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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to waveguide-binding sensors for
use in fluorescence assays, and, more particularly, to highly sensitive
fiber-optic waveguide-binding sensors that remotely sense fluorescence
radiation during assays in liquid solutions.
2. Description of the Related Art
The evanescent wave portion of an electromagnetic field produced by light
propagating through an optical waveguide characteristically penetrates a
few hundred nanometers into the medium surrounding the optical waveguide.
This evanescent wave can excite fluorescent molecules, e.g., fluorophores,
to fluoresce when they are bound by molecules near the optical waveguide
surface. The application of this phenomenon to an immunoassay sensor,
wherein the biological recognition (binding) of an antigen by antibodies
attached to the waveguide surface with concomitant displacement of
fluorescent-labeled antigen is measured as a change in fluorescence, was
first disclosed in A New Immunoassay Based on Fluorescence Excitation by
Internal Reflection Spectroscopy by Kronick and Little, 8 JOURNAL OF
IMMUNOLOGICAL METHODS, p. 235 (1975), incorporated by reference herein in
its entirety and for all purposes.
The use of optical fibers as a special class of waveguides for immunoassay
sensors is also known. For example, U.S. Pat. No. 4,447,546, incorporated
by reference herein in its entirety and for all purposes, discloses the
use of optical fibers as waveguides which capture and conduct fluorescence
radiation emitted by molecules near the optical fiber surfaces. However,
conventional waveguide-binding sensors for use with assays of aqueous
fluids have demonstrated inadequate sensitivity. Specifically, poor sensor
performance is attributed at least in part to the small size of the sample
being analyzed, typically, one or several monolayers in depth and the
small surface area of the optical waveguide. These factors limit the
number of fluorophores which may be excited. More serious sensor
performance degradation is attributable to the effects of a weak
evanescent wave which fails to excite enough fluorophores to produce
detectable levels of fluorescence and inadequate coupling of the
fluorescence into the waveguide for subsequent detection.
Increasing the strength of the evanescent wave penetrating into a fluid
sample to be assayed increases the amount of fluorescence, thereby,
increasing sensor sensitivity. Each mode (low and high order) propagating
in the fiber has a portion of its power in the evanescent wave. Higher
order modes have a larger percentage of their power in the evanescent wave
and so make a larger contribution to power in the evanescent wave.
However, these higher order modes are weakly guided, lossy, and can easily
leak at a discontinuity or a bending point along the waveguide.
The use of tapered optical fibers to increase the sensitivity of
fiber-optic assay systems is known. For example, U.S. Pat. Nos. 4,654,532
and 4,909,990, both incorporated by reference herein in their entirety and
for all purposes, disclose the use of optical fibers as sensors used in
conjunction with assays. In U.S. Pat. No. 4,654,532, an unclad, tapered
optical fiber that is completely isolated from the sample fluid.
The introduction of a tapered section of the optical waveguide, however,
fails to address certain important issues central to the sensitivity of
these sensors, especially in remote sensing applications. In particular,
the higher order modes propagating in the section of the waveguide where
the fluorophores are found (the distal end) contribute the most to power
in the evanescent wave and comprise the majority of the fluorescence
coupled back into the fiber. These higher order modes typically propagate
with greater loss than lower order modes.
For an incident beam of light of wavelength .lambda. traveling within a
cladded core and intersecting the edge of a cladded core at the core and
cladding boundary at an incident angle .theta. (measured from the normal
of the reflecting edge) wherein the core has an index of refraction of
n.sub.core and the cladding has an index of refraction of n.sub.cladding,
the thickness d.sub.p of the evanescent wave region contiguous and along
the outer edge of the core penetrating into the cladding is given by the
formula:
##EQU1##
If a portion of an optical fiber is unclad (i.e. the cladding is removed)
and the bare core is surrounded by a solution having an index of
refraction of n.sub.solution, then, for an incident beam of light of
wavelength .lambda. traveling within an uncladded core and intersecting
the edge of the uncladded core at the core and solution boundary at an
incident angle .zeta. (measured from the normal of the reflecting edge)
wherein the core has an index of refraction of n.sub.core and the solution
has an index of refraction of n.sub.solution, the thickness d.sub.p of the
evanescent wave region contiguous and along the outer edge of the core
penetrating into the solution is given by the formula:
##EQU2##
Typically, the evanescent wave region has a thickness d.sub.p of between
about 50-500 nm depending in part on the angle of incidence .theta. as
described by the equations above. Fluorescence radiation excited within
the evanescent wave region coupled into the core propagates in
higher-order modes and is susceptible to losses due to microbending and
V-number mismatch along the optical fiber.
U.S. Pat. No. 5,061,857, to Thompson et al., entitled Waveguide-Binding
Sensor for Use With Assays, filed Nov. 9, 1990 and issued Oct. 29, 1991
(the entirety of which is incorporated by reference herein for all
purposes) addresses concerns about poor V-number matching and the loss of
poorly guided fluorescence radiation along the length of the optical
fiber. The probe is inwardly tapered from the proximal to the distal end
at an angle such that the incident light beam of light traveling through
the fiber does not exceed the critical angle measured from the normal of
the reflecting edge to the incident beam of light. Thus, total internal
reflection (TIR) of the incident beam of light traveling within the
optical fiber is maintained. The fiber may also be variably doped along
its surface to similarly change the V-number along the length of the
fiber. Fluorescence coupled in from radii above the V-number matching
radius is lossy due to the V-number mismatch.
While the approach described in U.S. Pat. No. 5,061,857 improves over the
prior art, it still results in significant losses. Most of the signal in
this type of fiber optic sensor is generated at the tip. If the tip is
damaged, the entire probe is ruined. Also, a significant length of the
tapered portion is above the V-number matching radius. Thus, most of the
signal from this V-number mis-matched portion is lost.
SUMMARY OF THE INVENTION
It is an object of the present invention to improve the sensitivity of
fiber optic probes used in fluorescence assays.
It is another object of the present invention to enhance the power of
excitation radiation within the evanescent wave region along the length of
the sensing section of a fiber optic probe.
It is a further object of the present invention to propagate radiation, in
fiber optic probes, from lower order modes of excitation radiation to
higher order modes of excitation radiation with minimal loss, while
maximizing the effective sensing area of the fiber optic probe.
These and other objects are achieved by a fiber optic probe having a
sensing, distal end with a first inwardly tapered section and a second
inwardly tapered section distal to the first inwardly tapered section. The
first inwardly tapered section is more severely tapered, and extends for a
shorter length than the second inwardly tapered section. The first
inwardly tapered section tapers from a radius greater than the V-number
matching radius down to the V-number matching radius, at the intersection
of the first inwardly tapered section with the second inwardly tapered
section. Along the first inwardly tapered section, the angle of inward
taper may be maximized, such that the critical angle, in accordance with
Snell's law, measured from the normal of the reflecting edge to the
incident beam of light is not exceeded. Thus, total internal reflection
(TIR) of the incident beam of light traveling within the optical fiber is
maintained. In the second inwardly tapered section, the radius of the
probe remains at or below the V-number matching radius. The angle of
inward taper in the second inwardly tapered section is typically just that
which is sufficient to enhance the concentration of the excitation light
in the evanescent wave region.
BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation of the invention will be readily obtained by
reference to the following Description of the Preferred Embodiments and
the accompanying drawings in which like numerals in different figures
represent the same structures or elements, wherein:
FIG. 1 is a plan view of one prior art embodiment of a fiber optic probe.
FIG. 2 is a plan view of another prior art embodiment of a fiber optic
probe.
FIG. 3 is a plan view of a fiber optic probe according to the present
invention.
FIG. 4 is a plan view of the distal, unclad end of the fiber optic probe
according to the present invention.
FIG. 5 is a schematic layout of a typical fiber optic probe apparatus
according to the present invention.
FIG. 6 is a schematic layout of the sensing distal end of the fiber optic
probe having attached to it an antibody and a fluorescently labelled
antigen attached to the antibody attached to the optical fiber.
FIG. 7 is a schematic layout of the sensing distal end of the fiber optic
probe held within a capillary tube having tee connections as shown. Note
that the first and second tapered sections shown in FIGS. 3 and 4 are
present but not shown.
FIG. 8 is a schematic of a beam of incident light traveling through the
distal, unclad, combination tapered end of the optical fiber.
DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
As shown in FIG. 1, prior art fiber optic probe 10 includes a distal end
portion 12. At distal end portion 12, cladding 14 has been removed to
expose the surface of the fiber core 16. Just distal to the end of the
cladding, the radius of the fiber core abruptly changes from one that is
above the V-number matching radius (in section 18) to one that is at or
below the V-number matching radius (in section 20). Section 20, which is
at or below the V-number matching radius, is the sensing portion of fiber
optic probe 10.
The abrupt change in radius between sections 18 and 21) causes light to
escape through flat surface 22 where sections 18 and 21) meet, reducing
the radiation energy transmitted to the sensing portion. In addition,
higher order modes of radiation are absorbed or scattered along the
surface of sensing portion 20.
FIG. 2 shows yet another prior art fiber optic probe 21. At distal end
section 40, cladding 24 has been removed to expose the surface of the
fiber core 26. Just distal to the end of cladding 24, the radius of the
fiber core gradually changes, at a constant rate, from one that is above
the V-number matching radius (along section 28) to one that is at the
V-number matching radius (along line 29, the intersection of sections 28
and 23) to below the V-number matching radius (along section 23). Section
23 is now known to be the effective sensing portion of fiber optic probe
21. Because section 28 is above the V-number matching radius, it couples
inefficiently. Thus, a significant portion of the fluorescent signal is
still lost in optical probes employing a continuous taper. Additionally,
most of the fluorescent signal is generated at the very distal end
(section 23) of this probe. If this end of the probe is broken or
otherwise impaired, the entire probe must be discarded.
FIG. 3 shows a fiber optic probe 31 according to the present invention.
Along distal end portion 39 and 33, cladding 34 has been removed to expose
the surface of the fiber core 36. Just distal to the end of cladding 36,
the radius of the fiber core in section 39 continuously tapers down to the
V-number matching radius over a short distance. The radius of the section
33 tapers down slightly in the direction of tip 41. At the intersection of
sections 33 and 39 at line 69, the radius of the probe (r.sub.39 =r.sub.33
=r.sub.match) equals the V-number matching radius r.sub.match.
Referring to FIG. 8, the symbols within the figure represent the following:
(1) r.sub.0 is the radius of the unclad optical fiber at any point along
the fiber that is proximal to the first tapered section 39;
(2) a.sub.0 is the angle of the incident beam of light measured with
respect to the longitudinal axis of the optical fiber at any point along
the unclad, optical fiber that is proximal to the first tapered section
39;
(3) r.sub.z is the radius of the unclad optical fiber at any point along
the tapered, unclad, optical fiber within first and second tapered
sections 39 and 33, respectively, where the incident beam of light
intersects the reflecting edge;
(4) a.sub.z is the angle of the incident beam of light measured with
respect to the longitudinal axis of the optical fiber at any point along
the tapered, unclad, optical fiber within first and second tapered
sections 39 and 33, respectively;
(5) .theta. is the angle of incidence of the beam of light (measured from
the normal of the reflecting surface) traveling within tapered sections 39
or 33 at any point along the tapered, unclad, optical fiber within first
and second tapered sections 39 and 33, respectively;
(6) .beta. is the angle of inward taper of either tapered section 39 or 33
measured with respect to the longitudinal axis of the optical fiber at any
point along the tapered, unclad, optical fiber within first and second
tapered sections 39 and 33, respectively;
(7) L.sub.1 is the longitudinal length of the first tapered section 39; and
(8) L.sub.2 is the longitudinal length of the second tapered section 33,
all as shown in FIG. 8.
The following equations describe the relationships between the variables
described in (1)-(8) above and as shown in FIG. 8:
r.sub.0 sin (.alpha..sub.0)=r.sub.z sin (.alpha..sub.z),
tan (.alpha..sub.z)=r.sub.z /L.sub.1, and
L.sub.1 =r.sub.z /(tan (90-.theta.-.beta.)).
Typically, the longitudinal length, L.sub.1, of the first tapered section
39 is between about 0.1%-30% of the longitudinal length, L.sub.2, of the
second tapered section 33. Preferably, L.sub.1 is between about 1%-20% of
the length L.sub.2. More preferably, L.sub.1 is between about 2%-15% of
the length L.sub.2. Most preferably, L.sub.1 is between about 5%-12% of
the length L.sub.2. The taper angle B.sub.2 in section 33 should always be
more gradual than the taper angle B.sub.1 in section 39 (See FIG. 4).
To maintain total internal reflection, section 39 should be tapered
inwardly from the proximal to the distal end at a taper angle B.sub.1 such
that an incident beam of light traveling through the fiber does not fall
below the critical angle measured from the normal of the reflecting outer
edge of section 39 to the incident beam of light. The incident beam of
light is the excitation light traveling in the net direction from the
proximal end of the fiber 31 towards its distal end. Because section 39
does not efficiently couple fluorescent signals generated at its surface,
its length L.sub.1 is preferably minimized.
The equation for calculating the number of modes (V.sup.2 /2) that can be
propagated at any given point along the cladded fiber (FIG. 3; i.e. core
36 surrounded by cladding 34) is given by the following equation:
##EQU3##
where (1) V.sub.34 is the determines the number of modes that can be
propagated along the cladded optical fiber at the point on the fiber
wherein its radius is r.sub.34 from the center of the optical fiber to the
outermost core surface;
(2) n.sub.core is the index of refraction of the core of the fiber;
(3) n.sub.cladding is the index of refraction of the cladding;
(4) .lambda. is the wavelength of the fluorescent signal captured by the
fiber core at its distal, non-cladded end (sections 39 and 33) i.e. the
fluorescent signal emanating from the fluorophore in the evanescent wave
portion near the distal end (section 33) of the unclad fiber.
Note that at the distal, unclad end of the fiber (sections 43, 39 and 33),
the value of n.sub.cladding is substituted with the value of
n.sub.solution which is the index of refraction of the solution into which
the distal, sensing end of the fiber is immersed. In addition, at the
unclad, distal end of the fiber (sections 43, 39 and 33), the values of
r.sub.43, r.sub.39 and r.sub.33 are used instead of r.sub.34 when
calculating the V-number in sections 43, 39 and 33, respectively. Note
that r.sub.43 is the radius of the unclad fiber along section 43, r.sub.39
is the radius of the unclad, tapered fiber along section 39 and r.sub.33
is the radius of the unclad, tapered fiber along section 33. Thus, the
number of modes (V.sup.2 /2) that can be propagated at any given point
along the unclad fiber (sections 43, 39 and 33) can be calculated using
the following equations:
##EQU4##
For example, the number of modes within section 39 would be
(V.sub.39).sup.2 /2.
Maximum efficiency in the propagation of the captured fluorescent signal
from the distal, unclad sections of the optical fiber (sections 43, 39 and
33) into and along the proximal, cladded section (section 34) of the fiber
is accomplished when V.sub.34 .gtoreq.V.sub.43 .gtoreq.V.sub.39
.gtoreq.V.sub.33. Generally, without tapering, V.sub.34 would be less than
V.sub.33 (V.sub.34 <V.sub.33) and V.sub.39 (V.sub.34 <V.sub.39).
Therefore, it is necessary to reduce the value of V.sub.33 such that
V.sub.34 .gtoreq.V.sub.33. One way to reduce the value of V.sub.33 is to
reduce the radius from r.sub.34 (r.sub.34 =r.sub.43) to r.sub.33 such that
V.sub.34 .gtoreq.V.sub.33. In order to maximize the length L.sub.2 of the
unclad, distal portion 33 of the optical fiber and to have V.sub.34
.gtoreq.V.sub.33, a first short tapered section 39 is used to reduce the
radius from r.sub.39 where V.sub.34 <V.sub.39 to r.sub.39 where V.sub.34
=V.sub.39 at the distal, unclad end of the first short tapered section 39
at line 69. Here a first short tapered section 39 is used instead of a 90
degree step down to a value of r.sub.39 where V.sub.34 =V.sub.39 because
using a first short tapered section 39 avoids the problem of falling below
the critical angle by an incident beam of excitation radiation at the 90
degree step. The critical angle is measured from the normal drawn
perpendicular to the reflecting, unclad edge to the incident excitation
beam. Use of a step down instead of a first short taper down to r.sub.39
=R.sub.match (distal end of section 39) at line 69 where V.sub.34
=V.sub.39 causes the angle of incidence to fall below the critical angle
at the step 22 and results in a loss of the excitation light and further
causes unwanted excitation of the unbound fluorophore in the bulk
solution.
Thus, section 39 is tapered inwardly by gradually reducing r.sub.39 from a
value where V.sub.34 <V.sub.39 to a matching radius (r.sub.39
=r.sub.match) where V.sub.34 =V.sub.39 at line 69. The angle B.sub.1 of
taper is inward toward the center of the unclad fiber along the first
short tapered section 39 proceeding toward the distal, unclad end of the
fiber. The taper angle B.sub.1 along the first short tapered section 39 is
typically between about 0.01-30 degrees, preferably between about 0.05-25
degrees, more preferably between about 0.1-10 degrees and most preferably
between about 0.2-5 degrees measured from the outer untapered surface 43
of the unclad fiber (or a line parallel to the untapered, unclad surface
43) toward the center of the fiber along the taper outer edge of the
unclad fiber, the taper angle B.sub.1 opening toward the distal, unclad
end of the fiber.
Along the distal, unclad end of the fiber, a second tapered section 33
begins at the point at the distal end of the first short tapered section
39 at line 69 where r.sub.39 is such that V.sub.34 =V.sub.39. The second
tapered section 33 is tapered inwardly and the taper angle B.sub.2 opens
toward the distal, unclad end of the fiber. The second taper angle B.sub.2
is shallower than the angle B.sub.1 of the first short tapered section 39
which first short tapered section 39 is located adjacent to the second
tapered section 33 and which first short tapered section 39 is located
proximal to the second tapered section 33. The value of r.sub.33 located
at the intersection of the most proximal end of the second tapered section
33 and the most distal end of the first short tapered section 39 at line
69 is equal to r.sub.match such that V.sub.34 =V.sub.39 =V.sub.33 at line
69. The taper angle B.sub.2 along the unclad portion of the optical fiber
and along the second tapered section 33 opens toward the distal end of the
optical fiber, as does the taper angle B.sub.1 of the first short tapered
section 39 discussed earlier. The radius r.sub.33 equals r.sub.39 which
equals r.sub.match at the most proximal portion of the second tapered
section 33. The radius r.sub.3 is gradually reduced from a value of
r.sub.33 where V.sub.39 =V.sub.33 at line 69 to a value of r.sub.33 where
V.sub.39 >V.sub.33.
To maintain total internal reflection, section 33 should be tapered
inwardly from the proximal to the distal end at a taper angle B.sub.2 such
that an incident beam of light traveling through the fiber does not fall
below the critical angle measured from the normal of the reflecting outer
edge of section 33 to the incident beam of light. The incident beam of
light is the excitation light traveling in the net direction from the
proximal end of the fiber 31 towards its distal end.
With the value of V.sub.39 >V.sub.33 in the second tapered region 33,
except at the boundary at line 69 between the second tapered, uncladded
core section 33 and the first short tapered, uncladded core section 39
where V.sub.39 =V.sub.33, the maximum amount of the incoming fluorescent
signal captured from the fluorophore bound to molecules attached to the
distal, uncladded second tapered section 33 core surface, is transmitted
to the adjacent first short tapered section 39 uncladded core. Proximal to
the boundary line 69, V.sub.39 >V.sub.33. The incoming signal traveling
into the first short tapered section 39 uncladded core from the second
tapered section 33 uncladded core is further transmitted into the cladded,
untapered section of the fiber where V.sub.34 <V.sub.39 except at line 69
where V.sub.34 =V.sub.39 =V.sub.33. Except at line 69 where V.sub.34
=V.sub.39 =V.sub.33, the tapering in sections 39 and 33 is done such that
the condition V.sub.43 >V.sub.39 >V.sub.33 is also met.
Having described the invention, the following examples are given to
illustrate specific applications of the invention, including the best mode
now known to perform the invention. These specific examples are not
intended to limit the scope of the invention described in this
application.
EXAMPLES
Fluorimeter Configuration
The components used in conjunction with the biosensor were selected to
minimize noise and, thus, improve signal discrimination. A schematic of
the fiber optic fluorimeter utilized is shown in FIG. 5. The detection
optics were encased in a light-proof metal enclosure to reduce the effects
of ambient light and electromagnetic influence on the detector circuitry.
Table 1 shows the power drop across each component. The numbers represent
the power drop at 514 nm measured across the fiber optic fluorometer
components listed in Table 1 below.
TABLE 1
______________________________________
Component Power Drop (dB)
______________________________________
Mirror 0.02
Line Filter 1.55
Parabolic Mirror
0.32
Objective Lens 0.49
Chopper 3.01
Liquid & 3-66 Filters
34.78
KV550 Filter 35.60
______________________________________
Key components shown in FIG. 5 include optics for launching and collecting
the light, kinematic mounts (not shown), the off-axis parabolic mirror and
the emission filter.
A laser light source was selected for its moderate power, stability, narrow
excitation bandwidth and efficient light coupling into the fiber 31. The
exemplary rhodamine-based fluorescent labels used with the sensor were
excited at 514 nm and emit in the 570(.+-.50 nm) nm range where there is
little intrinsic fluorescence in most clinical and environmental samples.
A 514 nm laser beam from an air-cooled 50-mW argon ion laser 110
(Omnichrome 532, Chino, Calif.) was launched into the most proximal end of
the cladded fiber. The laser 110 was adjusted to a 12-mW output to
minimize bleaching of the fluorophores bound to the distal end of the
optical fiber 31. The line filter (Melles Griot) 130 removed plasma lines
from the laser source. The laser beam from laser 110, powered by laser
power supply 100, was reflected by mirror 120 into the line filter 130.
The laser beam passed through the line filter 130 and through an off-axis
parabolic mirror (Melles Griot) 140. The laser beam was focused by a
spherical lens (f/1, one inch focal length bioconvex lens; Newport
Corporation) 150 onto the proximal end of the optical fiber 31 through
chopper 160. The laser beam focused by the spherical lens 150 focused the
light onto the fiber, filling only a small portion of the fiber's
numerical aperture. Approximately, 8 degrees of the fiber's numerical
aperture of 23 degrees were filled. This simple lens 150 proved easier to
use than a microscope objective and provided ample room for the insertion
of the chopper 160 between it and the proximal end of the optical fiber
31. Positioning the chopper (Stanford Research Systems) 160 near the
proximal end of the fiber instead of at the laser 110 reduced the
background noise from scattered excitation light. See Table 2 below.
TABLE 2
______________________________________
Device Signal
Configuration
(mV) Noise (mV)
S/N.sup.b
N.sub.1 /N.sub.0.sup.c
______________________________________
NRL Device 0.99 0.095 (N.sub.0)
10.42 1.00
w/KV550 (Std)
Chopper Placed
1.13 0.13 8.69 1.36
by Laser
Minus Excitation
2.08 0.59 3.53 6.21
Filter
Liquid.fwdarw.3-66
0.93 0.20 4.65 2.10
filter.sup.a
3-66.fwdarw.Liquid filter
3.58 2.71 1.32 29
3-66 filter (only)
7.20 6.38 1.13 67
Liquid filter (only)
323 322 1.003 3389
______________________________________
.sup.a .fwdarw.indicates the light path toward the photodiode
.sup.b The signal (S) was obtained by placing fluorescent paper a
sufficient distance from the end of a cleaved fiber to produce a reading
of .about. 1mV with the standard configuration. The background noise leve
(N) was the voltage reading without the fluorescent paper.
.sup.c N.sub.1 /N.sub.0 is the background level produced by the
configuration under test divided by that of the standard configuration,
NRL Device w/KV550.
The chopper 160 and photodiode (EG&G Judson) 180 were connected to a
lock-in amplifier (LIA, Stanford Research Systems, Sunnyvale, Calif.) 210.
The photodiode 180 was selected rather than a photomultiplier tube because
of low cost, reliability and compatibility with the lock-in amplifier 210.
The chopper 160 was interfaced to the lock-in amplifier (LIA) 210 anal
computer 220 for phase sensitive detection via chopper controller 220. The
collected fluorescence signal from the distal end of the optical fiber 31
traveled the reverse path to the parabolic mirror 140 where it was
refocused through a longpass filter (KV550) 170 onto a silicon photodiode
180 which was also connected to the LIA 210.
All components were mounted in the device (See schematic FIG. 5) with
detachable kinematic mounts (not shown). The high degree of reliability
with which kinematic mounts can be removed and reinstalled permitted rapid
setup of the optics upon relocation. The fiber connectors (not shown) were
held in an x-y adjustable mount (not shown) to allow for precise
positioning of the fiber in the laser beam. The off-axis parabolic mirror
140, lens 150 and photodiode 180 were also mounted using x-y or x-y-z
adjustable mounts (not shown) to permit rapid alignment.
A design improvement maintained in this device was the perforated off-axis
parabolic mirror 140. The central perforation passes the laser excitation
while the mirror 140 focuses the fluorescence signal in a direction
orthogonal to the laser beam and onto the photodiode 180. The use of this
mirror 140 was found to improve the signal-to-noise ratio (SNR) not only
by providing an efficient collecting surface for returning fluorescent
light, but also by assisting in the removal of long wavelength plasma
lines, which are poorly collimated in comparison to the laser beam. In
addition, the parabolic mirror 140 added to the device's versatility,
because collection of various fluorescent wavelengths can be accomplished
without replacement of an expensive dichroic mirror. The focusing lens 150
that served to launch the laser light into the fiber 31 had a numerical
aperture greater than that of the cladded fiber 31. Thus, it also served
to collimate all of the returning fluorescence onto the parabolic mirror
140.
The parabolic mirror 140 focused the fluorescence signal onto a photodiode
180 through a KV550 long-pass filter (Schott-Glass) 170 that blocked any
stray excitation light. The KV550 filter 170 has a lower wavelength
absorption cutoff than the Corning 3-66 filter (alternative filter 170)
previously used, thus permitting more fluorescence signal to reach the
photodiode. The change to the KV550 filter also eliminated the need for
the liquid filter (5% K.sub.2 Cr.sub.2 O.sub.7), which was necessary to
reduce the fluorescence generated by stray light hitting the Corning 3-66
filter (alternative filter 170). Fluorescence from the 3-66 filter is
demonstrated by the 13-fold increase in background noise upon switching
the order of the liquid and 3-66 filters in the light path (Table 2,
supra). See L. C. Shriver-Lake, G. P. Anderson, J. P. Golden and F. S.
Ligler, The effect of Tapering the Optical Fiber on Evanescent Wave
Measurements 25 ANALYTICAL LETTERS 7, pp. 1183-1199 (1992), incorporated
by reference herein in its entirety and for all purposes. See J. P.
Golden, L. C. Shriver-Lake, G. P. Anderson, R. B. Thompson and F. S.
Ligler, Fluorometer and Tapered Fiber Optic Probe for Sensing in the
Evanescent Wave 31 OPTICAL ENGINEERING No. 7, pp. 1458-1462 (July 1992),
incorporated by reference herein in its entirety and for all purposes. See
G. P. Anderson, J. P. Golden and F. S. Ligler, A Fiber Optic Biosensor:
Combination Tapered Fibers Designed for Improved Signal Acquisition, 8
BIOSENSORS & BIOELECTRONICS, pp. 249-256 (1993), incorporated by reference
herein in its entirety and for all purposes. See J. P. Golden, S. Y.
Rabbany and G. P. Anderson, Ray Tracing Determination of Evanescent Wave
Penetration Depth in Tapered Fiber Optic Probes, Reprinted from CHEMICAL,
BIOCHEMICAL AND ENVIRONMENTAL FIBER SENSORS IV in 1796 SPIE PROCEEDINGS
SERIES, pp. 9-11 (Meeting 8-9 Sep. 1992 in Boston, Mass.; published April
1993), incorporated by reference herein in its entirety and for all
purposes. See J. P. Golden, G. P. Anderson, R. A. Ogert, K. A. Breslin and
F. S. Ligler, An Evanescent Wave Fiber Optic Biosensor: Challenges for
Real World Sensing, Reprinted from CHEMICAL, BIOCHEMICAL AND ENVIRONMENTAL
FIBER SENSORS IV in 1796 SPIE PROCEEDINGS SERIES, pp. 2-8 (Meeting 8-9
Sep. 1992 in Boston, Mass.; published April 1993), incorporated by
reference herein in its entirety and for all purposes. See R. B. Thompson
and L. Kondracki, Sensitivity Enhancement for Evanescent Wave-Excited
Fiber Optic Fluorescence Sensors, Reprinted from TIME RESOLVED LASER
SPECTROSCOPY IN BIOCHEMISTRY II, 1204 SPIE PROCEEDING SERIES, pp. 35-41
(1990), incorporated by reference herein in its entirety and for all
purposes.
Optical Probe Preparation
The fiber optic probe 31 used in this biosensor was made from a length of
step-index plastic clad silica optical fiber (200 .mu.m diameter core,
Quartz et Silice, Quartz Products, Tuckerton, Del.) 31 with a connector
(not shown) on the proximal end to facilitate replacement and alignment.
The distal end was modified to perform biochemical assays in the
evanescent wave. Lengths up to 60-m have been successfully tested, but 1-m
lengths were used for convenience. A metal SMA 905 connector (Amphenol
Fiber Products, Lisle, Ill.) and a small ferrule connector (Aurora Optics
Inc.) (both not shown) were tested. The metal SMA 905 connector was
attached to the fiber core with non-fluorescing epoxy (Epoxy Technologies,
Billerica, Mass.) and secured with a crimped metal sleeve and heat shrink
tubing (both not shown). The fiber was cleaved on the proximal end of the
connector (not shown) and polished using a Buehler Fib | | |
