is the refractive index of the medium (typically air) through
which the radiation is initially propagated so as to be incident upon an
end of the fiber, and NA is the so-called numerical aperture of the fiber.
The maximum acceptance angle B is simply defined as:
B = sin.sup.-1 NA (2)
and B=b when n.sub.2 =1 (e.g. n.sub.2 is for dry air). Thus, the numerical
aperture for a fiber is highest when the fiber core material has a very
high index and the medium surrounding it has a very low index, or n.sub.0
>> n.sub.1. For example, satisfactory sensitivities can be obtained where
a transparent fiber (glass, silica, polymer or the like) of ordinary index
of refraction is surrounded by an aqueous solution that typically has an
index of refraction in the vicinity of 1.33-1.35.
In known immunoassay apparatus, such as the one described in the
aforementioned U.S. Pat. No. 4447546 to Hirschfeld, an optical fiber is
supported within a capillary tube in approximately co-axial alignment
therewith. A fluid sample is introduced into the space formed between the
fiber and the tube and is drawn into and supported in the space by
capillary action. To maximize sensitivity and efficiency of such an
immunoassay apparatus, it is important that the fiber remain substantially
coaxially centered within the capillary tube. If the fiber contacts the
capillary wall, capillary action may be adversely affected, and total
internal reflection may not be achieved since radiation may pass out of
the fiber at the point of contact between the fiber and the capillary
wall. Loss of sensitivity typically occurs in the apparatus as a result of
such refraction.
Inasmuch as the intensity of the fluorescent signal tunnelling back into
the fiber is proportional to a very high power (ca. 9th) of the numerical
aperture (as defined in part by the refractive index of the sample in
which fluorescence is excited), it is important to try to preserve the
maximum possible numerical aperture throughout the system.
It is also important that the proximal end of the fiber into which optical
radiation is transmitted and from which fluorescent radiation is emitted
be supported in a fixed axial position with respect to the means for
transmitting optical radiation into the fiber. In the event the proximal
end of the fiber does not lie at a fixed position with respect to the
objective lens of the optical system associated with the immunoassay
apparatus, the amount and orientation of transmitted radiation entering
the rod may vary. This variation may adversely affect the accuracy and
sensitivity of the apparatus.
At least two techniques have been developed in known immunoassay apparatus
for locating an optical fiber within a capillary tube. The first technique
involves supporting the fiber in cantilever fashion at its distal end,
i.e. the end opposite the end where optical radiation is transmitted into
said fiber. The proximal end of an optical fiber supported by this
technique is displaceable both axially and radially. Such displacement
gives rise to the aforementioned loss of instrument sensitivity.
In the second technique, the proximal end of the optical fiber is supported
using a conventional fiber optic connector. Use of these connectors
typically involves covering the outer surface of the fiber adjacent its
proximal end with a cladding material typically consisting of a
transparent high molecular weight polymer. Known cladding materials
typically have a refractive index higher than that of the sample, e.g.
1.40 to 1.45, with the result that the numerical aperture of the fiber is
reduced to a level at which acceptable sensitivity levels cannot readily
be achieved with the apparatus.
The evanescent zone tends to increase in depth and the sensitivity of the
system also increases as the numerical aperture of the fiber increases.
Thus, it is preferred that the numerical aperture of the system be
maximized. Such maximization has heretofore been limited by the second of
the above-described techniques used to clamp and support the fiber.
Fiber-optic assay systems having a disposable optical fiber assembly are
useful in testing for the presence of harmful viruses. The optical fiber
assembly that receives the fluid sample containing the potentially harmful
viruses is readily disposable. Thus, to improve the efficiency and reduce
the cost of such important and widely-used assay procedures, it is
important that the fiber-optic assembly of the assay system be easily
replaceable and have a high numerical aperture.
A principal object of the present invention is therefore to provide an
improved fiber-optic assay system employing an optical rod or fiber
positioned within and spaced from an enclosure, which system comprises
means for mounting the rod within the enclosure so that the rod is
positioned and supported in a fashion maximizing the sensitivity of the
system. Other objects of the present invention are to provide such a
system in which the rod and enclosure may be readily inserted into and
removed from a base assembly in which the optics of the system are
located, with the rod being firmly supported and properly optically
aligned automatically upon the insertion of the rod into the base; and to
provide such a system in which the numerical aperture of the rod is
maximized by supporting the rod in alignment with the optics of the system
such that substantially none of the input optical radiation intersects the
mounting assembly for supporting the proximal end of the rod; to provide
an assay system designed to prevent a fluid assay sample contained within
the enclosure from escaping from the enclosure and the base assembly for
supporting the enclosure; and to include in the optical system of the
present invention a tapered fiber for increasing the power and numerical
aperture of radiation input into the optical fiber.
The foregoing and other objects of the present invention are achieved by an
assay system comprising an optical rod or fiber positioned within an
enclosure, and a base assembly including a holder for receiving the
proximal ends of the rod and enclosure. The holder comprises a concave
aperture that tapers frusto-conically to a bore having a seat formed
therein for supporting the proximal end of the fiber. The seat may be
designed to block input radiation intersecting the radially-outermost
portions of the proximal end of the fiber so as to eliminate stray light
production arising from edge defects in the rod. The holder also has a
sleeve portion for supporting and releasably locking the proximal end of
the enclosure. A centering device is attached to the distal end of the rod
for ensuring the rod remains substantially centered within the enclosure.
The centering device is designed to permit a fluid sample to be introduced
into the enclosure at the distal end thereof. A spring is attached to the
distal end of rod, preferably via the centering device, and the enclosure
for urging the rod into engagement with the seat in the holder. In
alternative embodiments of the invention, seal means are provided in the
base assembly and at the distal end of the enclosure for preventing an
assay sample disposed within the enclosure from escaping from the
enclosure. In another alternate embodiment of the invention the optical
system thereof includes a tapered fiber interposed between the optical
fiber and the radiation source of the optical system for increasing the
power of the radiation input into the optical fiber.
Other objects of the present invention will in part be obvious and will in
part appear hereinafter.
The invention accordingly comprises the apparatus possessing the
construction, combination of elements and arrangement of parts which are
exemplified in the following detailed disclosure, and the scope of the
application of which will be indicated in the claims.
For a fuller understanding of the nature and objects of the present
invention, reference should be had to the following detailed description
taken in connection with the accompanying drawings in which like numerals
in the several drawings are employed to denote like parts, and wherein:
FIG. 1 shows, in idealized, enlarged, longitudinal cross-section, a fiber
optic system embodying the principles of the present invention;
FIG. 2 is a side elevation view of the slidable spider portion of the
embodiment illustrated in FIG. 1;
FIG. 3 is an idealized, enlarged, fragmentary, longitudinal cross-sectional
view, of a portion of the embodiment illustrated in FIG. 1;
FIG. 4 is an idealized, longitudinal cross-sectional view of the fiber
optic system of FIG. 1 and the optical system with which it is adapted to
be used;
FIG. 5 is an idealized longitudinal cross-sectional view of a fiber optic
system embodying the principles of another embodiment of the present
invention and an optical system with which it is adapted to be used;
FIG. 6 is an idealized longitudinal cross-sectional view of a fiber optic
system embodying the principles of still another embodiment of the present
invention and an optical system with which it is adapted to be used;
FIG. 6a is identical to FIG. 6, except that a seal is shown positioned
between the proximal end of the tube and the base;
FIG. 6b is a fragment of an embodiment such as in FIG. 6, but shows
additional seals between the tube and base member;
FIG. 7 is an idealized longitudinal cross-sectional view of a fiber optic
system embodying the principles of yet another embodiment of the present
invention and an optical system with which it is adapted to be used;
FIG. 8 is an idealized longitudinal cross-sectional view of a fiber optic
system embodying the principles of still another embodiment of the present
invention and an optical system with which it is adapted to be used; and
FIG. 9 is an idealized, longitudinal cross-sectional view of the fiber
optic system of FIG. 4, including an alternate embodiment of optical
system thereof.
Referring to FIGS. 1, 2 and 4, there is shown exemplary apparatus 20 for
assaying a fluid sample, which apparatus incorporates the principles of
the present invention. Apparatus 20 includes optical rod or fiber 22,
hollow, elongated enclosure 24, fiber centering device 26 and base member
28, and is similar in many respects to the system shown in the aforesaid
U.S. Pat. No. 4,447,546.
Rod 22 is an elongated body extending from its proximal end or entrance
face 30 to a distal or terminal end 32. Rod 22 preferably has a
substantially circular cross-section. At proximal face 30 the rod surface
typically is planar, is disposed normally to the longitudinal axis of the
fiber and is preferably highly polished to minimize any blemishes or
surface defects that would tend to scatter incident and emitted radiation.
Alternatively, proximal face 30 of the rod may be configured in other
desired optical shapes to serve, for example, as a magnifying or matching
optical surface.
In a preferred embodiment, in which the fluorescence induced at the fiber
surface by excitation radiation launched down the fiber is collected or
observed at the same proximal end of the fiber at which the excitation
radiation is injected, it is desired to prevent stray radiation from going
back up the fiber from distal face 32 to proximal face 30. Consequently,
face 32 may be shaped to spill out light incident thereon internally, but
preferably is coated with a material matching the index of refraction of
the medium surrounding face 32, such material being both non-fluorescent
and absorbent with respect to the excitation radiation. Typically, an
epoxy resin loaded with carbon black serves such function.
Rod 22 is adapted to propagate along its length, by multiple total internal
reflection, optical excitation radiation entering proximal face 30 within
a conical acceptance angle (B) substantially symmetric with the long axis
of the fiber and defined herein before, as well known to those skilled in
the fiber optics art, in equation (1). Rod 22 may be any of a very large
number of substantially homogeneously materials optically transparent to
the excitation radiation, e.g. glassy materials such as glass, crystalline
materials such as quartz, sapphire and the like; synthetic polymers such
as polyolefins, polypropylenes and the like, and is preferably relatively
stiff. Where rod 22 is to be used in fluid assays as described
hereinafter, he index of refraction (n.sub.0) of the material forming rod
22 must be greater than n.sub.1, the index of refraction of the fluid
being assayed. The latter index is typically about 1.3 for an aqueous
solution. For purposes of an immunoassay apparatus, rod 22 has a length
ranging from 3cm to 5cm, with about 4cm being the preferred length. Rod 22
typically has a diameter in the range of from about 0.5mm to 1.5mm, with
about 1 mm being the preferred diameter. It should be understood, however,
that such length and diameter are merely exemplary and not limiting.
In an exemplary embodiment, it is intended that the operative portion of
the fiber surface be defined by the dimensions of an activated region at
which the assay is to be performed. To activate the surface of the
operative portion of rod 22, the latter is typically treated to provide
coating 34 such as is described in detail in U.S. Pat. No. 4,447,546 and
is incorporated herein by reference.
Enclosure 24 is preferably but not necessarily optically transparent, and
is formed of a material that is relatively insoluble and chemically
non-reactive with the fluid being assayed. Typically, enclosure 24 is
simply a glass tube having an inside diameter greater than the maximum
outside diameter of fiber 22, and preferably dimensioned to delimit a
predetermined volume surrounding at least activated coating 34 on fiber
22. In a preferred embodiment, the interspace between the coated surface
of fiber 22 and the inside wall of enclosure 24 is of capillary dimension.
Fiber centering device 26 comprises fixed spring mount 40, slidable spider
42 and tension spring 44. Spring mount 40 is secured to the outer surface
of tube 24, as by adhesive bonding or other suitable process. Spring mount
40 comprises a relatively rigid, radially-extending element to which one
end of tension spring 44 may be attached. As skilled practitioners will
appreciate, spring mount 40 may take a variety of forms, including, for
instance, an annulus having an outside diameter sized to frictionally
engage the interior of tension spring 44. Alternatively, a slot may be
formed in spring mount 40 for retaining the one end of tension spring 44.
Slidable spider 42 comprises a hollow body having at least one opening 43
formed in a sidewall thereof into which a fluid sample may be introduced.
An exemplary spider 42 having a substantially rectangular shape and
opposed side openings 43 is shown in FIG. 2. Aperture 46 is formed in one
end of spider 42, with the inside diameter of the aperture being slightly
greater than the outside diameter of tube 24 so that tube 24 is slidably
receivable in aperture 46. In this position, spider 42 is substantially
coaxially centered on tube 24. At an opposite end of spider 42, blind bore
48 is formed in enlarged portion 50 of the spider. The inside diameter of
bore 48 is selected so that the distal end of rod 22 may be secured in the
bore by force fit or adhesive bonding. Enlarged portion 50 may be formed
integrally with spider 42 or may comprise a separate element that is
press-fitted into an appropriate aperture provided in the end of spider 42
opposite opening 46. In any case, the longitudinal axis of bore 48 must
substantially coincide with the longitudinal axis of aperture 46. An
opposite end of tension spring 44 is secured to spider 42 adjacent
aperture 46 by suitable means, such as adhesive bonding or inserting the
opposite end of the spring into a spring retaining slot (not shown) formed
in the spider 42.
Tension spring 44 is preferably a coil spring having a suitable length and
spring coefficient. As described more fully hereinafter the length and
spring coefficient of spring 44 and length of rod 22 are selected so that
proximal end 30 protrudes a selected distance from tube 24 when spring 44
is unbiased. The spring coefficient of spring 44 is further selected so
that rod 22 may be suitably biased with respect to tube 24, as described
more fully hereinafter.
Referring now to FIGS. 3 and 4, base member 28 is made from a block of
relatively rigid material such as aluminum or a dense synthetic polymer. A
hard insert 54, made typically from stainless steel, titanium or the like,
is disposed in a cavity 56 formed at a central location on the bottom
surface of base member 28. An aperture extends through member 28 that
comprises large diameter portion 58 that terminates in frusto-conically
tapering portion 61 that tapers to reduced diameter portion 62. The latter
terminates in a radially-inwardly extending annular seat 64 having an
inside diameter that is a selected amount less than the outside diameter
of rod 22, as described more fully hereinafter. Thus, the opening defined
by seat 64 and reduced diameter portion 62 provide a bore and counterbore,
respectively, in the apex end of frusto-conically tapering portion 61. A
second frusto-conical portion 67 opens outwardly from seat 64 toward the
bottom surface of base member 28. Preferably, at least portion 62, seat
64, and frusto-conical portions 61 and 67 are disposed in insert 54.
The inside diameter of large diameter portion 58 is slightly greater than
the outside diameter of tube 24 so that the latter may be slidably
disposed in the large diameter portion. Similarly, the inside diameter of
reduced diameter portion 62 is slightly greater than the outside diameter
of rod 22 so that the latter may be slidably disposed in the reduced
diameter portion. Seat 64 is sized to engage only the radially-outermost
portion of proximal end 30 when rod 22 is inserted in reduced diameter
portion 62, so as to achieve maximum sensitivity in the apparatus, as
described hereinafter.
Base member 28 comprises means for securing tube 24 into portion 58, such
as set screw 70. Additionally, as described more fully hereinafter, means
are provided in base member 28, such as allen screw 72, for securing the
base member to the optical system with which the assay apparatus 20 is
adapted for use.
Referring now to FIG. 4, the present assay apparatus 20 is designed for use
with an optical system 100 comprising light source 102, photo detector
104, beam splitter 106, objective lens 108 and secondary lens 109. The
foregoing elements of optical system 100 are disposed in fixed optical
relationship to one another and to immunossay apparatus 20, as described
more fully hereinafter. By this relationship, light beam 110 generated by
light source 102 is folded by beam splitter 106 so as to pass through
objective lens 108 and into rod 22 through proximal end 30. Light source
102, beam splitter 106, objective lens 108 and secondary lens 109 are
selected and operated so that substantially all the rays of beam 110
intersect proximal end 30 at less than or equal to the maximum acceptance
angle at end 30. Of course, other optical elements may be used in place of
objective lens 108 for imaging the beam of input radiation, such as an
optical fiber.
Any light beams 112, the latter consisting of fluorescence excited by the
evanescent wave, emitted from proximal end 30 pass through objective lens
108 through beam splitter 106 and are focused by secondary lens 109 so as
to contact photo detector 104. Frusto-conical portion 67 is provided so
that light beams 112 are not intercepted by sections of insert 54 as they
travel toward objective lens 108.
Threaded aperture 114 may be provided in optical system 100 for receiving
Allen screw 72, whereby base 28 may be readily secured to and separated
from optical system 100 if desired.
To maximize the accuracy and efficiency of the present assay apparatus 20,
it is important that the flat face of proximal end 30 lie at a fixed
position with respect to objective lens 108. To this end, insert 54 is
formed so that when base member 28 is secured to optical system 100, as
shown in FIG. 4, seat 64 is fixed at a preselected focal or axial position
with respect to objective lens 108.
In operation of the embodiment of FIGS. 1-4, coating 34 of rod 22 is formed
from any of a number of activating reagents (such as a constituent of an
antibody-antigen complex that includes a fluorescent tag) and is
essentially subjected to the same procedures as are described in U.S. Pat.
No. 4,447,546. Rod 22 and tube 24, with centering device 26 affixed
thereto, are inserted together into aperture 58 until proximal end 30
engages seat 64. Frusto-conical portion 61 guides rod 22 into coaxial
alignment with portion 62 to facilitate the seating of proximal end 30.
Preferably, this operation is effected by grasping and pressing down on
spring mount 40 until proximal end 0 engages seat 64.
Tube 24 is then inserted farther into aperture 58 so as to bias tension
spring 44. Set screw 70 is then tightened to hold tube 24 in fixed
position in aperture 58. In this position, spider 42 ensures rod 22
remains substantially coaxially centered within tube 24. The length and
spring coefficient of spring 44 and length of rod 22 are selected so that
proximal end 30 engages seat 64 before tube 24 contacts frusto-conical
portion 61, whereby spring 44 holds proximal end 30 in relatively firm
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