U.S. patent application number 10/153516 was filed with the patent office on 2002-09-26 for high density array fabrication and readout method for a fiber optic biosensor.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Albertson, Donna, Gray, Joe, Pinkel, Daniel.
Application Number | 20020137090 10/153516 |
Document ID | / |
Family ID | 23778784 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137090 |
Kind Code |
A1 |
Pinkel, Daniel ; et
al. |
September 26, 2002 |
High density array fabrication and readout method for a fiber optic
biosensor
Abstract
The invention relates to the fabrication and use of biosensors
comprising a plurality of optical fibers each fiber having attached
to its "sensor end" biological "binding partners" (molecules that
specifically bind other molecules to form a binding complex such as
antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid,
biotin-avidin, etc.). The biosensor preferably bears two or more
different species of biological binding partner. The sensor is
fabricated by providing a plurality of groups of optical fibers.
Each group is treated as a batch to attach a different species of
biological binding partner to the sensor ends of the fibers
comprising that bundle. Each fiber, or group of fibers within a
bundle, may be uniquely identified so that the fibers, or group of
fibers, when later combined in an array of different fibers, can be
discretely addressed. Fibers or groups of fibers are then selected
and discretely separated from different bundles. The discretely
separated fibers are then combined at their sensor ends to produce
a high density sensor array of fibers capable of assaying
simultaneously the binding of components of a test sample to the
various binding partners on the different fibers of the sensor
array. The transmission ends of the optical fibers are then
discretely addressed to detectors--such as a multiplicity of
optical sensors. An optical signal, produced by binding of the
binding partner to its substrate to form a binding complex, is
conducted through the optical fiber or group of fibers to a
detector for each discrete test. By examining the addressed
transmission ends of fibers, or groups of fibers, the addressed
transmission ends can transmit unique patterns assisting in rapid
sample identification by the sensor.
Inventors: |
Pinkel, Daniel; (Walnut
Creek, CA) ; Gray, Joe; (San Francisco, CA) ;
Albertson, Donna; (Lafayette, CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
23778784 |
Appl. No.: |
10/153516 |
Filed: |
May 21, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10153516 |
May 21, 2002 |
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09642243 |
Aug 17, 2000 |
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6417506 |
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09642243 |
Aug 17, 2000 |
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08899000 |
Jul 24, 1997 |
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6146593 |
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08899000 |
Jul 24, 1997 |
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08448043 |
May 23, 1995 |
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5690894 |
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Current U.S.
Class: |
506/9 ; 427/2.11;
435/287.2; 435/6.1; 435/6.12; 435/7.1; 506/15; 506/16; 506/18;
506/23; 65/385 |
Current CPC
Class: |
B01J 2219/00605
20130101; B01J 2219/00621 20130101; G01N 21/6452 20130101; G02B
6/04 20130101; B01J 2219/00659 20130101; B01J 2219/00513 20130101;
G01N 33/54373 20130101; C12Q 1/6825 20130101; G01N 21/648 20130101;
B01J 2219/00626 20130101; B01J 2219/00524 20130101; G01N 2201/0833
20130101; G01N 21/7703 20130101; B01J 2219/00612 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 65/385; 435/7.1; 427/2.11 |
International
Class: |
C12Q 001/68; G01N
033/53; C12M 003/00; C03B 037/023; B05D 003/00; C12M 001/34 |
Goverment Interests
[0002] This invention was made with the Government support under
Grant No. CA 45919, awarded by the National Institute of Health and
under Grant No. DE-AC03-76SF0098, awarded by the Department of
Energy. The Government has certain rights in this invention.
Claims
What is claimed is:
1. A process for constructing a fiber optic bundle for sensing a
plurality of biological binding partners within a sample, the
process comprising the steps of: providing a plurality of optical
fibers with each fiber having a sensor end and a transmission end
wherein each fiber has attached to its sensor end a species of
biological binding partner; combining said fibers with differing
binding partners to form an optical fiber array wherein said fibers
have commonly aligned sensor ends for simultaneous assay of a
sample; and, addressing the transmission end of the combined
discrete fibers for interrogation to produce the fiber optic sensor
for the sample.
2. The process of claim 1, wherein said providing step comprises:
providing a plurality of optical fibers with each fiber having a
sensor end and a transmission end; placing the plurality of optical
fibers in a plurality of fiber groups, each fiber group with
commonly aligned sensor ends for simultaneous treatment; providing
a plurality of differing batches, each batch comprising a single
species of biological binding partner suitable for attachment under
treatment to the commonly aligned sensor ends of the optical fibers
of a selected group of optical fibers; placing and treating the
commonly aligned sensor ends of differing fiber groups in differing
batches with differing biological binding partners to produce a
plurality of groups of optical fibers with the sensor ends of the
optical fibers of each group having the same species of biological
binding partner and the sensor ends of optical fibers of differing
groups having differing species of biological binding partners; and
separating fibers from each group of fibers thereby providing a
plurality of optical fibers with each fiber having a sensor end and
a transmission end wherein each fiber has attached to its sensor
end a species of biological binding partner.
3. A process for constructing a fiber optic bundle for sensing a
plurality of binding partners within a sample according to claim 1
wherein: the binding partners are selected from a group consisting
of nucleic acid, antibody, peptide, lectin, biotin, and avidin.
4. A process for constructing a fiber optic bundle for sensing a
plurality of binding partners within a sample according to claim 1
wherein: the combining separated fibers with differing binding
partners with commonly aligned sensor ends includes randomly
gathering the sensor ends.
5. A process for constructing a fiber optic bundle for sensing a
plurality of binding partners within a sample according to claim 1
wherein: the combining separated fibers with differing binding
partners with commonly aligned sensor ends includes assembling said
sensor ends to form a tiered sensor face.
6. A process for constructing a fiber optic bundle for sensing a
plurality of binding partners within a sample according to claim 1
wherein: the combining separated fibers with differing binding
partners with commonly aligned sensor ends includes assembling said
sensor ends to form a planer sensor face.
7. A process for constructing a fiber optic bundle for sensing a
plurality of binding partners within a sample according to claim 1
wherein: the addressing the transmission end of the recombined
discrete fibers for interrogation includes addressing the
transmission ends of each of the discrete fibers to an optical
array.
8. A process for constructing a fiber optic bundle for sensing a
plurality of binding partners within a sample according to claim 1
wherein: transmission ends of the optical fibers of each group
having similar binding partners have similar markings for
distinguishing the transmission ends corresponding to binding
partners.
9. A process for constructing a fiber optic bundle for sensing a
plurality of binding partners within a sample according to claim 1
wherein: the providing a plurality of batches of differing binding
partners includes batches of nucleic acid binding partners to which
nucleic acids in the sample might hybridize.
10. A process for constructing a fiber optic bundle for sensing a
plurality of binding partners within a sample according to claim 9
wherein: the nucleic binding partners each correspond to specific
regions on chromosomes.
11. A sensor for detecting a multiplicity of analytes, the sensor
comprising in combination: a plurality of fibers, each fiber
including a sensot-end and a transmission end; the sensor end of at
least one first fiber having attached a first biological binding
partner; the sensor end of at least one second fiber having
attached a second biological binding partner; a transmission array
having first and second positions for addressing the transmission
ends of the first and second fibers; and means for addressing the
transmission ends of the first and second fibers to the
transmission array.
12. The sensor of claim 11, further comprising an optical
interrogation means adjacent the transmission ends for examining
the attachment of analytes at the sensor ends of said fibers.
13. The sensor of claim 11, wherein said sensor ends are arranged
to form a tiered sensor face.
14. The sensor of claim 11, wherein said first and second binding
partners are nucleic acids.
15. The sensor of claim 14, wherein the nucleic acids are mapped to
specific regions in human chromosomes.
16. The sensor of claim 14, wherein the nucleic acids are DNA.
17. The sensor of claim 14, wherein the nucleic acids are cDNA.
18. The sensor of claim 14, wherein the target nucleic acids are
about 1000 to about 1,000,000 nucleotides in complexity.
19. The sensor of claim 11, wherein said first and second binding
partners are antibodies.
20. A method for comparing copy number of nucleic acid sequences in
two or more collections of nucleic acid molecules, said method
comprising: (a) providing a biosensor wherein said biosensor
comprises a plurality of optical fibers, each fiber including
sensor end and a transmission end where the sensor ends of the
optical fibers bear target nucleic acids such that the sensor end
of at least one first fiber has attached a first target nucleic
acid and the sensor end of at least one second fiber has attached a
second target nucleic acid; (b) contacting said biosensor with (i)
a first collection of labelled nucleic acid comprising a sequence
substantially complementary to a target nucleotide sequence, and
(ii) at least a second labelled nucleic acid comprising a sequence
complementary to the target nucleotide sequence; wherein the first
and second labels are distinguishable from each other; and (c)
detecting the amount of binding of the first and second labelled
complementary nucleic acids to the target nucleic acids.
21. The method of claim 20, wherein the target nucleic acids are
DNA.
22. The method of claim 20, wherein the target nucleic acids are
cDNA.
23. The method of claim 20, wherein the target nucleic acids are
RNA.
24. The method of claim 20, wherein the target nucleic acids are
mapped to specific regions in human chromosomes.
25. The method of claim 20, wherein the target nucleic acids are
about 1000 to about 1,000,000 nucleotides in complexity.
26. The method of claim 20, wherein the complexity of the sequence
complementary to the target nucleic acid sequence is less than 1%
of the total complexity of the sequences in the sample.
27. The method of claim 20, wherein the first and second labels are
fluorescent labels.
28. The method of claim 20, wherein the first labeled nucleic acids
comprise mRNA or cDNA from a test cell and the second labeled
nucleic acids comprise mRNA are DNA from a reference cell.
29. The method of claim 20, wherein the first labeled nucleic acids
are from a test genome and the second labeled nucleic acids are
from a normal reference genome.
30. The method of claim 29, wherein the test genome comprises
nucleic acids from fetal tissue.
31. The method of claim 29, wherein the test genome comprises
nucleic acids from a tumor.
32. The method of claim 20, wherein the first and second
collections of nucleic acids are treated to inhibit the binding of
repetitive sequences.
33. The method of claim 32, wherein the first and second
collections of nucleic acids are mixed with unlabeled blocking
nucleic acids comprising repetitive sequences.
34. A detector for detecting an array of light sources, said
detector comprising a multiplicity of simple lenses joined together
to form a compound lens wherein each simple lens is positioned such
that each light source of said array of light sources is located at
the focal point of a single simple lens of said compound lens.
35. The detector of claim 34, further comprising a beam positioned
positioned to direct an excitation light through each simple lens
of said compound lens.
36. The detector of claim 35, further comprising an excitation
light source to provide said excitation light, said excitation
light source.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a Divisional of U.S. Ser. No. 08/448,043, filed on
May 23, 1995, which is incorporated herein by reference for all
purposes.
FIELD OF THE INVENTION
[0003] This invention relates to the fabrication and use of
biosensors comprising biological "binding partners" (molecules that
specifically bind other molecules to form a binding complex such as
antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid,
biotin-avidin, etc.) linked to optical fibers. Specifically,
batches of optical fibers are mass processed with the same species
of binding partner, singulated from their particular batch,
regrouped with like optical fibers from other batches having other
species of binding partners. Upon regrouping of the optical fibers,
high density arrays are formed which can simultaneously interrogate
samples for a multiplicity of analytes for sample identification
and processing.
BACKGROUND OF THE INVENTION
[0004] Biosensors are sensors that detect chemical species with
high selectivity on the basis of molecular recognition rather than
the physical properties of analytes. See, e.g., Advances in
Biosensors, A. P. F. Turner, Ed. JAI Press, London, (1991). Many
types of biosensing devices have been developed in recent years,
including enzyme electrodes, optical immunosensors, ligand-receptor
amperometers, and evanescent-wave probes. Updike and Hicks, Nature,
214: 986 (1967), Abdel-Latif et al., Anal. Lett., 21: 943 (111988);
Giaever, J. Immunol., 110: 1424 (1973); Sugao et al. Anal. Chem.,
65: 363 (1993), Rogers et al. Anal. Biochem., 182: 353 (1989).
[0005] Biosensors comprising a biological "binding molecule"
attached to an optical fiber are well known in the prior art, most
typically as evanescent wave detectors (see, for example, U.S. Pat.
No. 4,447,546 to Hirschfeld and U.S. Pat. No. 4,582,809 and
4,909,990 to Block et al.). In order to maximize sensitivity and
selectivity such biosensors typically utilize a single species of
biological binding molecule affixed to the face of the sensor.
[0006] Such "single-species" biosensors are limited in that they
have no inherent means to correct or calibrate for non-specific
binding. Thus, they must be calibrated against an external
standard. In addition, they are limited to the detection of a
single analyte.
[0007] Biosensors comprising two or more species of biological
binding partners overcome these limitations. A "multi-species"
biosensor in principle permits simultaneous detection of as many
different types of analytes as there are species of biological
binding partner incorporated into the sensor. In addition,
comparison of the amounts of a single analyte binding to multiple
species of binding partner provides a measure of non-specific
binding and thus acts as an intrinsic control for measurement
variability introduced by non-specific binding.
[0008] In addition, the inclusion of fibers bearing biological
binding partners specific for various analytes known to create a
background signal in a particular assay provides a means for
simultaneously measuring and substracting out the background
signal. The provision of a multiplicity of fibers bearing different
species of binding partner allows the detection of a multiplicity
of moieties contributing to a background, or other, signal and the
dissecting out of the contribution of each moiety to that
signal.
[0009] To be most useful, a multi-species biosensor requires that
the sensor provide a separate signal characterizing binding of
analytes to each of the various species of binding partner
comprising the probe. Thus each species of binding partner must be
individually "addressed".
[0010] In addition, a "sensor face" (the surface bearing the
biological binding partners) that has a relatively small surface
area will facilitate measurement of small sample volumes as less
sample material will be required to fully immerse the sensor face.
A small surface area detector will also prove advantageous for use
in in vitro measurements. Preparation of a detector bearing a large
number of different biological binding partners that occupies a
small area may be viewed as the preparation of a high density array
of biological binding partners.
[0011] The creation of high density arrays of biological binding
partners where each species of binding partner is uniquely
addressed presents formidable fabrication problems. One of the most
successful approaches, to date, is the large scale
photolithographic solid phase synthesis pioneered by Affymax Inc.
(see, e.g., Fodor et al. Science 251: 767-773 (1991) and U.S. Pat.
No. 5,143,854). In this approach arrays of peptides or nucleic
acids are chemically synthesized on a solid support. Different
molecules are simultaneously synthesized at different predetermined
locations on the substrate by the use of a photolithographic
process that selectively removes photolabile protecting groups on
the growing molecules in particular selected locations of the
substrate. The resulting array of molecules is "spatially
addressed". In other words the identity of each biological molecule
is determined by its location on the substrate.
[0012] The photolithographic approach, however, is limited to
molecules that can be chemically synthesized. Thus, it is typically
restricted to peptides shorter than about 50 amino acids and
nucleic acids shorter than about 150 base pairs. In addition, the
photolithographic approach typically produces such arrays on a
planar substrate (e.g. a glass slide) and provides no intrinsic
mechanism by which a signal produced by the binding of a particular
biological binding partner may be transmitted.
[0013] U.S. Pat. No. 5,250,264 to Walt et al. discloses a sensor
comprising a fiber optic array using a "plurality of different dyes
immobilized at individual spatial positions on the surface of the
sensor." Each dye is capable of responding to a different analyte
(e.g., pH, O.sub.2, CO.sub.2, etc.) and the sensor as a whole is
capable of providing simultaneous measurements of multiple
analytes.
[0014] Although the sensor disclosed by Walt et al. is not a
biosensor, the reference describes a means of fabricating a sensor
bearing a plurality of uniquely addressed "detection moieties". In
Walt et al. optical fibers are first assembled to form a bundle.
Transmission ends of a fiber or group of fibers of are then
specifically illuminated. Each illuminated fiber transmits the
light to its respective sensor end where the light
"photopolymerizes" a sensor dye mixture causing the dye to bind to
the sensor end. This process is repeated with different fibers for
different photopolymerized dyes. This repetition continues until a
sensor array is constructed.
[0015] This approach suffers from the limitations that it requires
photopolymerizable sensor dyes and thus is limited in the number of
different species per probe hy the number of different dye type. In
addition, this reference provides no means for attaching uniquely
addressed biological molecules (e.g. peptides, nucleic acids,
antibodies) to the sensor. Thus Walt et al. provide no means for
the fabrication of biosensors.
SUMMARY OF THE INVENTION
[0016] The present invention provides a novel means for fabricating
biosensors comprising a plurality of biological "binding partners"
(molecules that specifically bind other molecules to form a binding
complex such as antibody-antigen, lectin-carbohydrate, nucleic
acid-nucleic acid, biotin-avidin, etc.) linked to optical fibers.
In particular the method provides a means of preparing a high
density array of biological binding partners where each species of
binding partner is uniquely addressed. In contrast to certain
methods in the prior art the biological binding partners utilized
in the present invention are not limited to chemically synthesized
oligonucleotides or peptides, but rather include nucleic acids,
antibodies, proteins, lectins and other binding partners derived
from cells, tissues or organisms in their native state or otherwise
modified through the methods of recombinant DNA technology.
[0017] In particular, the biosensors of the present invention
comprise a multiplicity of optical fibers bundled together to form
an optical fiber array. The sensor end of each optical fiber or
group of optical fibers comprising the optical fiber array bear a
particular species of biological binding partner. Optical signals
produced by binding of an analyte to a biological binding partner
are conducted along the respective optical fibers to a transmission
end which may be attached to a detector. Detection of the signal
from the fibers corresponding to each species of biological binding
partner provides a simultaneous measurement of the binding of a
multiplicity of analytes.
[0018] The present invention provides a method of fabrication of
fiber optic biosensors. The method involves providing a
multiplicity of optical fibers which are grouped into a plurality
of separate fiber groups or batches. Each fiber has a sensor end
and a transmission end and the fibers in each group are oriented so
that the sensor ends are commonly aligned. Each group of fibers is
then treated to attach a single species of biological binding
partner to the sensor ends of the constituent fibers.
Alternatively, a multiplicity of species of biological binding
partner may be attached to each group as long as the multiplicity
of species of biological binding partners attached to one fiber
group is different than the multiplicity of species attached to the
other fiber groups.
[0019] Fibers or groups of fibers are then selected and discretely
separated from their respective batches. One or more of the
discretely separated fibers from each group are then recombined at
their sensor ends with other fibers from other batches to produce
an optical fiber array. The sensor ends may be arranged in a
substantially planar orientation or may be tiered to form a tiered
sensor face. The optical fiber array contains fibers capable of
assaying simultaneously the binding of components of a test sample
to the various binding partners on the different fibers of optical
fiber array.
[0020] The batch identity of each fiber is maintained during the
bundling process, preferably at or adjacent to the transmission end
of the fibers. These transmission ends are then discretely
addressed to detectors--such as a multiplicity of optical sensors.
The location and spatial array of the transmission ends
corresponding to particular biological binding partners are
distinct from one another and known.
[0021] Thus, the invention provides for the fabrication of a high
density array of biological binding partners in which each binding
partner is uniquely addressed. An optical signal, produced by
binding of the binding partner to its substrate to form a binding
complex, is conducted through the optical fiber or group of fibers
to a detector for each discrete test. Thus, binding of a molecule
to a particular biological binding partner is specifically
detectable. By examining the addressed transmission ends of fibers,
or groups of fibers, the addressed transmission ends can transmit
unique patterns assisting in rapid sample identification of
analytes by the sensor.
[0022] In one embodiment, the fiber optics might bear nucleic acid
binding partners to which nucleic acids in the test sample might
hybridize. As used herein, the terms polymer in either single- or
double-stranded form, and unless otherwise limited, would encompass
known analogs of natural nucleotides that can function in a similar
manner as naturally occurring nucleotides.
[0023] In one particularly preferred embodiment, the biosensor
comprises a plurality of fibers, each fiber including an sensor end
and a transmission end, the sensor end of at least one first fiber
having attached a first biological binding partner and the sensor
end of at least one second fiber having attached a second
biological binding partner, a transmission array having first and
second positions addressing the transmission ends of the first and
second fibers, means for addressing the transmission ends of the
first and second fibers to the transmission array, optical
interrogation means adjacent the transmission ends for examining
the comparative attachment of analytes at the sensor ends of the
fibers. The sensor ends of the first and second fibers may be
arranged to form a tiered sensor face. The first and second binding
partners may be nucleic acids, for example, DNA and cDNA, and the
nucleic acids may be mapped to specific regions on one or more
human chromosomes. In a particularly preferred embodiment, the
target nucleic acids are about 1,000 to 1,000,000 nucleotides in
complexity.
[0024] Nucleic acid bearing arrays are particularly useful in
Comparative Genomic Hybridization (CGH) assays to detect
chromosomal abnormalities; in particular increases or decreases in
copy number of particular chromosomal regions. In one example of
this approach, a first collection of (probe) nucleic acids is
labeled with a first label, while a second collection of (probe)
nucleic acids is labeled with a second label. A biosensor, as
described above, is one in which the biological binding partners
are target nucleic acids. (As used herein the term "target nucleic
acids" typically refers to nucleic acids attached to the optical
fibers comprising the fiber optic array, while "probe nucleic
acids" are those nucleic acids free in solution that hybridize with
the target nucleic acids.) The ratio of hybridization of the
nucleic acids is determined by the ratio of the two (first and
second) labels binding to each fiber in the array. Where there are
chromosomal deletions or multiplications, differences in the ratio
of the signals from the two labels will be detected. Identification
of the specific optical fibers in the array giving rise to these
ratios will indicate the nucleic acid sequence the probe bears and
thus the nucleic acid sequence that is altered.
[0025] The target nucleic acids (the nucleic acids attached to the
optical fibers) may include DNA and cDNA and may be mapped to
specific regions in human chromosomes. In addition, the target
nucleic acids are preferably about 1,000 to 1,000,000 nucleotides
in complexity. The complexity of the sequence complementary to the
target nucleic acid is preferably less than 1% of the total
complexity of the sequences in the sample.
[0026] The first and second labels are preferably fluorescent
labels. In a particularly preferred embodiment, the first probe
nucleic acids comprise mRNA or cDNA from a test cell and the second
probe nucleic acids comprise mRNA or cDNA from a reference cell. In
another preferred embodiment, the first probe nucleic acids are
from a test genome and the second probe nucleic acids are from a
reference genome. The test genome may comprise nucleic acids from
fetal tissue or from a tumor.
[0027] According to one aspect of the invention, arrays of optical
fibers are disclosed where the interrogating end of each fiber in
the array comprises a multiplicity of biological "binding
partners." Each binding partner is attached to one or more optical
fibers specifically addressed or identified at the transmitting end
as being connected to the particular binding partner. With the
transmission ends properly addressed and interrogated, measurement
occurs.
[0028] In one specific embodiment, arrays of optical fibers bearing
nucleic acid molecules are disclosed. These optical fibers at their
interrogating ends have specific nucleic acids such as nucleic
acids having a certain minimum length (e.g. 400 bp), or being
derived from particular libraries (e.g. evenly spaced along a
particular chromosome or representing a particular gene).
[0029] It is an advantage of the disclosed apparatus and process
that the constructed array can be tailored to rapid screening of
extensive arrays of biological binding partners. Using already
identified information, arrays can be assembled which can
simultaneously and rapidly survey samples nucleic acid variations
across entire genomes. For example, a fiber optic sensor bearing
30,000 target nucleic acids, each containing 100 kb of genomic DNA
could give complete coverage of the human genome.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a schematic illustration showing a group of fibers
with the sensor ends bound together for joint treatment to attach a
binding partner and the transmission ends discretely marked to
enable the fibers of the illustrated group to be distinguished from
fibers of similar groups when subsequent separation of the fibers
later occurs from the group.
[0031] FIG. 2 illustrates a plurality of differing batches of
treatment solution with the sensor ends of the group of fibers of
FIG. 1 being immersed for treatment in one of the fiber
batches;
[0032] FIG. 3 illustrates differing groups of previously treated
fibers lying side-by-side with fibers being singulated from each
group for gathering into a common high density array.
[0033] FIG. 4 illustrates an assembled high density array at the
sensor and transmitting ends only with disposition of the sensor
ends in a tiered disposition for transillumination by interrogating
light and the detector ends identified and discretely addressed to
a sensor array, the sensor array here illustrated having a
corresponding array of condensing lenses for relaying fiber
illumination to a detector surface;
[0034] FIG. 5 illustrates a comparative genomic hybridization
process being carried out with two samples for comparison having
been previously tagged with differing fluorophores and being added
together in a common batch.
[0035] FIGS. 6A and 6B illustrate an expanded detail of the common
batch of FIG. 5 being transilluminated at the tiered sampling
fibers at the sensor end of the array for the excitation of
fluorophores attached to the binding partners without undue direct
illumination of the fibers themselves.
[0036] FIGS. 7A and 7B illustrate a detector that may be used in
conjunction with an optical fiber array or with any other array of
light sources (e.g. an array of hybridized fluorescent probes).
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0037] The present invention is a marked improvement in fiber optic
biosensors, methods for fabricating biosensors, and methods for
performing qualitative and quantitative measurements of biological
molecules using a unique fiber optic biosensor. In particular, the
present invention provides for a novel method of construction of a
biosensor comprising a high-density array of biological binding
partners.
[0038] The biosensors of the present invention generally comprise a
bundle of coalligned optical fibers. Each individual optical fiber
or group of fibers within the biosensor bears a single species of
biological binding partner. As used herein biological binding
partners are molecules that specifically recognize and bind other
molecules thereby forming a binding complex. Typical binding
complexes include, but are not limited to, antibody-antigen,
lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin,
receptorreceptor ligand, etc.
[0039] The terms "specifically recognize and bind" refers to the
binding of a biological binding partner to a particular molecule
and to no other molecule to which the biological binding partner is
normally exposed. In the case of nucleic acids, specific binding is
by hybridization and the terms "specific hybridization" or
"specifically hybridizes with" refers to hybridization in which a
probe nucleic acid binds substantially to target nucleic acid and
does not bind substantially to other nucleic acids present in the
biosensor under defined stringency conditions. One of skill will
recognize that relaxing the stringency of the hybridizing
conditions will allow sequence mismatches to be tolerated. The
degree of mismatch tolerated can be controlled by suitable
adjustment of the hybridization conditions.
[0040] The term "species", as used herein, refers to a biological
binding partner capable of specifically binding a particular target
molecule. Thus, for example, biological binding partners may both
be nucleic acids, but if they have different nucleotide sequences,
so that they specifically hybridize to different molecules, they
are considered different species. Similarly, two antibodies
specific for different epitopes are considered different
species.
[0041] In a preferred embodiment, the biosensor bears two or more
different species of biological binding partner. The use of two or
more species of binding partner permits the simultaneous detection
of two or more analytes in a test sample with the number of
detectable analytes limited only by the number of different
biological binding partners present on the biosensor. The biosensor
may optionally include additional optical fibers lacking biological
binding partners. These additional fibers may bear moieties for the
detection of various physical parameters of the test sample, such
as temperature or pH, or alternatively may lack any moiety and
simply serve as an optical conduit for visualization thereby
serving as an endoscope for guiding the insertion of the biosensor
probe in various in vivo applications.
[0042] A biosensor bearing a plurality biological binding partners
permits the simultaneous assay of a multiplicity of analytes in a
sample. In addition, the measurement of binding of a single analyte
to a number of different species of biological binding partners
provides a control for non-specific binding. A comparison of the
degree of binding of different analytes in a test sample permits
evaluation of the relative increase or decrease of the different
analytes. Finally, because of the small cross-sectional area of
optical fibers, the bundling together into an optical array a
number of optical fibers, each bearing a different biological
binding partner, provides an effective mechanism for the
fabrication of high density arrays of biological binding partners
for suitable for a wide variety of in vivo and in vitro assays.
I. Organization of the Biosensor
[0043] The unique fiber optic biosensor of the present invention,
its organization, its construction and its component parts are
illustrated by FIGS. 1-6 respectively. Each discrete fiber optic
biosensor is comprised of a plurality of fiber optic strands 10
disposed coaxially along their lengths to form a single, discrete
construction. The biosensor thus comprises an optical fiber array
14, the smallest common repeating unit within which is a single
fiber optical strand 10.
[0044] A preferred fiber optical biosensor is illustrated by FIG.
4. As seen therein an individual fiber optical strand 10 comprises
a single optical fiber having a rodlike shaft and two fiber ends
designated a sensor end 11 and a transmission end 12 each of which
provides a substantially planar end surface. The optical fiber
strand 10 is typically composed of glass or plastic and is a
flexible rod able to convey light energy introduced at either of
its ends 11, 12. Such optical fibers 10 are conventionally known
and commercially available. Alternatively, the user may himself
prepare optical fibers in accordance with the conventional
practices and techniques reported by the scientific and industrial
literature. Accordingly, the optical fiber 10 is deemed to be
conventionally known and available as such.
[0045] It will be appreciated that FIGS. 1-6 are illustrations in
which the features have been purposely magnified and exaggerated
beyond their normal scale in order to provide both clarity and
visualization of extreme detail. Typically, the conventional
optical fiber has a cross section diameter of 5-500 micrometers and
is routinely employed in lengths ranging between centimeters (e.g.
in the laboratory) to kilometers (e.g. in field
telecommunications). Typically, however, when utilized in a
biosensor, the optical fibers will preferably range in length from
centimeters to about a meter.
[0046] Although the optical fiber 10 is illustrated via FIGS. 14 as
a cylindrical extended rod having substantially circular end
surfaces, there is no requirement or demand that this specific
configuration be maintained. To the contrary, the optical fiber may
be polygonal or asymmetrically shaped along its length, provide
special patterns and shapes at the sensor end or transmission end
and need not present an end surface that is substantially planar.
Nevertheless, in a preferred embodiment, the optical fiber is
substantially cylindrical.
[0047] Each optical fiber 10 may be individually clad axially along
its length. The cladding may be any material which has a lower
refractive index and prevents the transmission of light energy
photons from the optical fiber 10 to the external environment. The
cladding may thus be composed of a variety of different chemical
formulations including various glasses, silicones, plastics,
cloths, platings and shielding matter of diverse chemical
composition and formulation. Methods of cladding including
deposition, extrusion, painting and covering are scientifically and
industrially available and any of these known processes may be
chosen to meet the requirements and convenience of the user.
[0048] The user has a variety of choices at his discretion
regarding the configuration of the sensor end 11 of the optical
fiber 10. As indicated above, the sensor end 11 may present a
surface that is substantially planar and smooth. Alternatively the
sensor end 11 may provide an end surface which is essentially
convex or concave.
[0049] It will be appreciated that the range and variety of
dimensional and configurational variation of the optical fiber 10
is limited only by the user's ability to subsequently dispose and
immobilize a biological binding partner on the intended sensor end
11 of the strand. The use of concave or convex sensor ends 11 will
provide greater surface area upon which to immobilize a biological
binding partner thereby increasing efficiency (the signal to noise
ratio per optical fiber) of the biosensor.
[0050] While the single repeating component of the fiber optic
biosensor is the individual optical fiber 10, it is the aggregation
of a plurality of such fibers to form a discrete optical fiber
array 14 that permits the simultaneous detection of a multiplicity
of analytes. When the optical fibers are aggregated to form a
discrete optical fiber array 14, the coalligned sensor ends 11 of
the fibers are aggregated to form a sensor face 13. A typical
biosensor is illustrated in FIG. 4 and FIGS. 6A and 6B in which the
sensor face 13 appears in exaggerated, highly simplified views
without regard to scale. The optical fiber array 14 comprises a
unitary rod-like collective body forming a sensor face 13 and a and
a transmission face 15.
[0051] In practice, it is estimated that there are typically
1000-3000 fiber optical strands in a conventional imaging fiber of
0.5 mm diameter and nearly 1 million strands per square millimeter.
The total number of individual fiber optic strands forming the
optical fiber array 14 of the present invention will be
approximately as great; the total number varying with the
cross-sectional diameter of each optical fiber, the pattern of
packing of the individual optical fibers in the collective body,
and the thickness of cladding material, when employed. It will be
appreciated that a 1 square millimeter biosensor, containing nearly
1 million strands in which groups of about 33 optical fibers are
each labeled with a different species of biological binding partner
will produce a sensor face 13 comprising approximately 30,000
different species of biological binding partner in 1 square
millimeter. As explained above, such a sensor could provide nucleic
acid biological binding partners covering the entire human genome
at 10 megabase intervals.
[0052] The sensor face 13 need not be arranged as a planar surface.
Rather, the individual optical fibers may be "tiered" so as to
protrude from the optical array varying distances. This will
maximize the exposure of each optical fiber sensor end 11 both to
the sample fluid and to a transilluminating light source 19 as
shown in FIGS. 6A and 6B.
[0053] In a preferred embodiment, the sensor ends 11 of the optical
fibers comprising the optical fiber array 14 will be bundled
together in a random or haphazard pattern to form the sensor face
13. Alternatively, the placement of the sensor ends 11 may be
highly ordered with the sensor end of each fiber occupying a
specific predetermined location in the sensor face 13. As indicated
above, the sensor ends 11 of the optical fibers 10 forming the
sensor face 13 or the optical fiber array 14 have attached a
biological binding partner.
[0054] Each optical fiber or group of fibers comprising the optical
fiber array 14 may bear a different species of biological binding
molecule. Although the use of a single species of biological
binding partner per optical fiber or group of fibers is preferred,
alternatively, each optical fiber or group of fibers may bear a
multiplicity of biological binding partners as long as that
multiplicity differs from the biological binding partners or
multiplicity of biological binding partners present on other fibers
or groups of fibers comprising the optical fiber array 14. The
fibers bearing like species of binding partner may be physically
grouped together thereby producing distinct regions of the sensor
face 13 characterized by the presence of a particular biological
binding partner or alternatively the fibers bearing different
binding partners may be intermingled, the sensor face 13 presenting
a relatively uniform or haphazard or random distribution of species
of biological binding partners.
[0055] The transmission face 15 of the optical array may present a
substantially planar optical array lacking any further attachments.
However, in a preferred embodiment, the transmission face 15 will
be permanently or removeably attached to a detector 20, as
illustrated in FIG. 4. The detector may comprise one or more lenses
for focusing and enhancement of an optical signal transmitted along
the optical fibers comprising the optical fiber array 14. The
detector may additionally comprise a device for transforming the
optical signal into a digital or analog electrical signal.
Preferred detectors include phototubes (photomultipliers) or charge
coupled devices (CCDs). A single photomultiplier or CCD element may
be arranged to measure the aggregate signal provided by the entire
transmission face 15 of the biosensor. Alternatively, a CCD (or
other) camera may be focused at the transmission face of the
biosensor to simultaeously read signals from all of the optical
fibers while permitting individual evaluation of the signal from
each fiber or group of fibers. In another embodiment, multiple CCD
elements or phototubes are used to each detect a signal
representing binding of a single species of biological binding
partner present at the sensor face 13 of the biosensor. Thus the
detector is preferably arranged to read the signal from single
optical fibers 10 or from groups of optical fibers where all of the
optical fibers 10 in a group bear the same species of biological
binding partner.
[0056] In addition to detecting optical signals from a fiber optic
array, the detector 20, may be generally used to amplify and detect
optical signals from any array of light sources. Thus, for example,
the array of light sources may be an array of fluorescent spots as
due to hybridization of fluorescently labeled probes hybridized to
arrays of target nucleic acids. Similarly, the array may be of
fluorescently labeled antibodies bound to an array of proteins to
which the antibodies bind, or conversely fluorescently labeled
proteins bound to an array of antibodies.
[0057] In a preferred embodiment suitable for such applications,
illustrated in FIGS. 7A and 7B, the detector 20 may comprise a
compound objective lens 31 that consists of an array of single
lenses 32. The single lenses are spaced so that each lens is
focused on a location 34 in the array where fluorescence is to be
measured. The detector may optionally include a beam splitter 35 a
second lens 36 an optical filter 37 and a detection device such as
a camera 38. The beam splitter is then used to direct an excitation
illumination 39 upon the array of light sources. The resulting
fluorescence at each spot is then focused through the compound
objective lens 31 optionally focused by a second lens, optionally
filtered by an optical filter and then detected either visually or
by a detection means such as a camera.
[0058] The compound objective may be cast, pressed, etched, or
ground out of glass, plastic, quartz, or other materials well known
as suitable for lens manufacture. The compound lens may be formed
as a single piece, or alternatively may be assembled by attaching
together simple lenses to form a compound objective.
II. Fabrication of the Biosensor
[0059] FIGS. 1-4 illustrate a method of fabrication of a biosensor
comprising a plurality of optical fibers bearing biological binding
partners. In general the method involves providing a plurality of
optical fibers with each fiber having a sensor end and a
transmission end with a particular species of biological binding
partner attached to the sensor end of each fiber. Fibers with
differing binding partners are combined to form an optical fiber
array wherein said fibers have commonly aligned sensor ends for
simultaneous assay of a sample. The transmission ends of the
combined discrete fibers are addressed for interrogation to produce
the fiber optic sensor.
[0060] FIG. 1, illustrates a particularly preferred embodiment that
details one method of providing the optical fibers with attached
biolgocial binding partners. A plurality of optical fibers 10 are
provided, each fiber having a sensor end 11 and a transmission end
12. The fibers are arranged together to form a plurality of fiber
groups or bundles 16, as shown in FIG. 2, with the fibers in each
bundle disposed coaxially alongside each other with the sensor ends
11 of each fiber commonly aligned at the same end of the bundle.
The fibers comprising each bundle may be optionally marked 17 to
permit their identification when subsequently removed from the
bundle.
[0061] As shown in FIG. 2, each bundle of fibers is separately
treated to attach a particular species of biological binding
partner 18 to the sensor ends 11 of the optical fibers comprising
the particular bundle. Many methods for immobilizing biological
binding partners 18 on a variety of solid surfaces are known in the
art. In general, the desired component may be covalently bound or
noncovalently attached through nonspecific binding.
[0062] In preparing the sensor end 11 for attachment of the binding
partner, a plurality of different materials may be employed,
particularly as laminates, to obtain various properties. For
example, proteins (e.g., bovine serum albumin) or mixtures of
macromolecules (e.g., Denhardt's solution) can be employed to avoid
non-specific binding, simplify covalent conjugation, enhance signal
detection or the like.
[0063] If covalent bonding between a biological binding partner and
the surface of the sensor end 11 is desired, the surface will
usually be polyfunctional or be capable of being
polyfunctionalized. Functional groups which may be present on the
surface and used for linking can include carboxylic acids,
aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl
groups, mercapto groups and the like.
[0064] Covalent linkage of the binding partner to the sensor end
may be direct or through a covalent linker. Generally linkers are
either hetero- or homo-bifunctional molecules that contain two or
more reactive sites that may each form a covalent bond with the
respective binding partner. Linkers suitable for joining biological
binding partners are well known to those of skill in the art. For
example, biological binding partners may be joined by a peptide
linker, by a straight or branched chain carbon chain linker, or by
a heterocyclic carbon. Heterobifunctional cross linking reagents
such as active esters of N-ethylmaleimide have been widely used.
See, for example, Lerner et al. Proc. Nat. Acad. Sci. (USA), 78:
3403-3407 (1981) and Kitagawa et al. J. Biochem., 79: 233-236
(1976), which are incorporated herein by reference.
[0065] The manner of linking a wide variety of compounds to various
surfaces is well known and is amply illustrated in the literature.
Proteins, for example, may be joined to linkers or to functional
groups on the sensor end 11 by coupling through their amino or
carboxyl termini, or through side groups of various constituent
amino acids. Thus, coupling through a disulfide linkage to a
cystein is common.
[0066] Similarly, methods for immobilizing nucleic acids by
introduction of various functional groups to the molecules is known
(see, e.g., Bischoff et al., Anal. Biochem. 164:336-344 (1987);
Kremsky et al., Nuc. Acids Res. 15:2891-2910 (1987) which are
incorporated herein by reference). Modified nucleotides can be
placed on the target using PCR primers containing the modified
nucleotide, or by enzymatic end labeling with modified
nucleotides.
[0067] Referring to FIG. 3, after the biological binding partners
18 are attached to the sensor faces 11 of the optical fibers 10
comprising each bundle, individual fibers, or groups of fibers, are
separated from each bundle. In FIG. 3, only four fiber bundles
F.sub.1-F.sub.4 are illustrated. In the process of being separated
from each of the respective fiber bundles F.sub.1-F.sub.4 are
individual fibers 10.sub.a-10.sub.d. These respective fibers are
being regrouped into optical fiber array 14.
[0068] The individual fibers or groups of fibers, may be marked
prior to separation from the original bundle to facilitate
identification of the binding partner bound to a particular fiber
or group during later assembly steps. The separated fibers, or
groups of fibers, are recombined with fibers or groups of fibers,
separated from different bundles to form an optical fiber array 14
comprising a plurality of fibers or groups of fibers where each
fiber or group of fibers bears a different species of biological
binding partner.
[0069] FIG. 4 illustrates that members of the optical fiber array
14 are oriented such that the sensor ends 11 of all of the
constituent optical fibers 10 are commonly aligned at the same end
of the optical fiber array 14 thereby forming a sensor face 13. The
fibers may be arranged in a substantially planar configuration or
tiered, as illustrated in FIGS. 6A and 6B.
[0070] The fibers may be bundled at the sensor face 13 in a
substantially random or haphazard manner with the relative location
of a the sensor end 11 of a particular fiber within the sensor face
13 being determined by chance. Alternatively, the fibers may be
positioned within the fiber array in a highly ordered manner such
that the location of any particular optical fiber 10 in the sensor
face 13 is predetermined.
[0071] The transmission ends 12 of the optical fibers comprising
the optical fiber array 14 are addressed to permit interrogation
and detection of binding events to the biological binding partners
attached to the sensor face 13. Addressing is accomplished by any
of a number of means well known to those of skill in the art. In a
preferred embodiment, the transmission ends 12 of individual
optical fibers, or groups of optical fibers all bearing the same
species of biological binding partner, are spatially addressed.
This comprises localizing the optical fibers or bundles of optical
fibers at fixed locations relative to the other optical fibers or
bundles of optical fibers comprising the optical fiber array 14,
see e.g. FIG. 4. Most typically this may be accomplished by
attaching the fiber array to a fiber optic connector and ferrule
(e.g. see AMP, Inc. Harrisburg, Pa.).
[0072] Alternatively, the transmission ends 12, may be addressed by
attaching the transmission end of each optical fiber 10 or bundle
of optical fibers bearing a particular biological binding partner
to an individual detector. Each detector is subsequently known to
be associated with a particular biological binding partner and
there is no need to preserve a fixed spatial relationship between
any of the transmission ends 12.
[0073] Detection of a signal from the biosensor (optical array) may
be accomplished by visual inspection of the transmission face 15 of
the optical fiber array 14 or by the use of one or more detectors
20. As indicated above, the transmission face may be permanently or
removably attached to a single optical lens or system of multiple
optical lenses. The lens or lenses may be arranged to focus an
optical signal from the entire transmission face 15 or from
selected subregions of the transmission face. In a preferred
embodiment, lenses will be arranged to each focus an optical signal
from the portion of the transmission face 15 corresponding to a
single biological binding partner. In the extreme case, the signal
for each optical fiber comprising the optical fiber array 14 will
be individually focused.
[0074] Again with a lens or lens system present, the signal may be
simply detected visually. However, in a preferred embodiment, the
use of detectors is contemplated. Preferred detectors are devices
that convert an optical signal into a digital or analog electrical
signal. Typically detectors are of two general types: phototubes
and charge coupled devices (CCDs). A single photomultiplier or CCD
element may be utilized to measure the aggregate signal provided by
the entire transmission face 15 of the biosensor. More preferably,
however, multiple CCD elements or phototubes are used to each
detect a signal representing binding of a single species of
biological binding partner present at the sensor face 13 of the
optical fiber array 14.
[0075] The detector system may be employed with a computerized data
acquisition system and analytical program. In this embodiment,
providing a fully automated, computer controlled processing
apparatus and measurement system, the data obtained from the
biosensor is processed into immediately useful information. By
using such fully automated, computerized apparatus and analytical
systems, not only are a variety of different measurements made and
diverse parameters measured concurrently within a single fluid
sample, but also many different fluid samples may be analyzed
individually seriatim for detection of multiple analytes of
interest concurrently--each individual fluid sample following its
predecessor in series.
II. Methods of Use
[0076] A variety of in vitro measurements and analytical
determinations may be made using a fiber optic biosensor prepared
in accordance with the present invention. In vitro applications and
assay techniques may be performed concurrently using one or
multiple fluid samples. Each concurrently conducted measurement or
determination for different analytes of interest is made
individually, accurately and precisely. The observed results are
then correlated and/or computed to provide precise information
regarding a variety of different parameters or ligands
individually.
[0077] The fiber optic biosensor of the present invention may also
be employed in a variety of different in vivo conditions with both
humans and animals. The present invention provides accurate and
precise measurements and determinations using a single discrete
fiber optic biosensor rather than the conventional bundle of
different sensors joined together for limited purposes. The present
invention thus provides a minimum-sized diameter sensor for in vivo
catheterization: a minimum intrusion into the bloodstream or
tissues of the living subject for assay purposes, and a minimum of
discomfort and pain to the living subject coupled with a maximum of
accuracy and precision as well as a multiplicity of parameter
measurement in both qualitative and/or quantitative terms.
[0078] The biosensor of the present invention may be utilized for
the detection of a wide variety of analytes, depending on the
particular biological binding partner selected. As indicated above,
biological binding partners are molecules that specifically
recognize and bind other molecules thereby forming a binding
complex. Typical binding complexes include, but are not limited to,
antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid,
biotin-avidin, receptor-receptor ligand, etc. Either member of the
binding complex may be used as the biological binding member
attached to the sensor end 11 of the optical fibers comprising the
biosensor. Thus, for example, where it is desired to detect an
antibody in a sample, the corresponding antigen may be attached to
the sensor end. Conversely, where it is desired to detect the
antigen in the sample, the antibody may be attached to the sensor
end.
[0079] The selection of binding partners for a particular assay is
well known to those of skill in the art. Typically, where proteins
are to be detected, antibodies are most preferred as the biological
binding partner. Where enzymatic substrates are to be detected,
enzymes are preferred biological binding partners, and where
nucleic acids are to be detected, nucleic acid binding partners are
most preferred. Thus, for example, fiber optic biosensors have been
described that utilize enzymes such as xanthine oxidase and
peroxidase to detect hypoxanthine and xanthine (Hlavay et al.,
Biosensors and Bioelectronics, 9(3):189-195 (1994), that use
alkaline phosphatase to detect organophosphorous-based pesticides
(Gao et al. Proceedings--Lasers and Electro-Optics Society, Annula
Meeting, 8(4): abstract 20782 (1994), and that use antibodies or
DNA780 binding proteins (Anderson, et al., Fiber Optic Medical and
Fluorescent Sensors and Applications, Proc. S.P.I.E. 1648: 3943
(1992).
[0080] Of course the biosensor may be designed to simultaneously
detect several different classes of analyte. Thus the sensor may
bear a combination of two or more different classes of biological
binding partner. The sensor face 13 will bear one or more binding
partners selected, for example, from the group consisting of
nucleic acids, proteins, antibodies, carbohydrates, biotin, avidin,
and lectins.
[0081] In the simplest application, the biosensor of the present
invention may be utilized to detect a single analyte in a test
sample. The test sample may be in vivo, in culture, or in vitro.
The assay may register simple presence or absence of the analyte or
may quantify the amount of analyte present in the sample.
[0082] The assay may be run in either a direct or a competitive
format. In a direct format, the amount of analyte is determined
directly measuring the analyte bound to the biological binding
partner. In a competitive format, a known analyte is present in the
sample and the test analyte is detected by its ability to compete
the known analyte from the biological binding partners present on
the sensor face 13.
[0083] In a preferred method of use, the optical fibers 10
comprising the optical fiber array 14 conduct an optical signal
indicative of the binding between a biological binding partner on
the sensor face 11 and the analyte in the sample. The optical
signal may be produced by a number of means known to those of skill
in the art. Typically the optical signal is generated by a
fluorescent, luminescent, or calorimetric label present at the
sensor end 11 of the optical fiber 10. Typically the concentration
of label at the sensor end of the optical fiber is a function of
the concentration of analyte that specifically binds to the
biological binding partner present on that sensor end.
[0084] Methods of providing a label whose concentration is a
function of the amount of an analyte specifically associated with a
biological binding partner are well known to those of skill in the
art. In the simplest approach, the analyte itself is labeled.
Binding of the analyte to the binding partner then brings the label
into proximity with the sensor end 11 to which the binding partner
is attached. Alternatively, a labeled "blocking" analyte may be
provided in the test sample or pre-bound to the biosensor.
Displacement of the labeled "blocking" analyte by the unlabeled
test analyte in the sample produces a reduction of label bound to
the sensor end where the reduction is proportional to the
concentration of unlabeled analyte in the test sample.
[0085] Other approaches may use a second biological binding partner
that itself is labeled. The first biological binding partner
attached to the sensor end binds and thereby immobilizes the
analyte. The second, labelled binding partner then binds to the
analyte immobilized on the sensor end thereby bringing the label in
close proximity to the sensor end where it may be detected.
[0086] Luminescent labels are detected by measuring the light
produced by the label and conducted along the optical fiber.
Luminescent labels typically require no external illumination.
[0087] In contrast colorimetric or fluorescent labels typically
require a light source. Colorimetric labels typically produce an
increase in optical absorbance and/or a change in the absorption
spectrum of the solution. Colorimetric labels are measured by
comparing the change in absorption spectrum or total absorbance of
light produced by a fixed light source. In the present invention,
the change in light absorbance or absorption spectrum is preferably
detected through the optical fibers comprising the biosensor. The
change in absorbance, or absorption spectrum, may be measured as a
change in illumination from an absolutely calibrated light source,
or alteratively may be made relative to a second "reference light
source". The light source may be external to the biosensor or may
be provided as an integral component. In one embodiment, some of
the constituent optical fibers will conduct light from the signal
and/or reference source to the sensor face. For maximum sensitivity
the light used to measure absorbance, or absorption spectrum,
changes will be directed directly at the sensor face of the
biosensor.
[0088] Fluorescent labels produce light in response to excitation
by a light source. The emitted light, characteristically of a
different (lower) wavelength than the excitation illumination, is
detected through the optical fiber to which the fluorescent label
has become bound.
[0089] The excitation illumination may be provided by an integral
component of the biosensor or by a separate light source according
to a number of methods well known to those of skill in the art.
Evanescent wave systems involve introducing a light beam at the
transmission end 12 of the optical fiber. This light beam is
conducted along the fiber until it reaches the sensor end 11 of the
fiber where it generates in the test solution an electromagnetic
waveform known as the evanescent wave component. The evanescent
wave component may be sufficient to excite a fluorophore and
produce a fluorescent signal. (See, for example U.S. Pat. No.
4,447,546 and U.S. Pat. No. 4,909,990 which are incorporated herein
by reference).
[0090] In another embodiment, the excitation illumination is
provided external to the biosensor. It is particularly preferred
that the illumination be provided as a "transillumination" normal
to the sensor ends 11 of the optical fibers (see, e.g. FIG. 4).
This provides an increased signal to noise ratio as, in this
configuration, most of the excitation illumination will not be
conducted along the optical fibers. The individual optical fibers
10 comprising the biosensor may be tiered, for example, as shown in
FIGS. 6A and 6B, to prevent individual fibers from shadowing each
other when transilluminated.
[0091] To optimize a given assay format one of skill can determine
sensitivity of fluorescence detection for different combinations of
optical fiber, sensor face configuration, fluorochrome, excitation
and emission bands and the like. The sensitivity for detection of
analyte by various optical fiber array configurations can be
readily determined by, for example, using the biosensor to measure
a dilution series of fluorescently labeled analytes. The
sensitivity, linearity, and dynamic range achievable from the
various combinations of fluorochrome and biosensor can thus be
determined. Serial dilutions of pairs of fluorochromes in known
relative proportions can also be analyzed to determine the accuracy
with which fluorescence ratio measurements reflect actual
fluorochrome ratios over the dynamic range permitted by the
detectors and biosensor.
Use in Comparative Genomic Hybridization
[0092] In a particularly preferred embodiment, the biosensor of the
present invention will be used in a Comparative Genomic
Hybridization (CGH) assay. Comparative genomic hybridization (CGH)
is a recent approach used to detect the presence and identify the
chromosomal location of amplified or deleted nucleotide sequences.
(See, Kallioniemi et al., Science 258: 818-821 (1992), WO 93/18186,
and copending application U.S. Ser. No. 08/353,018, filed on Dec.
9, 1994, which are incorporated herein by reference).
[0093] In the traditional implementation of CGH, genomic DNA is
isolated from normal reference cells, as well as from test cells
(e.g., tumor cells). The two nucleic acids (DNA) are labelled with
different labels and then hybridized in situ to metaphase
chromosomes of a reference cell. The repetitive sequences in both
the reference and test DNAs may be removed or their hybridization
capacity may be reduced by some means such as an unlabeled blocking
nucleic acid (e.g. Cot-1). Chromosomal regions in the test cells
which are at increased or decreased copy number can be quickly
identified by detecting regions where the ratio of signal from the
two DNAs is altered. For example, those regions that have been
decreased in copy number in the test cells will show relatively
lower signal from the test DNA than the reference compared to other
regions of the genome. Regions that have been increased in copy
number in the test cells will show relatively higher signal from
the test DNA.
[0094] In one embodiment, the present invention provides a CGH
assay in which the biosensor of the present invention replaces the
metaphase chromosome used as the hybridization target in
traditional CGH. Instead, the biological binding partners present
on the biosensor are nucleic acid sequences selected from different
regions of the genome. The biosensor itself becomes a sort of
"glass chromosome" where hybridization of a nucleic acid to a
particular binding partner is informationally equivalent to
hybridization of that nucleic acid to the region on a metaphase
chromosome from which the biological binding partner is derived. In
addition, nucleic acid binding partners not normally contained in
the genome, for example virus nucleic acids, can be employed.
[0095] More particularly, in a CGH assay, the biosensor may be
utilized in methods for quantitatively comparing copy numbers of at
least two nucleic acid sequences in a first collection of nucleic
acid molecules relative to the copy numbers of those same sequences
in a second collection, as illustrated in FIG. 5. The method
comprises labeling the nucleic acid molecules in the first
collection 25 and the nucleic acid molecules in the second
collection 26 with first and second labels, respectively thereby
forming at least two collections of nucleic acid probes. The first
and second labels should be distinguishable from each other.
[0096] As used herein, the term "probe" is thus defmed as a
collection of nucleic acid molecules (either RNA or DNA) capable of
binding to a target nucleic acid of complementary sequence through
one or more types of chemical bonds, usually through hydrogen bond
formation. The probes are preferably directly or indirectly
labelled as described below. They are typically of high complexity,
for instance, being prepared from total genomic DNA or MRNA
isolated from a cell or cell population.
[0097] The probes 30 thus formed are contacted, either
simultaneously or serially, to a plurality of target nucleic acids,
present on the sensor face 13 of the biosensor of array 14 under
conditions such that nucleic acid hybridization to the target
nucleic acids can occur. Here a tranilluminating light source 19 is
utilized. After contacting the probes to the target nucleic acids
the amount of binding of each probe, and ratio of the binding of
the probes is determined for each species of target nucleic acid.
Typically the greater the ratio of the binding to a target nucleic
acid, the greater the copy number ratio of sequences in the two
probes that bind to nucleic acid. Thus comparison of the ratios of
bound labels among target nucleic acid sequences permits comparison
of copy number ratios of different sequences in the probes.
[0098] In a preferred embodiment, the sequence complexity of each
target nucleic acid in the biosensor is much less than the sequence
complexity of the first and second collections of labeled nucleic
acids. The term "complexity" is used here according to standard
meaning of this term as established by Britten et al. Methods of
Enzymol. 29:363 (1974). See, also Cantor and Schimmel Biophysical
Chemistry: Part III at 1228-1230 for further explanation of nucleic
acid complexity.
[0099] The methods are typically carried out using techniques
suitable for fluorescence in situ hybridization. Thus, the first
and second labels are usually fluorescent labels.
[0100] To inhibit hybridization of repetitive sequences in the
probes to the target nucleic acids, unlabeled blocking nucleic
acids (e.g., Cot-1 DNA) can be mixed with the probes. Thus, the
invention focuses on the analysis of the non-repetitive sequences
in a genome. However, use of repetitive sequences as targets on the
biosensor and omiting the blocking nucleic acids would permit
relative copy number determinations to be made for repetitive
sequences.
[0101] In a typical embodiment, one collection of probe nucleic
acids is prepared from a test cell, cell population, or tissue
under study; and the second collection of probe nucleic acids is
prepared from a reference cell, cell population, or tissue.
Reference cells can be normal non-diseased cells, or they can be
from a sample of diseased tissue that serves as a standard for
other aspects of the disease. For example, if the reference probe
is genomic DNA isolated from normal cells, then the copy number of
each sequence in that probe relative to the others is known (e.g.,
two copies of each autosomal sequence, and one or two copies of
each sex chromosomal sequence depending on gender). Comparison of
this to a test probe permits detection in variations from normal.
Alternatively the reference probe may be prepared from genomic DNA
from a primary tumor which may contain substantial variations in
copy number among its different sequences, and the test probe may
prepared from genomic DNA of metastatic cells from that tumor, so
that the comparison shows the differences between the primary tumor
and its metastasis. Further, both probes may be prepared from
normal cells. For example comparison of mRNA populations between
normal cells of different tissues permits detection of differential
gene expression that is a critical feature of tissue
differentiation. Thus in general the terms test and reference are
used for convenience to distinguish the two probes, but they do not
imply other characteristics of the nucleic acids they contain.
Target Nucleic Acids
[0102] The target nucleic acids comprising the biological binding
partners attached to the sensor ends 11 of the optical fibers 10
and the probes may be, for example, RNA, DNA, or cDNA. The nucleic
acids may be derived from any organism. Usually the nucleic acid in
the target sequences and the probes are from the same species.
[0103] The "target nucleic acids" comprising biological binding
partners typically have their origin in a defmed region of the
genome (for example a clone or several contiguous clones from a
genomic library), or correspond to a functional genetic unit, which
may or may not be complete (for example a full or partial cDNA).
The target nucleic acids can also comprise inter-Alu or Degenerate
Oligonucleotide Primer PCR products derived from such clones. If
gene expression is being analyzed, a target element can comprise a
full or partial cDNA.
[0104] The target nucleic acids may, for example, contain specific
genes or, be from a chromosomal region suspected of being present
at increased or decreased copy number in cells of interest, e.g.,
tumor cells. The target nucleic acid may also be an mRNA, or cDNA
derived from such mRNA, suspected of being transcribed at abnormal
levels.
[0105] Alternatively, target nucleic acids may comprise nucleic
acids of unknown significance or location. The array of such target
nucleic acids comprising the sensor face 13 of a biosensor of the
present invention could represent nucleic acids derived from
locations that sample, either continuously or at discrete points,
any desired portion of a genome, including, but not limited to, an
entire genome, a single chromosome, or a portion of a chromosome.
The number of target elements and the complexity of the nucleic
acids in each would determine the density of sampling. For example
an biosensor bearing 300 different species of target nucleic acid
(biological binding partners), each target nucleic acid being DNA
from a different genomic clone, could sample the entire human
genome at 10 megabase intervals. An array of 30,000 elements, each
containing 100 kb of genomic DNA could give complete coverage of
the human genome.
[0106] Similarly, an array of target nucleic acids comprising
nucleic acids from anonymous cDNA clones would permit
identification of those that might be differentially expressed in
some cells of interest, thereby focusing attention on study of
these genes.
[0107] In some embodiments, previously mapped clones from a
particular chromosomal region of interest are used as targets. Such
clones are becoming available as a result of rapid progress of the
worldwide initiative in genomics.
[0108] Mapped clones can be prepared from libraries constructed
from single chromosomes, multiple chromosomes, or from a segment of
a chromosome. Standard techniques are used to clone suitably sized
fragments in vectors such as cosmids, yeast artificial chromosomes
(YACs), bacterial artificial chromosomes (BACs) and P1 phage.
[0109] While it is possible to generate clone libraries, as
described above, libraries spanning entire chromosomes are also
available commercially. For instance, chromosome-specific libraries
from the human and other genomes are available from Clonetech
(South San Francisco, Calif.) or from The American Type Culture
Collection (see, ATCC/NIH Repository of Catalogue of Human and
Mouse DNA Probes and Libraries, 7th ed. 1993).
[0110] If necessary, clones described above may be genetically or
physically mapped. For instance, FISH and digital image analysis
can be used to identify and map the locations on a chromosome to
which specific cosmid inserts hybridize. This method is described,
for instance, in Lichter et al., Science, 247:64-69 (1990). The
physically mapped clones can then be used to more finally map a
region of interest identified using CGH or other methods.
[0111] One of skill will recognize that each target nucleic acids
may be selected so that a number of nucleic acids of different
length and sequence represent a particular region on a chromosome.
Thus, for example, a the sensor face 13 of the biosensor may bear
more than one copy of a cloned piece of DNA, and each copy may be
broken into fragments of different lengths. One of skill can adjust
the length and complexity of the target sequences to provide
optimum hybridization and signal production for a given
hybridization procedure, and to provide the required resolution
among different genes or genomic locations. Typically, the target
sequences will have a complexity between about 1 kb and about 1
Mb.
Preparation of Probe Nucleic Acids
[0112] As with target nucleic acids (those attached to the fiber
optic sensor), a wide variety of nucleic acids can be used as probe
nucleic acids in the methods of the present invention. The probes
may be comprise, for example, genomic DNA representing the entire
genome from a particular organism, tissue or cell type or may
comprise a portion of the genome, such as a single chromosome.
[0113] To compare expression levels of a particular gene or genes,
the probe nucleic acids can be derived from mRNA or cDNA prepared
from an organism, tissue, or cell of interest. For instance, test
cDNA or MRNA, along with MRNA or cDNA from normal reference cells,
can be hybridized to an array of target nucleic acids on the sensor
comprising clones from a normalized cDNA library. In addition,
probes made from genomic DNA from two cell populations can be
hybridized to a target cDNA array to detect those cDNAs that come
from regions of variant DNA copy number in the genome.
[0114] The methods of the invention are suitable for comparing copy
number of particular sequences in any combination of two or more
populations of nucleic acids. One of skill will recognize that the
particular populations of sample nucleic acids being compared is
not critical to the invention. For instance, genomic or cDNA can be
compared from two related species. Alternatively, levels of
expression of particular genes in two or more tissue or cell types
can be compared. As noted above, the methods are particularly
useful in the diagnosis of disease.
[0115] Standard procedures can be used to isolate nucleic acids
(either DNA or MRNA) from appropriate tissues (see, e.g., Sambrook,
et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1985)). Conventional methods
for preparation of cDNA from mRNA can also be used.
[0116] The particular cells or tissue from which the nucleic acids
are isolated will depend upon the particular application.
Typically, for detection of abnormalities associated with cancer,
genomic DNA is isolated from tumor cells. For prenatal detection of
disease, fetal tissue will be used.
[0117] If the tissue sample is small, so that a small amount of
nucleic acids is available, amplification techniques such as the
polymerase chain reaction (PCR) using degenerate primers can be
used. For a general description of PCR, see, PCR Protocols, Innis
et al. eds. Academic Press, 1990. In addition, PCR can be used to
selectively amplify sequences between high copy repetitive
sequences. These methods use primers complementary to highly
repetitive interspersed sequences (e.g., Alu) to selectively
amplify sequences that are between two members of the Alu family
(see, Nelson et al., Proc. Nati. Acad. Sci. USA 86:6686
(1989)).
[0118] CGH, at the cytogenetic level, facilitates the search for
disease genes by identifying regions of differences in copy number
between a normal and tumor genome, for example. For instance, CGH
studies have been applied to the analysis of copy number variation
in breast cancer (see, e.g., Kallioniemi et al. Proc. Natl. Acad.
Sci. USA 91:2156-2160 (1994)).
[0119] In CGH, the resolution with which a copy number change can
be mapped is on the order of several megabases. With the present
invention the resolution is a function of the length of the genomic
DNA segments comprising the target nucleic acid sequences and the
difference in map position between neighboring clones. Resolution
of more than a factor of 10 better than with standard CGH can be
achieved with the present invention. This improved localization
will facilitate efforts to identify the critical genes involved in
a disease, and permit more sensitive detection of abnormalities
involving a small region of the genome, such as in microdeletion
syndromes.
Labeling Nucleic Acid Probes
[0120] As noted above, the nucleic acids which are hybridized to
the target nucleic acids are preferably labeled to allow detection
of hybridization complexes. The nucleic acid probes used in the
hybridization described below may be detectably labeled prior to
the hybridization reaction. Alternatively, a detectable label may
be selected which binds to the hybridization product. As noted
above, the target nucleic acid array is hybridized to two or more
probe nucleic acids, either simultaneously or serially. Thus, the
probes are each preferably labeled with a separate and
distinguishable label.
[0121] The particular label or detectable group attached to the
probe nucleic acids is selected so as to not significantly
interfere with the hybridization of the probe to the target
sequence. The detectable group can be any material having a
detectable physical or chemical property. Such detectable labels
have been well-developed in the field of nucleic acid
hybridizations and in general most any label useful in such methods
can be applied to the present invention. Thus a label is any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical
means.
[0122] However, preferred labels produce an optical signal. Thus,
particularly useful labels in the present invention include
fluorescent dyes (e.g., fluorescein isothiocyanate, texas red,
rhodamine, and the like) and labels that produce a colorimetric
signal such as various enzymes (e.g., horse radish peroxidase,
alkaline phosphatase and others commonly used in an ELISA).
[0123] The nucleic acids can be indirectly labeled using ligands
for which detectable anti-ligands are available. For example,
biotinylated nucleic acids can be detected using labeled avidin or
streptavidin according to techniques well known in the art. In
addition, antigenic or haptenic molecules can be detected using
labeled antisera or monoclonal antibodies. For example,
N-acetoxy-N-2-acetylaminofluorene-labelled or digoxigenin-labelled
probes can be detected using antibodies specifically immunoreactive
with these compounds (e.g., FITC-labeled sheep anti-digoxigenin
antibody (Boehringer Mannheim)). In addition, labeled antibodies to
thymidine-thymidine dimers can be used (Nakane et al. ACTA
Histochem. Cytochem. 20:229 (1987)).
[0124] Generally, labels which are detectable in as low a copy
number as possible, thereby maximizing the sensitivity of the
assay, and yet be detectable above any background signal are
preferred. A label is preferably chosen that provides a localized
signal, thereby providing spatial resolution of the signal from
each target element.
[0125] The labels may be coupled to the DNA in a variety of means
known to those of skill in the art. In a preferred embodiment the
probe will be labeled using nick translatiotor random primer
extension (Rigby, et al. J. Mol. Biol., 113: 237 (1977) or
Sambrook, et al., Molecular Cloning--A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1985)).
Hybridization of Labeled Nucleic Acids to Targets
[0126] The copy number of particular nucleic acid sequences in two
probes are compared by hybridizing the probes to one or more target
nucleic acid arrays (biosensors). The hybridization signal
intensity, and the ratio of intensities, produced by the probes on
each of the target elements is determined. Typically the greater
the ratio of the signal intensities on a target nucleic acid the
greater the copy number ratio of sequences in the two probes that
bind to that target sequence. Thus comparison of the signal
intensity ratios among target elements permits comparison of copy
number ratios of different sequences in the probes.
[0127] Standard hybridization techniques are used to probe a target
nucleic acid array. Suitable methods are described in references
describing CGH techniques (Kallioniemi et al., Science 258: 818-821
(1992) and WO 93/18186). Several guides to general techniques are
available, e.g., Tijssen, Hybridization with Nucleic Acid Probes,
Parts I and II (Elsevier, Amsterdam 1993). For a description of
techniques suitable for in situ hybridizations see, Gall et al.
Meth. Enzymol., 21:470-480 (1981) and Angerer et al. in Genetic
Engineering: Principles and Methods Setlow and Hollaender, Eds. Vol
7, pgs 43-65 (plenum Press, N.Y. 1985).
[0128] Generally, nucleic acid hybridizations utilizing the
biosensors of the present invention comprise the following major
steps: (1) prehybridization treatment to increase accessibility of
target DNA, and to reduce nonspecific binding; (2) hybridization of
the mixture of nucleic acids to the nucleic acid targets on the
biosensor; (3) posthybridization washes to remove nucleic acid
fragments not bound in the hybridization and (4) detection of the
hybridized nucleic acid fragments. The reagent used in each of
these steps and their conditions for use vary depending on the
particular application.
[0129] In some applications it is necessary to block the
hybridization capacity of repetitive sequences. A number of methods
for removing and/or disabling the hybridization capacity of
repetitive sequences are known (see, e.g., WO 9/18186).
[0130] For instance, bulk procedures can be used. In many genomes,
including the human geome, a major portion of shared repetitive DNA
is contained within a few families of highly repeated sequences
such as Alu. These methods exploit the fact that hybridization rate
of complementary sequences increases as their concentration
increases. Thus, repetitive sequences, which are generally present
at high concentration will become double stranded more rapidly than
others following denaturation and incubation under hybridization
conditions. The double stranded nucleic acids are then removed and
the remainder used in hybridizations. Methods of separating single
from double stranded sequences include using hydroxyapatite or
immobilized complementary nucleic acids attached to a solid
support. Alternatively, the partially hybridized mixture can be
used and the double stranded sequences will be unable to hybridize
to the target.
[0131] Alternatively, unlabeled sequences which are complementary
to the sequences whose hybridization capacity is to be inhibited
can be added to the hybridization mixture. This method can be used
to inhibit hybridization of repetitive sequences as well as other
sequences. For instance, "Cot-1" DNA can be used to selectively
inhibit hybridization of repetitive sequences in a sample. To
prepare Cot-1 DNA, DNA is extracted, sheared, denatured and
renatured to a C.sub.0t-1 (for description of reassociation
kinetics and C.sub.0t values, see, Tijssen, supra at pp 48-54).
Because highly repetitive sequences reanneal more quickly, the
resulting hybrids are highly enriched for these sequences. The
remaining single stranded (i.e., single copy sequences) is digested
with S1 nuclease and the double stranded Cot-1 DNA is purified and
used to block hybridization of repetitive sequences in a sample.
Although Cot-1 DNA can be prepared as described above, it is also
commercially available (BRL). Reassociation to large C.sub.0t
values will result in blocking DNA containing reptitive sequences
that are present at lower copy number.
Analysis of Detectable Signals from Hybridizations
[0132] Standard methods for detection and analysis of signals
generated by labeled probes can be used. In particular, the optical
signal produced by binding of a labeled probe to a particular
binding partner will be carried along the optical fibers 10, to
which that binding partner is attached. As indicated above, the
optical signal may be visualized directly or transduced into an
analog or digital electronic signal by means of a detector 20. To
facilitate the display of results and to.improve the sensitivity of
detecting small differences in fluorescence intensity, a detector
and a digital signal analysis system is preferably used. The
detector may be equipped with one or more filters to pass the
emission wavelengths while filtering out excitation wavelengths
thereby increasign the signal to noise ratio. The use of filters
will also facilitate distinguishing between binding events
involving the two, or more, differently labeled probes. Such
detector/filter/signal processing systems are well known to those
of skill in the art.
* * * * *