U.S. patent application number 09/840012 was filed with the patent office on 2002-09-05 for fiber optic sensor with encoded microspheres.
This patent application is currently assigned to Trustees of Tufts College. Invention is credited to Michael, Karri Lynn, Walt, David R..
Application Number | 20020122612 09/840012 |
Document ID | / |
Family ID | 25224936 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020122612 |
Kind Code |
A1 |
Walt, David R. ; et
al. |
September 5, 2002 |
Fiber optic sensor with encoded microspheres
Abstract
A microsphere-based analytic chemistry system and method for
making the same is disclosed in which microspheres or particles
carrying bioactive agents may be combined randomly or in ordered
fashion and dispersed on a substrate to form an array while
maintaining the ability to identify the location of bioactive
agents and particles within the array using an optically
interrogatable, optical signature encoding scheme. In a preferred
embodiment, a modified fiber optic bundle or array is employed as a
substrate to produce a high density array. The disclosed system and
method have utility for detecting target analytes and screening
large libraries of bioactive agents. In a preferred embodiment the
methods include detecting a change in an optical property around a
microsphere on an array.
Inventors: |
Walt, David R.; (Lexington,
VA) ; Michael, Karri Lynn; (Somerville, MA) |
Correspondence
Address: |
Robin M. Silva, Esq.
FLEHR HOHBACH TEST ALBRITTON & HERBERT LLP
Suite 3400
Four Embarcadero Center
San Francisco
CA
94111-4187
US
|
Assignee: |
Trustees of Tufts College
|
Family ID: |
25224936 |
Appl. No.: |
09/840012 |
Filed: |
April 20, 2001 |
Related U.S. Patent Documents
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Application
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09840012 |
Apr 20, 2001 |
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09450829 |
Nov 29, 1999 |
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6266459 |
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09450829 |
Nov 29, 1999 |
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08818199 |
Mar 14, 1997 |
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6023540 |
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Current U.S.
Class: |
385/12 ;
436/172 |
Current CPC
Class: |
B01J 2219/00659
20130101; B01J 2219/00677 20130101; Y10S 435/808 20130101; G01N
15/1459 20130101; G01N 33/54346 20130101; G01N 15/1468 20130101;
G01N 2015/1465 20130101; G01N 2035/0097 20130101; B01J 2219/00605
20130101; G01N 15/1456 20130101; B01J 2219/0061 20130101; B01J
2219/00585 20130101; B01J 2219/00466 20130101; B82Y 30/00 20130101;
B01J 2219/00621 20130101; B01J 2219/00725 20130101; G01N 2021/6484
20130101; Y10S 359/90 20130101; C12Q 1/6837 20130101; G01N 21/6428
20130101; B01J 2219/005 20130101; B01J 2219/00612 20130101; B01J
2219/00648 20130101; B01J 2219/00524 20130101; G01N 2015/1438
20130101; B01J 2219/00596 20130101; G01N 21/6452 20130101; G01N
21/7703 20130101; B01J 2219/00317 20130101; C40B 40/10 20130101;
B01J 2219/00626 20130101; B01J 2219/00704 20130101; B01J 2219/00637
20130101; G01N 2021/7786 20130101; B01J 2219/0063 20130101; B01J
2219/0074 20130101 |
Class at
Publication: |
385/12 ;
436/172 |
International
Class: |
G02B 006/26; G01N
021/76 |
Claims
What is claimed is:
1. An analytic chemistry system, comprising a population of beads
including separate subpopulations, each subpopulation carrying
chemical functionality which changes an optical signature of the
beads in the presence of targeted analytes, beads in each
subpopulation having an optical signature which is encoded with a
description of the chemical functionality carried by that
subpopulation.
2. The system described in claim 1, wherein the beads are encoded
using dyes.
3. The system described in claim 2, wherein the dyes are entrapped
within the beads and the chemical functionality is on surfaces of
the beads.
4. The system described in claim 1, wherein the beads are encoded
using fluorescent dyes.
5. The system described in claim 1, wherein the beads are encoded
by controlling a ratio of at least two dyes.
6. The system described in claim 1, wherein the chemical
functionality changes the optical signature by producing an
optically active chemical in the presence of targeted analytes.
7. The system described in claim 1, wherein the optical signature
is changed by the chemical functionalities of the beads by the
presence or absence of a fluorescent signal.
8. The system described in claim 1, wherein the chemical
functionalities of the beads support sites for hybridization.
9. The system described in claim 1, wherein the beads are affixed
to a distal end of an optical fiber bundle.
10. The system described in claim 1, wherein the beads are located
within etched wells at terminal ends of optical fibers of the
bundle.
11. A chemical analysis method, comprising preparing separate
subpopulations of beads, each subpopulation carrying chemical
functionalities that change optical signatures of the beads in the
presence of targeted analytes; encoding optical signature of the
beads in each subpopulation with a description of the chemical
functionalities carried by that subpopulation; combining the
subpopulations to produce a system; applying the system; detecting
changes in the optical signatures indicative of a presence of the
targeted analytes; and decoding optical signature of the beads to
identify the chemical functionalities.
12. The method described in claim 11, wherein encoding the optical
signatures with the chemical functionalities comprises doping the
beads with fluorescent dyes.
13. The method described in claim 11, wherein encoding the optical
signatures with chemical functionalities comprises attaching
encoding dyes to the beads.
14. The method described in claim 11, wherein encoding the optical
signatures with the chemical functionalities comprises controlling
a ratio of at least two dyes carried by each bead.
15. The method described in claim 11, further comprising: encoding
the beads with the chemical functionalities by entrapping dyes
within or attaching dyes to the beads; and applying the chemical
functicnalities to the beads.
16. The method described in claim 11, further comprising enabling
the chemical functionalities to produce an optically active species
in the presence of targeted analytes to change the optical
signature.
17. The method described in claim 11, further comprising changing
the optical signature by the presence or absence of a fluorescent
signal from the beads.
18. The method described in claim 11, further comprising enabling
the chemical functionalities to hybridize.
19. An analytic chemistry sensor, comprising: a bundle of optical
fibers; a population of beads carrying chemical functionalities at
a distal end of the fiber optic bundle, light from individual bead
being coupled into separate or groups of separate fibers of the
bundle for transmission to the proximal end of the bundle.
20. The sensor described in claim 19, wherein each one of the beads
is located within separate wells formed at terminal ends of optical
fibers of the bundle.
21. The sensor described in claim 20, wherein the wells are formed
by anisotropic etching of the cores of the optical fibers with
respect to the cladding.
22. The sensor described in claim 19, further comprising a light
source for exciting optically active chemicals bound to the
chemical functionalities.
23. The sensor described in claim 19, wherein the population of
beads includes separate subpopulations, each subpopulation carrying
a different chemical functionality and an optically interrogatable
code descriptive of the chemical functionality.
24. The sensor described in claim 23, further comprising a light
source for exciting optically active chemicals bound to the
chemical functionalities.
25. The sensor described in claim 23, wherein code of each
subpopulation comprises fluorescent dyes.
26. The sensor described in claim 23, further comprising a filter
and a frame capturing camera for detecting optical signatures
indicative of a status of the chemical functionalities and optical
signatures indicative of the encoding of the beads.
27. A method for constructing and using an analytic chemistry
sensor, comprising: forming wells at terminal ends of optical
fibers within a bundle; distributing beads carrying chemical
functionalities within the wells; and monitoring a status of the
chemical functionalities from a proximal end of the bundle.
28. The method described in claim 27, wherein forming the wells
comprises anisotropically etching of cores of the optical fibers
with respect to cladding.
29. The method described in claim 27, further comprising forming a
population of beads in the wells from separate subpopulations, each
subpopulation carrying a different chemical functionality and an
optically interrogatable code descriptive of the chemical
functionality.
30. The method described in claim 29, further comprising randomly
distributing the subpopulations within the wells.
31. The method described in claim 29, further comprising serially
adding the subpopulations to the wells.
Description
BACKGROUND OF THE INVENTION
[0001] The use of optical fibers and optical fiber strands in
combination with light absorbing dyes for chemical analytical
determinations has undergone rapid development, particularly within
the last decade. The use of optical fibers for such purposes and
techniques is described by Milanovich et al., "Novel Optical Fiber
Techniques For Medical Application", Proceedings of the SPIE 28th
Annual International Technical Symposium On Optics and
Electro-Optics, Volume 494, 1980; Seitz, W. R., "Chemical Sensors
Based On Immobilized Indicators and Fiber Optics" in C.R.C.
Critical Reviews In Analytical Chemistry, Vol. 19, 1988, pp.
135-173; Wolfbeis, O. S., "Fiber Optical Fluorosensors In
Analytical Chemistry" in Molecular Luminescence Spectroscopy,
Methods and Applications (S. G. Schulman, editor), Wiley &
Sons, New York (1988); Angel, S. M., Spectroscopy 2 (4):38 (1987);
Walt, et al., "Chemical Sensors and Microinstrumentation", ACS
Symposium Series, Vol. 403, 1989, p. 252, and Wolfbeis, O. S.,
Fiber Optic Chemical Sensors, Ed. CRC Press, Boca Raton, Fla.,
1991, 2nd Volume.
[0002] When using an optical fiber in an in vitro/in vivo sensor,
one or more light absorbing dyes are located near its distal end.
Typically, light from an appropriate source is used to illuminate
the dyes through the fiber's proximal end. The light propagates
along the length of the optical fiber; and a portion of this
propagated light exits the distal end and is absorbed by the dyes.
The light absorbing dye may or may not be immobilized; may or may
not be directly attached to the optical fiber itself; may or may
not be suspended in a fluid sample containing one or more analytes
of interest; and may or may not be retainable for subsequent use in
a second optical determination.
[0003] Once the light has been absorbed by the dye, some light of
varying wavelength and intensity returns, conveyed through either
the same fiber or collection fiber(s) to a detection system where
it is observed and measured. The interactions between the light
conveyed by the optical fiber and the properties of the light
absorbing dye provide an optical basis for both qualitative and
quantitative determinations.
[0004] Of the many different classes of ligh: absorbing dyes which
conventionally are employed with bundles of fiber strands and
optical fibers for different analytical purposes are those more
common compositions that emit light after absorption termed
"fluorophores" and those which absorb light and internally convert
the absorbed light to heat, rather than emit it as light, termed
"chromophores."
[0005] Fluorescence is a physical phenomenon based upon the ability
of some molecules to absorb light (photons) at specified
wavelengths and then emit light of a longer wavelength and at a
lower energy. Substances able to fluoresce share a number of common
characteristics: the ability to absorb light energy at one
wavelength .lambda..sub.ab; reach an excited energy state; and
subsequently emit light at another light wavelength,
.lambda..sub.ab. The absorption and fluorescence emission spectra
are individual for each fluorophore and are often graphically
represented as two separate curves that are slightly overlapping.
The same fluorescence emission spectrum is generally observed
irrespective of the wavelength of the exciting light and,
accordingly, the wavelength and energy of the exciting light may be
varied within limits; but the light emitted by the fluorophore will
always provide the same emission spectrum. Finally, the strength of
the fluorescence signal may be measured as the quantum yield of
light emitted. The fluorescence quantum yield is the ratio of the
number of photons emitted in comparison to the number of photons
initially absorbed by the fluorophore. For more detailed
information regarding each of these characteristics, the following
references are recommended: Lakowicz, J. R., Principles of
Fluorescence Spectroscopy, Plenum Press, New York, 1983;
Freifelder, D., Physical Biochemistry, second edition, W. H.
Freeman and Company, New York, 1982; "Molecular Luminescence
Spectroscopy Methods and Applications: Part I" (S. G. Schulman,
editor) in Chemical Analysis, vol. 77, Wiley & Sons, Inc.,
1985; The Theory of Luminescence, Stepanov and Gribkovskii, Iliffe
Books, Ltd., London, 1968.
[0006] In comparison, substances which absorb light and do not
fluoresce usually convert the light into heat or kinetic energy.
The ability to internally convert the absorbed light identifies the
dye as a "chromophore." Dyes which absorb light energy as
chromophores do so at individual wavelengths of energy and are
characterized by a distinctive molar absorption coefficient at that
wavelength. Chemical analysis employing fiber optic strands and
absorption spectroscopy using visible and ultraviolet light
wavelengths in combination with the absorption coefficient allow
for the determination of concentration for specific analyses of
interest by spectral measurement. The most common use of absorbance
measurement via optical fibers is to determine concentration which
is calculated in accordance with Beers' law; accordingly, at a
single absorbance wavelength, the greater the quantity of the
composition which absorbs light energy at a given wavelength, the
greater the optical density for the sample. In this way, the total
quantity of light absorbed directly correlates with the quantity of
the composition in the sample.
[0007] Many of the recent improvements employing optical fiber
sensors in both qualitative and quantitative analytical
determinations concern the desirability of depositing and/or
immobilizing various light absorbing dyes at the distal end of the
optical fiber. In this manner, a variety of different optical fiber
chemical sensors and methods have been reported for specific
analytical determinations and applications such as pH measurement,
oxygen detection, and carbon dioxide analyses. These developments
are exemplified by the following publications: Freeman, et al.,
Anal Chem. 53:98 (1983); Lippitsch et al., Anal. Chem. Acta. 205:1,
(1988); Wolfbeis et al., Anal. Chem. 60:2028 (1988); Jordan, et
al., Anal. Chem. 59:437 (1987); Lubbers et al., Sens. Actuators
1983; Munkholm et al., Talanta 35:109 (1988); Munkholm et al.,
Anal. Chem. 58:1427 (1986); Seitz, W. R., Anal. Chem. 56:16A-34A
(1984); Peterson, et al., Anal. Chem. 52:864 (1980): Saari, et al.,
Anal. Chem. 54:821 (1982); Saari, et al., Anal. Chem. 55:667
(1983); Zhujun et al., Anal. Chem. Acta. 160:47 (1984); Schwab, et
al., Anal. Chem. 56:2199 (1984); Wolfbeis, O. S., "Fiber Optic
Chemical Sensors", Ed. CRC Press, Boca Raton, Fla., 1991, 2nd
Volume; and Pantano, P., Walt, D.R., Anal. Chem., 481A-487A, Vol.
67, (1995).
[0008] More recently, fiber optic sensors have been constructed
that permit the use of multiple dyes with a single, discrete fiber
optic bundle. U.S. Pat. Nos. 5,244,636 and 5,250,264 to Walt, et
al. disclose systems for affixing multiple, different dyes on the
distal end of the bundle, the teachings of each of these patents
being incorporated herein by this reference. The disclosed
configurations enable separate optical fibers of the bundle to
optically access individual dyes. This avoids the problem of
deconvolving the separate signals in the returning light from each
dye, which arises when the signals from two or more dyes are
combined, each dye being sensitive to a different analyte, and
there is significant overlap in the dyes' emission spectra.
SUMMARY OF THE INVENTION
[0009] The innovation of the two previous patents was the placement
of multiple chemical functionalities at the end of a single optical
fiber bundle sensor. This configuration yielded an analytic
chemistry sensor that could be remotely monitored via the typically
small bundle. The drawback, however, was the difficulty in applying
the various chemistries associated with the chemical
functionalities at the sensor's end; the functionalities were built
on the sensor's end in a serial fashion. This was a slow process,
and in practice, only tens of functionalities could be applied.
[0010] The present design is based on two synergistic inventions:
1) the development of a bead-based analytic chemistry system in
which beads, also termed microspheres, carrying different chemical
functionalities may be mixed together while the ability is retained
to identify the functionality of each bead using an optically
interrogatable encoding scheme; and 2) an optical fiber bundle
sensor in which the separate beads or microspheres may be optically
coupled to discrete fibers or groups of fibers within the bundle.
Each invention has separate applications but, when implemented
together, yields an optical fiber sensor that can support large
numbers, thousands or more, of separate chemical functionalities,
which is relatively easy to manufacture and use.
[0011] In general, according to one aspect, the invention concerns
an analytic chemistry system that comprises a population of beads
or microspheres. Within the population are separate subpopulations,
each of which carries chemical functionality which changes an
optical signature of the beads in a presence of targeted analytes.
This signature change can occur via many different mechanisms. A
few examples include the binding of a dye-tagged analyte in the
bead, the production of a dye species on or near the beads, the
destruction of an existing dye species, a change in optical signal
upon analyte interaction with dye on bead, or any other optical
interrogatable event. Although the subpopulations may be randomly
mixed together, the chemical functionality on each bead is
determined via an optical signature which is encoded with a
description o f the chemical functionality. As a result, by
observing whether the optical signature of a particular bead is
exhibiting a change, or not, and then decoding the signature for
the functionality of the bead, the presence or not of the analyte
targeted by the functionality may be determined.
[0012] In specific embodiments, the beads are encoded using dyes
that are preferably entrapped within the beads, the chemical
functionality being added on surfaces. The dyes may be chromophores
or phosphors but are preferably fluorescent dyes, which due to
their strong signals provide a good signal-to-noise ratio for
decoding. The encoding can be accomplished in a ratio of at least
two dyes, although more encoding dimensions may be added in the
size of the beads, for example.
[0013] According to another aspect, the invention also concerns an
analytic chemistry fiber optic bundle sensor. This sensor has a
population of beads carrying chemical functionalities at, on or
near, a distal end of the bundle. The ability to monitor optical
signature changes associated with individual or multiple beads is
provided by coupling those signature changes into separate optical
fibers or groups of fibers of the bundle for transmission to the
proximal end where analysis is performed either manually, by the
user, or automatically, using image processing techniques.
[0014] In the preferred embodiment, each one of the beads is
located within separate wells formed at terminal ends of optical
fibers of the bundle. These microwells are formed by anisotropic
etching of the cores of the optical fibers with respect to the
cladding.
[0015] Also in the preferred embodiment, the population of beads
includes separate subpopulations, typically randomly distributed in
an array across the bundle end, each subpopulation carrying a
different chemical functionality and an optically interrogatable
code descriptive of the chemical functionality.
[0016] Although each sensor is different insofar that it has a
different distribution of the subpopulations of beads within its
wells, only those beads that exhibit a positive optical response or
signature change need to be decoded. Therefore, the burden is
placed on the analysis rather than on sensor manufacture. Moreover,
since the beads and fibers in the array can be monodisperse, the
fluorescent regions arising from signal generation are extremely
uniform and can be analyzed automatically using commercially
available microscopy analysis software, such image processing
software is capable of defining different spectral regions
automatically and counting the number of segments within each
region in several seconds.
[0017] The above and other features. of the invention including
various novel details of construction and combinations of parts,
and other advantages, will now be more particularly described with
reference to the accompanying drawings and pointed out in the
claims. It will be understood that the particular method and device
embodying the invention are shown by way of illustration and not as
a limitation of the invention. The principles and features of this
invention may be employed in various and numerous embodiments
without departing from the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In the accompanying drawings, reference characters refer to
the same parts throughout the different views. The drawings are not
necessarily to scale; emphasis has instead been placed upon
illustrating the principles of the invention. Of the drawings:
[0019] FIG. 1 is a schematic diagram illustrating the optical
signature encoding and chemical functionalizing of the microspheres
according to the present invention;
[0020] FIG. 2 is a process diagram describing the preparation,
encoding, and functionalizing of the microspheres of the present
invention;
[0021] FIG. 3 is a schematic diagram illustrating a microsphere
system including microspheres with different chemical
functionalities and encoded descriptions of the
functionalities;
[0022] FIG. 4 is a schematic diagram of the inventive fiber optic
sensor and associated instrumentation and control system;
[0023] FIGS. 5A and 5B are micrographs illustrating the preferred
technique for attaching or affixing the microspheres to the distal
end of the optical fiber bundle;
[0024] FIG. 6 is a process diagram describing well formation in the
optical fiber bundle and affixation of the microspheres in the
wells;
[0025] FIGS. 7A and 7B are micrographs showing the array of
microspheres in their corresponding wells prior and subsequent to
physical agitation, tapping and air pulsing, demonstrating the
electrostatic binding of the microspheres in the wells;
[0026] FIGS. 8A, 8B, and 8C are micrographs from alkaline
phosphatase microspheres when exposed to fluorescein diphosphate,
at the fluorescein emission wavelength, at an encoding wavelength
for DiIC, and at an encoding wavelength for TRC, respectively;
[0027] FIGS. 9A and 9B are micrographs showing the optical signal
from .beta.-galactosidase microspheres when exposed to fluorescein
.beta.-galactopyranoside at the fluorescein emission wavelength and
at an encoding wavelength for DiIC, respectively; and
[0028] FIG. 10A and 10B are micrographs showing the optical
response from xrabbit antibody microspheres prior to and post,
respectively, exposure to fluorescein labeled antigens.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0029] 1. Microspheres:
[0030] FIG. 1 illustrates the construction of a bead or microsphere
10 according to the principles of the present invention. In common
with the prior art, the microsphere 10 is given a chemical
functionality 12, which is typically applied to the microsphere's
surface. The chemical functionality is designed so that in the
presence of the analyte(s) to which it is targeted, an optical
signature of the microsphere, possibly including region surrounding
it, is changed.
[0031] The bead or microsphere need not be spherical, irregular
beads may be used. They are typically constructed from plastic or
ceramic, and bead sizes ranging from nanometers, e.g., 500 nm, to
millimeters, e.g., 1 mm, may be used. Moreover, various bead
chemistries may be used. Beads or microspheres comprising
methylstyrene, polystyrene, acrylic polymer, latex, paramagnetic,
thoria sol, carbon graphityed, and titanium dioxide are various
possibilities. "Microsphere Detection Guide" from Bangs
Laboratories, Fishers Ind. is a helpful guide.
[0032] The inventive microsphere has an optical signature that is
encoded with a description of the chemical functionality. In the
preferred embodiment, reporter dyes 14 are added to the microsphere
10 with the encoding occurring in the ratio of two or more dyes.
The reporter dyes 14 may be chromophore-type. Fluorescent dyes,
however, are preferred because the strength of the fluorescent
signal provides a better signal-to-noise ratio when decoding.
Additionally, encoding in the ratios of the two or more dyes,
rather than single dye concentrations, is preferred since it
provides insensitivity to the intensity of light used to
interrogate the reporter dye's signature and detector
sensitivity.
[0033] FIG. 2 is a process diagram illustrating the preparation of
the microspheres. In step 50, an aliquot of stock microspheres are
vacuum filtered to produce a dry cake. In one implementation,
microspheres of methylstyrene (87%) and divinylbenzene (13%) are.
used that have a 3.1 micrometer (.mu.m) diameter.
[0034] The microspheres may be purchased with the desired chemical
functionalities already present. A large selection of such
pre-prepared microspheres are currently available from a number of
commercial vendors. Alternatively, "blank" microspheres may be used
that have surface chemistries that facilitate the attachment of the
desired functionality by the user. Some examples of these surface
chemistries for blank microspheres are listed in Table I.
1 TABLE I Surface chemistry Name: NH.sub.2 Amine COOH Carboxylic
Acid CHO Aldehyde CH.sub.2--NH.sub.2 Aliphalic Amine CO NH.sub.2
Amide CH.sub.2--C1 Chloromethyl CONH--NH.sub.2 Hydrazide OH
Hydroxyl SO.sub.4 Sulfate SO.sub.3 Sulfonate Ar NH.sub.2 Aromatic
Amine
[0035] The dry cake is then broken apart and a dye solution added
to it in step 52 to encode optical signatures of the microspheres
with information concerning the intended surface chemical
functionalities. Dyes may be covalently bonded to the microspheres'
surface, but this consumes surface binding sites desirably reserved
for the chemical functionalities. Preferably, the microspheres are
placed in a dye solution comprising a ratio of two or more
fluorescent reporter dyes dissolved in an organic solvent that will
swell the microspheres, e.g., dimethylformamide (DMF). The length
of time the microspheres are soaked in the dye solution will
determine their intensity and the broadness of the ratio range.
Longer times yield higher intensities, but broader ratio
ranges.
[0036] In an exemplary two dye system, Texas Red Cadaverine (TRC)
is used, which is excited at .lambda..sub.ab=580 mm and emits at
.lambda..sub.em=630 mm, in combination with indodicarbocyanine
(DiIC): 610/670 (.lambda..sub.ab/.lambda..sub.em). Generally, dyes
are selected to be compatible with the chemistries involved in the
analysis and to be spectrally compatible. The emission wavelengths
of the dyes should not overlap the regions of the optical spectrum
in which the chemical functionalities induce changes in the
microsphere signatures. This avoids deconvolution problems
associated with determining signal contributions based on the
presence of both the analyte and the encoding dye ratios
contributing to an overlapping emission spectral region.
[0037] Examples of other dyes that can be used are Oxazin
(662/705), IR-144 (745/825), IR-140 (776/882), IR-125 (786/800)
from Exiton, and Bodipy 665/676 from Molecular Probes, and
Naphthofluorescein (605/675) also from Molecular Probes. Lanthide
may also be used. Fluorescent dyes emitting in other than the near
infrared may also be used. Chromophore dyes are still another
alternative that produce an optically interrogatable signature, as
are more exotic formulations using Raman scattering-based dyes or
polarizing dyes, for example. The ability of a particular dye pair
to encode for different chemical functionalities depends on the
resolution of the ratiometric measurement. Conservatively, any dye
pair should provide the ability to discriminate at least twenty
different ratios The number of unique combinations of two dyes made
with a particular dye set is shown in the following Table II.
2 TABLE II Number of Combinations dyes in set possible 3 3 4 6 5 10
6 15
[0038] Thus, using six dyes and twenty distinct ratios for each dye
pair, 300 separate chemical functionalities may be encoded in a
given population of microspheres. Combining more than two dyes
provides additional diversity in the encoding combinations.
[0039] In step 54, the microspheres are vacuum filtered to remove
excess dye. The microspheres are then washed in water or other
liquid that does not swell the microspheres, but in which the dyes
are still soluble. This allows the residual dye to be rinsed off
without rinsing the dye out of the microspheres.
[0040] In step 56, the chemical functionality is attached to the
microsphere surface chemistries if not already present. It should
be understood that surface chemistries may be present throughout
the microsphere's volume, and not limited to the physical
circumferential surface.
[0041] In the prior art, a large spectrum of chemical
functionalities have been manifest on microspheres that produce
optically interrogatable changes in the presence of the targeted
analyte. These functionalities include four broad classifications
of microsphere sensors: 1) basic indicator chemistry sensors; 2)
enzyme-based sensors; 3) immuno-based sensors; and 3)
geno-sensors.
3TABLE III TARGE ANALYTE CHEMICAL FUNCTIONALITY NOTES
(.lambda..sub.AB/.lambda..sub.EM) pH Sensors based on:
seminaphthofluoresceins e.g., carboxy-SNAFL seminaphthorhodafluors
e.g., carboxy-SNARF 8-hydroxypyrene-1,3,6- trisulfonic acid
fluorescein CO2 Sensors based On: seminaphthofluoresceins e.g.,
carboxy-SNAFL seminaphthorhodafluors e.g., carbody-SNARF
8-hydroxypyrene-1,3,6- trisulfonic acid Metal Ions Sensors
desferriozamine B e.g., Fe based on: cyclen derivative e.g., Cu, Zn
derivatized peptides e.g., FITC-Gly-Gly-His, and FITC-Gly His, Cu,
Zn fluorexon (calcine) e.g., Ca, Mg, Cu, Pb, Ba calcine blue e.g.,
Ca, Mg, Cu methyl calcine blue e.g., Ca, Mg, Cu ortho-dianisidine
e.g., Zn tetracetic acid (ODTA) bis-salicylidene e.g., Al
ethylenediamine (SED) N-(6-methozy-8-quinolyl- e.g., Zn
p-toluenesulfonamine (TSQ) Indo-1 e.g., Mn, Ni Fura-2 e.g., Mn, Ni
Magesium Green e.g., Mg, Cd, Tb O.sub.2 Siphenylisobenzofuran
409/476 Methoxy-vinyl pyrene 352/401 Nitrite diaminonaphthaline
340/377 NO luminol 355/411 dihydrohodamine 289/none Ca.sup.2+
Bis-fura 340/380 Calcium Green visible light/530 Fura-2 340/380
Indo-1 405/485 Fluo-3 visible light/525 Rhod-2 visible light/570
Mg.sup.2+ Mag-Fura-2 340/380 Mag-Fura-5 340/380 Mag-Indo-1 405/485
Magnesium Green 475/530 Magnesium Orange visible light/545
Zn.sup.2+ Newport Green 506/535 TSQ Methoxy-Quinobyl 334/385
Cu.sup.+ Phen Green 492/517 Na.sup.+ SBFI 339/565 SBFO 354/575
Sodium Green 506/535 K.sup.+ PBFI 336/557 CL.sup.- SPQ 344/443 MQAE
350/460
[0042] Each of the chemicals listed in Table III directly produces
an optically interrogatable signal or optical signature change in
the presence of the targeted analyte.
[0043] Enzyme-based microsphere sensors have also been demonstrated
and could be manifest on microspheres. Examples include:
4 TABLE IV SENSOR TARGET CHEMICAL FUNCTIONALITY Glucose Sensor
glucose oxidase (enz.) + O.sub.2-sensitive dye (see Table I)
Penicillin Sensor penicillinase (enz.) + pH-sensitive dye (see
Table I) Urea Sensor urease (enz.) + pH-sensitive dye (see Table I)
Acetylcholine Sensor acetylcholinesterase (enz.) + pH-sensitive dye
(see Table I)
[0044] Generally, the induced change in the optical signature due
to the presence of the enzyme-sensitive chemical analyte occurs
indirectly in this class of chemical functionalities. The
microsphere-bound enzyme, e.g., glucose oxidase, decomposes the
target analyte, e.g., glucose, consume a co-substrate, e.g.,
oxygen, or produce some by-product, e.g., hydrogen peroxide. An
oxygen sensitive dye is then used to trigger the signature
change.
[0045] Techniques for immobilizing enzymes on microspheres, are
known in the prior art. In one case, NH.sub.2 surface chemistry
microspheres are used. Surface activation is achieved with a 2.5%
glutaraldebyde in phosphate buffered saline (10 mM) providing a pH
of 6.9. (138 mM NaCl, 2.7 mM, KCl). This is stirred on a stir bed
for approximately 2 hours at room temperature. The microspheres are
then rinsed with ultrapure water plus 0.01% tween 20 (surfactant)
-0.02%, and rinsed again with a pH 7.7 PBS plus 0.01% tween 20.
Finally, the enzyme is added to the solution, preferably after
being prefiltered using a 0.45 .mu.m amicon micropure filter.
[0046] Immuno-based microsphere sensors have been demonstrated for
the detection for environmental pollutants such as pesticides,
herbicides, PCB's and PAH's. Additionally, these sensors have also
been used for diagnostics, such as bacterial (e.g., leprosy,
cholera, lyme disease, and turburculosis), viral (e.g., HIV, herpes
simplex, cytomegalovirus), fungal (e.g., aspergillosis,
candidiasis, cryptococcoses), Mycoplasmal (e.g., mycoplasmal
pneumonia), Protozoal (e.g., amoebiasis, toxoplasmosis),
Rickettsial (e.g., Rock Mountain spotted fever), and pregnancy
tests.
[0047] Microsphere genosensors have also been demonstrated These
are typically constructed by attaching a probe sequence to the
microsphere surface chemistry, typically via an NH.sub.2 group. A
fluorescent dye molecule, e.g., fluorescein, is attached to the
target sequence, which is in solution. The optically interrogatable
signature change occurs with the binding of the target sequences to
the microsphere. This produces a higher concentration of dye
surrounding the microsphere than in the solution generally. A few
demonstrated probe and target sequences, see Ferguson, J. A. et al.
Nature Biotechnology, Vol. 14, December 19996, are listed below in
Table V.
5TABLE V PROBE SEQUENCES TARGET SEQUENCES B-glo(+) (segment of
human B- B-glo(+)-CF globin)5'-NH.sub.2--(CH.sub.2).sub.8-)TT TTT
TTT 5'-Fluorescein-TC AAC GTG TCA ACT TCA TCC ACG TTC ACC-3 GAT GAA
GTT C-3' IFNG (interferon gamma 1)5'-NH.sub.2-- IFNG-CF
(CH.sub.2).sub.8-T.sub.1- 2-TGG CTT CTC TTG GCT 5'-Fluorescein-AG
TAA CAG GTT ACT-3' CCA AGA GAA CCC AAA- 3' IL2
(interleukin-2)5'-NH.sub.2--(CH.sub.2)- .sub.8- IL2-CF T.sub.12-TA
ACC GAA TCC CAA ACT CAC 5'-Fluorescein-CT GGT GAG CAG-3' TTT GGG
ATT CTT GTA-3' IL4 (interleukin-4)5'NH.sub.2--(CH.sub.2).sub.8-
IL4-CF T.sub.12-CC AAC TGC TTC CCC CTC TGT- 5'-Fluorescein-AC AGA
GGG 3' GGA AGC AGT TGG-3' IL6 (interleukin-6)
5'NH.sub.2--(CH.sub.2).sub.8- IL6-CF T12-GT TGG GTC AGG GGT GGT TAT
5'-Fluorescein-AR TAA CCA T-3' CCC CTG ACC CAA C-3'
[0048] Alternatively, upon binding of the target sequences, an
intercalating dye (e.g., ethidium bromide) can be added
subsequently to signal the presence of the bound target to the
probe sequence.
[0049] FIG. 3 illustrates the construction of an analytic chemistry
system 100 from microspheres that have different chemical
functionalities. Subpopulations of microspheres are represented as
10a, 10b, 10c carrying respective probe sequences 60a, 60b, 60c, as
exemplary functionalities. These subpopulations, once manufactured,
are combined and mixed with each other. The resulting combined
population contains microspheres with randomly distributed
individual chemical functionalities.
[0050] Typically, mixing microspheres with different
functionalities results in the loss of information regarding the
selectivity for each of the corresponding target sequences. In a
solution of microspheres with each of the probe sequences 60a, 60b,
and 60c, it is possible to determine that at least one of the
target sequences 62a, 62b, and 62c is present when a fluorescent
marker dye 64 concentration is observed on the microspheres 10.
There is no way, however, to determine which probe sequence 60a,
60b, and 60c is generating the activity since the information
concerning which microsphere contained which probe sequence was
lost when the subpopulations were mixed.
[0051] In contrast, in the present invention, each microsphere in
each subpopulation is encoded with a common optical signature. In
the illustrated example, the subpopulation represented by
microsphere 10a has a two reporter dye ratio of 10:1; the
subpopulation of microspheres 10b has a ratio of 1:1 of the same
reporter dyes, and subpopulation of microspheres 10c has a ratio of
1:10 of the reporter dyes.
[0052] Thus, the randomly mixed subpopulations of microspheres are
useful as an analytic chemistry based on each of the carried
chemical functionalities 60a-60c separately. The 100 is added to an
analyte of interest to which some of the chemical functionalities
may interact. Any interaction changes the optical response of the
corresponding microspheres by, for example, binding a fluorescent
dye 64 to the microspheres. By identifying the chemical
functionalities of the microsphere in which the optical signature
has changed, using the encoded dye combinations, information
regarding the chemical identity and concentration of the analyte
may be gained based upon the interaction or noninteraction of each
functionality contained in the probe 100.
[0053] The microspheres exhibiting activity or changes in their
optical signature may be identified by utilizing a somewhat
"manual" approach of observing the individual microspheres through
a microscope. Decoding can also be performed manually, depending on
the particular reporter dyes used. It may be helpful to use optical
aids such as light filters to observe the light from the
microspheres at emission wavelengths of the reporter dyes. While
this approach is possible, in the preferred embodiment, the
analytic chemistry microsphere is used with the inventive optical
fiber sensor.
[0054] 2. Optical Fiber Sensor
[0055] FIG. 4 is a schematic block diagram showing the inventive
optical fiber sensor 200 and associated control 210. The fiber
optic sensor 200 comprises a fiber optic bundle 202, that is
constructed from separately clad fibers so that light does not mix
between fibers. The microsphere 100 is attached to the bundle's
distal end 212, with the proximal end 214 being received by a
z-translation stage 216 and x-y micropositioner 218. These two
components act in concert to properly position the proximal end 214
of the bundle 202 for a microscope objective lens 220. Light
collected by the objective lens 220 is passed to a reflected light
fluorescence attachment with three pointer cube slider 222. The
attachment 222 allows insertion of light from a 75 Watt Xe lamp 224
through the objective lens 220 to be coupled into the fiber bundle
202. The light from the source 224 is condensed by condensing lens
226, then filtered and/or shuttered by filter and shutter wheel
228, and subsequently passes through a ND filter slide 230.
[0056] Light returning from the distal end 212 of the bundle 202 is
passed by the attachment 222 to a magnification changer 232 which
enables adjustment of the image size of the fiber's proximal or
distal end. Light passing through the magnification changer 232 is
then shuttered and filtered by a second wheel 234. The light is
then imaged on a charge coupled device (CCD) camera 236. A computer
238 executes imaging processing software to process the information
from the CCD camera 236 and also possibly control the first and
second shutter and filter wheels 228, 234. The instrumentation
exclusive of the fiber sensor 200, i.e., to the left of the
proximal end of the bundle 202 is discussed more completely by
Bronk, et al., Anal. Chem. 1995, Vol. 67, number 17, pp.
2750-2752.
[0057] The microsphere system 100 may be attached to the distal end
of the optical fiber bundle using a variety of compatible
processes. It is important that the microspheres are located close
to the end of the bundle. This ensures that the light returning in
each optical fiber predominantly comes from only a single
microsphere. This feature is necessary to enable the interrogation
of the optical signature of individual microspheres to identify
reactions involving the microsphere's functionality and also to
decode the dye ratios contained in those microspheres. The adhesion
or affixing technique, however, must not chemically insulate the
microspheres from the analyte.
[0058] FIGS. 5A and 5B are micrographs of the distal end 212 of the
bundle 202 illustrating the preferred technique for attaching the
microspheres 10 to the bundle 202. Wells 250 are formed at the
center of each optical fiber 252 of the bundle 202. As shown in
FIG. 5B, the size of the wells 250 are coordinated with the size of
the microspheres 10 so that the microspheres 10 can be placed
within the wells 250. Thus, each optical fiber 252 of the bundle
202 conveys light from the single microsphere 10 contained in 5 its
well. Consequently, by imaging the end of the bundle 202 onto the
CCD array 236, the optical signatures of the microspheres 10 are
individually interrogatable.
[0059] FIG. 6 illustrates how the microwells 250 are formed and
microspheres 10 placed in the wells. A 1 mm hexagonally-packed
imaging fiber contains approximately 20,600 individual optical
fibers that have cores approximately 3.7 .mu.m across (Part No.
ET26 from Galileo Fibers). Typically, the cores of each fiber are
hexagonally shaped as a result of glass hardness and drawing during
fiber fabrication. In some cases, the shape can be circular,
however.
[0060] In step 270, both the proximal and distal ends 212, 214 of
the fiber bundle 202 are successively polished on 12 .mu.m, 9
.mu.m, 3 .mu.m, 1 .mu.m, and 0.3 .mu.m lapping films. Subsequently,
the ends can be inspected for scratches on an atomic force
microscope. In step 272, etching is performed on the distal end 212
of the bundle 202. A solution of 0.2 grams NH.sub.4F (ammonium
fluoride) with 600 .mu.l dH.sub.2O and 100 .mu.l of HF
(hydrofluoric acid), 50% stock solution, may be used. The distal
end 212 is etched in this solution for a specified time, preferably
approximately 80 seconds.
[0061] Upon removal from this solution, the bundle end is
immediately placed in deionized water to stop any further etching
in step 274. The fiber is then rinsed in running tap water. At this
stage, sonication is preferably performed for several minutes to
remove any salt products from the reaction. The fiber is then
allowed to air dry.
[0062] The foregoing procedure produces wells by the anisotropic
etching of the fiber cores 254 favorably with respect to the
cladding 256 for each fiber of the bundle 202. The wells have
approximately the diameter of the cores 254, 3.7 .mu.m. This
diameter is selected to be slightly larger than the diameters of
the microspheres used, 3.1 .mu.m, in the example. The preferential
etching occurs because the pure silica of the cores 254 etches
faster in the presence of hydrofluoric acid than the
germanium-doped silica claddings 256.
[0063] The microspheres are then placed in the wells 250 in step
276 according to a number of different techniques. The placement of
the microspheres may be accomplished by dripping a solution
containing the desired randomly mixed subpopulations of the
microspheres over the distal end 212, sonicating the bundle to
settle the microspheres in the wells, and allowing the microsphere
solvent to evaporate. Alternatively, the subpopulations could be
added serially to the bundle end. Microspheres 10 may then be fixed
into the wells 250 by using a dilute solution of sulfonated Nafion
that is dripped over the end. Upon solvent evaporation, a thin film
of Nafion was formed over the microspheres which holds them in
place. This approach is compatible for fixing microspheres for pH
indication that carry FITC functionality. The resulting array of
fixed microspheres retains its pH sensitivity due to the
permeability of the sulfonated Nafion to hydrogen ions. This
approach, however, can not be employed generically as Nafion is
impermeable to most water soluble species. A similar approach can
be employed with different polymers. For example, solutions of
polyethylene glycol, polyacrylamide, or polyhydroxymethyl
methacrylate (polyHEMA) can be used in place of Nafion, providing
the requisite permeability to aqueous species.
[0064] An alternative fixation approach employs microsphere
swelling to entrap each microsphere 10 in its corresponding
microwell 250. In this approach, the microspheres are first
distributed into the microwells 250 by sonicating the microspheres
suspended in a non-swelling solvent in the presence of the
microwell array on the distal end 212. After placement into the
microwells, the microspheres are subsequently exposed to an aqueous
buffer in which they swell, thereby physically entrapping them,
analogous to muffins rising in a muffin tin.
[0065] One of the most common microsphere formations is tentagel, a
styrene-polyethylene glycol co-polymer These microspheres are
unswollen in nonpolar solvents such as hexane and swell
approximately 20-40% in volume upon exposure to a more polar or
aqueous media. This approach is extremely desirable since it does
not significantly compromise the diffusional or permeability
properties of the microspheres themselves.
[0066] FIGS. 7A and 7B show polymer coated microspheres 12 in wells
250 after their initial placement and then after tapping and
exposure to air pulses. FIGS. 7A and 7B illustrate that there is no
appreciable loss of microspheres from the wells due to mechanical
agitation even without a specific fixing technique. This effect is
probably due to electrostatic forces between the microspheres and
the optical fibers. These forces tend to bind the microspheres
within the wells. Thus, in most environments, it may be unnecessary
to use any chemical or mechanical fixation for the
microspheres.
[0067] In alternative embodiments, additional encoding parameters
can be added, such as microsphere size. If a number of sensors
needed exceeds a few hundred, it is possible to use microspheres of
different sizes to expand the encoding dimensions of the
microspheres. Optical fiber arrays can be fabricated containing
pixels with different fiber diameters. With different diameters,
the largest wells can be filled with the largest microspheres and
then moving onto progressively smaller microspheres in the smaller
wells until all size wells are then filled. In this manner, the
same dye ratio could be used to encode microspheres of different
sizes thereby expanding the number of different oligonucleotide
sequences or chemical functionalities present in the array.
[0068] Experimental Results
[0069] Enzyme-Based Sensor
[0070] Subpopulation A
[0071] Chemical functionality: Alkaline phosphatase
[0072] Target substrate: fluorescein diphosphate (FDP)
[0073] Reported dye ratio: 1:1 ratio of DiIC:TRC, where DiIC is
1,1',3,3,3',3'-hexamethyl-indodicarbocyanine iodide and TRC is
Texas Red cadaverine
[0074] A range of ratios of light intensities are selected that are
representative of the optical signature for the dye ratio of the
subpopulation based on the quantum yield of the two dyes. The
optical signature for this subpopulation is:
[0075] iIC .lambda. intensity-ave.DiIC background=0.847.+-.0.23
[0076] TRC .lambda. intensity-ave.TRC background
[0077] Subpopulation B
[0078] Chemical functionality: B-Galactosidase;
[0079] Target substrate=fluorescein di-B-galactopyranoside
(FDG)
[0080] Reporter dye ratio: 10:1 ratio of DiIC:TRC which translates
to an optical signature of:
[0081] DiIC A intensity-ave.DiIC background=4.456.+-.1.27
[0082] TRC .lambda. intensity-ave.TRC background
[0083] Subpopulation C
[0084] Chemical functionality: B-glucuronidase
[0085] Target substrate=fluorescein di-B-D-glucuronide
(FDGicu).
[0086] Reporter dye ratio: 1:10 ratio of DiIC:TRC, which translates
to an optical signature of:
[0087] DiIC .lambda. intensity-ave. DiIC
background=0.2136.+-.0.03
[0088] TRC .lambda. intensity-ave. TRC background
[0089] When the microsphere populations are in the presence of one
or more of the substrates, the respective enzymes on the
microspheres catalyze the breakdown of the substrates producing
fluorescein which is fluorescent, emitting light at 530 nanometers
when excited at 490 nm. The production of fluorescein localized to
particular beads is then monitored. In this approach, the
localization of fluorescein around the microspheres is increased by
using a substrate solution of 90% glycerol and 10% substrate. The
glycerol prevents the generated fluorescein from diffusing away
from the microsphere reaction sites.
[0090] During the experiment, images in the encoded wavelengths are
first taken. Since both DiIC and TRC are excited at 577 nm. Each
microsphere's emissions at 670 nm, indicative of the presence of
DiIC and 610 nm indicative of the presence of TRC were recorded
using a 595 nm dichroic and an acquisition time of 5 seconds for
the CCD 236. Next, the distal end 212 of the fiber bundle is placed
in a buffer and another image taken while illuminating the beams
with 490 nm light. Emissions in the 530 nm fluorescein wavelengths
were recorded with a 505 nm dichroic. In this case, a CCD
acquisition time of one second was used. This process provides a
background normalizing image. The buffer was removed and the fiber
allowed to dry to avoid substrate solution dilution.
[0091] The substrate solution is then introduced and CCD images
acquired every 30 seconds to a minute for 30 minutes While
illuminating the microspheres with 490 nm light and collecting
emissions in the 530 nm range. Fiber is then placed back in the
buffer solution and another background image captured. Those beads
that generate a signal indicative of fluorescein production are
decoded. Depending in the ratio of the intensity of light from the
two reporter dyes, DiIC:TRC, the chemical functionality of the
optically active beads may be decoded according to the following
table.
6 0.617-1.08 alkaline phosphatase bead 3.188-5.725
.beta.-galactosidase bead 0.183-0.243 .beta.-glucunonidese bead
[0092] This process is then repeated for the remaining two
substrates.
[0093] FIGS. 8A-8C are images generated by the CCD 236 when the
bead populations are exposed to fluorescein diphosphate. FIG. 8A
illustrates the signals from the alkaline phosphatase microspheres
when excited at 490 nm and recording emissions at 530 nm, emissions
at this wavelength being indicative of fluorescein production. FIG.
8B shows the image captured by the CCD when the microspheres are
excited at 577 nm and emissions at 670 nm are recorded. This
wavelength is an encoding wavelength indicative of the
concentration of DiIC on the microspheres. Finally, FIG. 8C shows
the image when the microspheres are excited with 577 nm light and
emissions in the 610 nm range are recorded being indicative of the
concentration of TRC in the microspheres.
[0094] In a similar vein, FIGS. 9A and 9B are images when the
microspheres are exposed to fluorescein d-.beta.-galactosidase.
FIG. 9A shows emissions at 530 nm indicative of the fluorescein
production; and FIG. 9B shows light emitted at the 670 nm range
indicative of the presence of DiIC.
[0095] These micrographs, FIGS. 8A-8C and 9A-9B illustrate
fluorescein production around the microspheres may be detected as
an optical signature change indicative of reactions involving the
chemical functionality of the microspheres. The micrographs also
illustrate that the optical signatures may be decoded to determine
the chemical functionalities on each microsphere.
[0096] Genosensor
[0097] Three separate subpopulations of beads were used. In
subpopulation A, xrabbit antibodies (Ab) were affixed to the
surface of the microspheres; in subpopulation B, xgoat antibodies
were affixed to the microspheres; and in subpopulation C, xmouse
antibodies were affixed to the microspheres. These three separate
subpopulations were identified using a DiIC:TRC encoding ratio
similar to that in the previously described experiment.
[0098] For the first step of the experiment, images at the encoded
wavelengths were captured using 577 nm excitation and looking for
emissions at 610 and 670 nm. After this decoding, the fiber was
placed in a buffer and an image taken at 530 nm with 490 nm
excitation. This provided a background normalizing signal at the
fluorescein emission wavelength. Next, the fiber was placed in
rabbit IgG antigen (Ag) which is fluorescein labeled. Images were
then captured every few minutes at the 530 nm emission wavelength
for fluorescein. FIGS. 10A and 10B are micrographs showing the
image captured by the CCD prior to and subsequent to exposure to a
rabbit antigen, which clearly show reaction of the selected
micropheres within the population.
[0099] Note, if the fluorescein background from the antigen
solution is too high to see the antibody-antigen signal, the fiber
bundle may be placed in a buffer. This removes the background
florescence leaving only the Ab-Ag signal.
[0100] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims.
* * * * *