U.S. patent application number 10/238284 was filed with the patent office on 2003-02-13 for test systems and sensors for detecting molecular binding events.
This patent application is currently assigned to Signature BioScience Inc.. Invention is credited to Hefti, John.
Application Number | 20030032067 10/238284 |
Document ID | / |
Family ID | 23441192 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032067 |
Kind Code |
A1 |
Hefti, John |
February 13, 2003 |
Test systems and sensors for detecting molecular binding events
Abstract
A bio-assay test system includes a test fixture, a measurement
system, and a computer. The test fixture includes a bio-assay
device having a signal path and a retaining structure configured to
place a sample containing molecular structures in electromagnetic
communication with the signal path. The measurement system is
configured to transmit test signals to and to receive test signals
from the signal path at one or more predefined frequencies. The
computer is configured to control the transmission and reception of
the test signals to and from the measurement system.
Inventors: |
Hefti, John; (San Francisco,
CA) |
Correspondence
Address: |
Richard L. Neeley
Kelvan Patrick Howard
Signature BioScience, Inc.
475 Brannan Street
San Francisco
CA
94107
US
|
Assignee: |
Signature BioScience Inc.
1450 Rollins Road
Burlingame
CA
94010
|
Family ID: |
23441192 |
Appl. No.: |
10/238284 |
Filed: |
September 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10238284 |
Sep 10, 2002 |
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09365978 |
Aug 2, 1999 |
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6485905 |
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09365978 |
Aug 2, 1999 |
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09243194 |
Feb 1, 1999 |
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6368795 |
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60073445 |
Feb 2, 1998 |
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Current U.S.
Class: |
435/7.9 ;
205/777.5; 435/287.2; 435/6.1; 435/6.18; 702/19 |
Current CPC
Class: |
G01N 33/54373 20130101;
B82Y 10/00 20130101; Y10S 436/805 20130101; H01J 49/04 20130101;
G01N 33/5438 20130101; C12Q 1/001 20130101; Y10S 436/806
20130101 |
Class at
Publication: |
435/7.9 ;
435/287.2; 702/19; 205/777.5; 435/6 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/542; G06F 019/00; G01N 033/48; G01N 033/50; C12M
001/34 |
Claims
What is claimed is:
1. A bio-assay test system comprising: a test fixture comprising: a
bio-assay device comprising a signal path; and a retaining
structure configured to place a sample comprising molecular
structures in electromagnetic communication with the signal path; a
measurement system configured to transmit test signals to and
receive test signals from the signal path at one or more predefined
frequencies; a computer coupled to the measurement system
configured to control the transmission and reception of the test
signals to and from the measurement system.
2. The single path test system of claim 1, wherein the measurement
system comprises a vector network analyzer configured to compare
the magnitude and phase response of the received test signal to the
magnitude and phase response of the transmitted test signal.
3. The single path test system of claim 2, wherein the test signals
comprise signals in the range of 5 Hz to 300 MHz.
4. The single path test system of claim 2, wherein the test signals
comprise signals in the range of 45 MHz to 40 GHz.
5. The single path test system of claim 2, wherein the test signals
comprise signals in the range of 33 GHz to 110 GHz.
6. The single path test system of claim 2, wherein the bio-assay
device comprises a transmission line.
7. The single path test system of claim 2, wherein the bio-assay
device comprises a meandered transmission line.
8. The single path test system of claim 2, wherein the bio-assay
device comprises a ring resonator circuit.
9. The single path test system of claim 2, wherein the bio-assay
device comprises a capacitive gap circuit.
10. The single path test system of claim 2, wherein the bio-assay
device comprises a dielectric signal path.
11. The single path test system of claim 2, wherein the retaining
structure comprises an O-ring removeably compressed around a
portion of the signal path, the O-ring configured to hold the
sample solution in contact with the signal path.
12. The single path test system of claim 2, further comprising: an
input connector coupled between the measurement system and a first
port of the signal path; and an output connector coupled between
the measurement system and a second port of the signal path.
13. A bio-assay array test system, comprising: a test fixture
comprising: a bio-assay device comprising a plurality of signal
paths; and a plurality of retaining structures configured to place
a sample comprising molecular structures in electromagnetic
communication with each of the plurality of signal paths; a
measurement system having at least one output port configured to
transmit test signals to and at least one input port configured to
receive test signals from one or more of the plurality of signal
paths at one or more predefined frequencies; and a computer coupled
to the measurement system and configured to control the
transmission and reception of the test signals to and from the
measurement system.
14. The bio-assay array test system of claim 13, wherein the
measurement system comprises one output port and one input port,
and wherein the bio-assay array comprises N input ports coupled to
the plurality of signal paths and M output ports coupled to the
plurality of signal paths, the bio-assay system further comprising:
a 1.times.N input switch having an input coupled to the measurement
system output port and an output coupled to the N signal path input
ports; and a M.times.1 output switch having an input coupled to the
M signal path output ports and an output coupled to the measurement
system input port.
15. The bio-assay array test system of claim 13, wherein each of
the plurality of bio-assay arrays comprises a transmission
line.
16. The bio-assay array test system of claim 13, wherein at least
one of the plurality of bio-assay arrays comprises a meandered
transmission line.
17. The bio-assay array test system of claim 13, wherein at least
one of the plurality of bio-assay arrays comprises a ring resonator
circuit.
18. The bio-assay array test system of claim 13, wherein at least
one of the plurality of bio-assay arrays comprises a capacitive gap
circuit.
19. The bio-assay array test system of claim 13, wherein at least
one of the plurality of bio-assay arrays comprises a dielectric
signal path.
20. The bio-assay array test system of claim 13, wherein at least
one of the plurality of bio-assay arrays comprises an
electronically switched transistor.
21. The bio-assay array test system of claim 13, wherein at least
one of the plurality of bio-assay arrays comprises an optically
switched transistor.
22. The bio-assay array test system of claim 13, wherein the test
signals comprise signals in the range of 5 Hz to 300 MHz.
23. The bio-assay array test system of claim 13, wherein the test
signals comprise signals in the range of 45 MHz to 40 GHz.
24. The bio-assay array test system of claim 13, wherein the test
signals comprise signals in the range of 30 GHz to 110 GHz.
25. A bio-assay device, comprising a signal path having an input
port and an output port; and a retaining structure configured to
place a sample comprising molecular structures in electromagnetic
communication with at least a portion of the signal path.
26. The bio-assay device of claim 25, wherein the signal path
comprises a continuous transmission line.
27. The bio-assay device of claim 25, wherein the signal path
comprises a meandered continuous transmission line.
28. The bio-assay device of claim 25, wherein the signal path
comprises a resonant cavity circuit.
29. The bio-assay device of claim 25, wherein the signal path
comprises a capacitive gap circuit.
30. The bio-assay device of claim 25, wherein the signal path
comprises a dielectric signal path.
31. A bio-assay array device, comprising a plurality of signal
paths, each having an input port and an output port; and a
respective plurality of retaining structures configured to place a
sample comprising molecular structures in electromagnetic
communication with at least a portion of each of the plurality of
signal paths.
32. The bio-assay array device of claim 31, wherein each signal
path comprises an electrically-switched transistor.
33. The bio-assay array device of claim 31, wherein each signal
path comprises an optically-switched transistor.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. application Ser. No. 09/243,194, entitled "Method and
Apparatus for Detecting Molecular Binding Events," filed Feb. 1,
1999, which claims the benefit of U.S. Provisional Application No.
60/073,445, entitled "Detection of Molecular Binding Events on a
Conductive Surface," filed Feb. 2, 1998.
[0002] Further, the following applications are herein incorporated
by reference in their entirety for all purposes:
[0003] "A Sensitive Detection of Dispersions in Aqueous-based,
Surface-bound Macromolecular Structures Using Microwave
Spectroscopy," Ser. No. 60/134,740, filed May 18, 1999;
[0004] "Methods of Nucleic Acid Analysis," Atty Docket
019501-000600, filed concurrently herewith; and
[0005] Methods for Analyzing Protein Binding Events," Atty Docket
019501-000700, also filed currently herewith.
BACKGROUND
[0006] Virtually every area of the biomedical sciences is in need
of a system to assay chemical and biochemical reactions and
determine the presence and quantity of particular analytes. This
need ranges from the basic science research lab, where biochemical
pathways are being mapped out and their functions correlated to
disease processes, to clinical diagnostics, where patients are
routinely monitored for levels of clinically relevant analytes.
Other areas include pharmaceutical research and drug discovery
applications, DNA testing, military applications such as biowarfare
monitoring, veterinary, food, and environmental applications. In
all of these cases, the presence and quantity of a specific analyte
or group of analytes, needs to be determined.
[0007] For analysis in the fields of pharmacology, genetics,
chemistry, biochemistry, biotechnology, molecular biology and
numerous others, it is often useful to detect the presence of one
or more molecular structures and measure interactions between
molecular structures. The molecular structures of interest
typically include, but are not limited to, cells, antibodies,
antigens, metabolites, proteins, drugs, small molecules, enzymes,
nucleic acids, and other ligands and analytes. In medicine, for
example, it is very useful to determine the existence of a cellular
constituents such as receptors or cytokines, or antibodies and
antigens which serve as markers for various disease processes,
which exists naturally in physiological fluids or which has been
introduced into the system. In genetic analyses, fragment DNA and
RNA sequence analysis is very useful in diagnostics, genetic
testing and research, agriculture, and pharmaceutical development.
Because of the rapidly advancing state of molecular cell biology
and understanding of normal and diseased systems, there exists an
increasing need for methods of detection, which do not require
labels such as fluorophores or radioisotopes, are quantitative and
qualitative, specific to the molecule of interest, highly sensitive
and relatively simple to implement. Many known targets such as
orphan drug receptors, and many more targets becoming available,
have no known affinity ligands, so that unlabeled means of
detecting molecular interactions are highly desirable. In addition,
the reagent costs for many labeled assay technologies are quite
expensive, in addition to the economic and environmental costs of
disposing of toxic fluorophores and radioisotopes.
[0008] Numerous methodologies have been developed over the years to
meet the demands of these fields, such as Enzyme-Linked
Immunosorbent Assays (ELISA), Radio-Immunoassays (RIA), numerous
fluorescence assays, mass spectroscopy, colorimetric assays, gel
electrophoresis, as well as a host of more specialized assays. Most
of these assay techniques require specialized preparations,
especially attaching a label or greatly purifying and amplifying
the sample to be tested. To detect a binding event between a ligand
and an antiligand, a detectable signal is required which relates to
the existence or extension of binding. Usually the signal is
provided by a label that is conjugated to either the ligand or
antiligand of interest. Physical or chemical effects which produce
detectable signals, and for which suitable labels exist, include
radioactivity, fluorescence, chemiluminescence, phosphorescence and
enzymatic activity to name a few. The label can then be detected by
spectrophotometric, radiometric, or optical tracking methods.
Unfortunately, in many cases it is difficult or even impossible to
label one or all of the molecules needed for a particular assay.
Also, the presence of a label may make the molecular recognition
between two molecules not function for many reasons including
steric effects. In addition, none of these labeling approaches
determines the exact nature of the binding event, so for example
active site binding to a receptor is indistinguishable from
non-active-site binding such as allosteric binding, and thus no
functional information is obtained via the present detection
methodologies. Therefore, a method to detect binding events that
both eliminates the need for the label as well as yields functional
information would greatly improve upon the above mentioned
approaches.
[0009] Other approaches for studying biochemical systems have used
various types of dielectric measurements to characterize certain
classes of biological systems such as tissue samples and cellular
systems. In the 1950's, experiments were conducted to measure the
dielectric properties of biological tissues using standard
techniques for the measurement of dielectric properties of
materials known at the time. Since then various approaches to
carrying out these measurements have included frequency domain
measurements, and time domain techniques such as Time Domain
Dielectric Spectroscopy. In these approaches, the experiments were
commonly carried out using various types of coaxial transmission
lines, or other transmission lines and structures of typical use in
dielectric characterization of materials. This included studies to
look at the use and relevance of the dielectric properties of a
broad range of biological systems: The interest has ranged from
whole tissue samples taken from various organs of mammalian
species, to cellular and sub-cellular systems including cell
membrane and organelle effects. Most recently, there have been
attempts to miniaturize the above-mentioned techniques (see e.g.,
U.S. Pat. Nos. 5,653,939; 5,627,322 and 5,846,708) for improved
detection of changes in the dielectric properties of molecular
systems. These configurations have several drawbacks, including
some substantial limitations on the frequencies useable in the
detection strategy, and a profound limitation on the sensitivity of
detecting molecular systems, as well as being expensive to
manufacture.
[0010] In general, limitations exist in the areas of specificity
and sensitivity of most assay systems. Cellular debris and
non-specific binding often cause the assay to be noisy, and make it
difficult or impossible to extract useful information. As mentioned
above, some systems are too complicated to allow the attachment of
labels to all analytes of interest, or to allow an accurate optical
measurement to be performed. Further, a mentioned above, most of
these detection technologies yield no information on the functional
nature of the binding event. Therefore, a practical and economical
universal enabling which can directly monitor without a label, in
real time, the presence of analytes or the extent, function and
type of binding events and other interactions that are actually
taking place in a given system would represent a significant
breakthrough.
[0011] More specifically, the biomedical industry needs an improved
general platform technology which has very broad applicability to a
variety of water-based or other fluid-based physiological systems,
such as nucleic acid binding, protein-protein interactions, small
molecule binding, as well as other compounds of interest. Ideally,
the assay should not require highly specific probes, such as
specific antibodies and exactly complementary nucleic acid probes;
it should be able to work in native environments such as whole
blood, cytosolic mixtures, as well as other naturally occurring
systems; it should operate by measuring the native properties of
the molecules, and not require additional labels or tracers to
actually monitor the binding event; for some uses it should be able
to provide certain desired information on the nature of the binding
event, such as whether or not a given compound acts as an agonist
or an antagonist on a particular drug receptor, and not function
simply as a marker to indicate whether or not the binding event has
taken place. For many applications, it should be highly
miniaturizable and highly parallel, so that complex biochemical
pathways can be mapped out, or extremely small and numerous
quantities of combinatorial compounds can be used in drug screening
protocols. In many applications, it should further be able to
monitor in real time a complex series of reactions, so that
accurate kinetics and affinity information can be obtained almost
immediately. Perhaps most importantly, for most commercial
applications it should be inexpensive and easy to use, with few
sample preparation steps, affordable electronics and disposable
components, such as surface chips for bio-assays that can be used
for an assay and then thrown away, and be highly adaptable to a
wide range of assay applications.
[0012] It is important to note that other industries have similar
requirements for detection, identification or additional analysis.
While most applications involve the use of biological molecules,
virtually any molecule can be detected if a specific binding
partner is available or if the molecule itself can attach to the
surface as described below.
[0013] The present invention fulfills many of the needs discussed
above and other needs as well.
SUMMARY OF THE INVENTION
[0014] The present invention provides test systems and bio-assay
devices which can be used to detect and identify molecular binding
events. In one embodiment, the invention provides a test system
having a test fixture, a measurement system, and a computer. The
test fixture includes a bio-assay device having a signal path and a
retaining structure configured to place a sample containing
molecular structures in electromagnetic communication with the
signal path. The measurement system is configured to transmit test
signals to and to receive test signals from the signal path at one
or more predefined frequencies. The computer is configured to
control the transmission and reception of the test signals to and
from the measurement system.
[0015] The invention will be better understood when considered in
light of the foregoing drawings and detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates one embodiment of a bio-assay system in
accordance with the present invention.
[0017] FIG. 2 illustrates one possible embodiment of a single path
test system in accordance with the present invention.
[0018] FIGS. 3A-3F illustrate various views of a test fixture in
accordance with the present invention.
[0019] FIG. 4A illustrates a top view of a standard microstrip
transmission line bio-assay for use with the test fixture of FIG.
3.
[0020] FIG. 4B illustrates a top view of a meandered transmission
line bio-assay for use with the test fixture of FIG. 3.
[0021] FIG. 4C illustrates a top view of a ring resonator bio-assay
for use with the test fixture of FIG. 3.
[0022] FIG. 4D illustrates a top view of a capacitive gap bio-assay
for use with the test fixture of FIG. 3.
[0023] FIG. 4E illustrates a side view of a dielectric signal path
bio-assay for use with the test fixture of FIG. 3.
[0024] FIG. 5 illustrates one possible embodiment of an N.times.M
array test system in accordance with the present invention.
[0025] FIGS. 6A-B illustrate various views of an N.times.M array
test fixture in accordance with the present invention.
[0026] FIG. 7A illustrates one embodiment of a bio-assay array in
accordance with the present invention.
[0027] FIG. 7B illustrates one embodiment of an array element in
accordance with the present invention comprising a
series-connected, electronically switched Field Effect
Transistor.
[0028] FIG. 7C illustrates one embodiment of an array element in
accordance with the present invention comprising a
series-connected, optically switched Field Effect Transistor.
[0029] FIG. 7D illustrates one embodiment of an array in accordance
with the present invention comprising two paths of two,
serially-connected FET devices.
[0030] FIG. 7E illustrates the circuit equivalent model of the
array shown in FIG. 7D in accordance with the present
invention.
[0031] FIG. 7F illustrates one embodiment of a two-dimensional
bio-assay array in accordance with the present invention.
[0032] FIG. 8 is an example of the effects of a protein binding
non-specifically to the dielectric signal path of the bio-assay
device illustrated in FIG. 4E.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0033] Table of Contents
[0034] I. Definitions
[0035] II. General Overview
[0036] III. Single Path Test System and Bio-Assays
[0037] A. Test System
[0038] B. Test Fixture
[0039] C. Bio-Assay Devices
[0040] IV. Array Test System and Bio-Assays
[0041] A. Test System
[0042] B. Test Fixture
[0043] C. Bio-Assay Devices
[0044] V. Applications
[0045] A. Drug Discovery Application
[0046] B. Nucleic Acid Chemistry Application
[0047] I. Definition of Terms
[0048] As used herein, the terms biological "binding partners" or
"ligand/antiligand" or "ligand/antiligand complex" refers to
molecules that specifically recognize other molecules to form
proximal complexes such as antibody-antigen, lectin-carbohydrate,
nucleic acid-nucleic acid, protein-protein, protein-small molecule
such as drug-receptor, etc. Biological binding partners need not be
limited to pairs of single molecules. Thus, for example, a single
ligand may be bound by the coordinated action of two or more
"anti-ligands".
[0049] As used herein, the term "ligand" or "analyte" or "marker"
refers to any molecule being detected. It is detected through its
interaction with an antiligand, which specifically or
non-specifically binds the ligand, or by the ligand's
characteristic dielectric properties. The ligand is generally
defined as any molecule for which there exists another molecule
(i.e. an antiligand) which specifically or non-specifically binds
to said ligand, owing to recognition, chemical or otherwise, of
some portion of said ligand. The antiligand, for example, can be an
antibody and the ligand a molecule such as an antigen which binds
specifically to the antibody. In the event that the antigen is
bound to the surface and the antibody is the molecule being
detected, for the purposes of this document the antibody becomes
the ligand and the antigen is the antiligand. The ligand may also
consist of nucleic acids, proteins, lipids, small molecules,
membranes, carbohydrates, polymers, cells, cell membranes,
organelles and synthetic analogues thereof.
[0050] Suitable ligands for practice of this invention include, but
are not limited to antibodies (forming an antibody/epitope
complex), antigens, nucleic acids (e.g. natural or synthetic DNA,
RNA, gDNA, cDNA, mRNA, tRNA, etc.), lectins, sugars (e.g. forming a
lectin/sugar complex), glycoproteins, receptors and their cognate
ligand (e.g. growth factors and their associated receptors,
cytokines and their associated receptors, signaling receptors,
etc.), small molecules such as drug candidates (either from natural
products or synthetic analogues developed and stored in
combinatorial libraries), metabolites, drugs of abuse and their
metabolic by-products, co-factors such as vitamins and other
naturally occurring and synthetic compounds, oxygen and other gases
found in physiologic fluids, cells, cellular constituents cell
membranes and associated structures, other natural products found
in plant and animal sources, other partially or completely
synthetic products, and the like.
[0051] As used herein, the term "antiligand" refers to a molecule
which specifically or nonspecifically binds another molecule (i.e.,
a ligand). The antiligand is also detected through its interaction
with a ligand to which it specifically binds or by its own
characteristic dielectric properties. As used herein, the
antiligand is usually immobilized on the surface, either alone or
as a member of a binding pair that is immobilized on the surface.
In some embodiments, the antiligand may consist of the molecules on
the signal path, on a dielectric surface or in a dielectric volume,
or a conductive surface. The antiligand may further be attached by
one or more linkers to a surface or matrix proximal to, or
incorporated in, the signal path. Alternatively, once an antiligand
has bound to a ligand, the resulting antiligand/ligand complex can
be considered an antiligand for the purposes of subsequent binding
or other subsequent interactions.
[0052] As used herein, the term "specifically binds" when referring
to a protein or polypeptide, nucleic acid, or receptor or other
binding partners described herein, refers to a binding reaction
which is determinative of the cognate ligand of interest in a
heterogeneous population of proteins and/or other biologics. Thus,
under designated conditions (e.g. immunoassay conditions in the
case of an antibody, or stringent conditions in the case of nucleic
acid binding), the specified ligand binds to its particular
"target" (e.g. a hormone specifically binds to its receptor, or a
given nucleic acid sequence binds to its complementary sequence)
and does not bind in a significant amount to other molecules
present in the sample or to other molecules to which the ligand or
antibody may come in contact in an organism or in a sample derived
from an organism.
[0053] As used herein, the terms "isolated" "purified" or
"biologically pure" refer to material which is substantially or
essentially free from components that normally accompany it as
found in its native state.
[0054] As used herein, the term "nucleic acid" refers to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogs of natural nucleotides that can function in a similar
manner as naturally occurring nucleotides.
[0055] As used herein, the terms "polypeptide", "peptide" and
"protein" are used interchangeably to refer to a monomer or polymer
of amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residue is an artificial chemical
analogue of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers.
[0056] As used herein, the term "antibody" refers to a protein
consisting of one or more polypeptides substantially encoded by
immunoglobulin genes or fragments of immunoglobulin genes. The
recognized immunoglobulin genes include the kappa, lambda, alpha,
gamma, delta, epsilon and mu constant region genes, as well as
myriad immunoglobulin variable region genes. Light chains are
classified as either kappa or lambda. Heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0057] A typical immunoglobulin (antibody) structural unit is known
to comprise a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (VL) and variable heavy chain (VH) refer to
these light and heavy chains respectively.
[0058] Antibodies exist as intact immunoglobulins or as a number of
well-characterized fragments produced by digestion with various
peptidases. Thus, for example, pepsin digests an antibody below the
disulfide linkages in the hinge region to produce F(ab)'.sub.2, a
dimer of Fab which itself is a light chain joined to VH-CH1 by a
disulfide bond. The F(ab)'.sub.2 may be reduced under mild
conditions to break the disulfide linkage in the hinge region
thereby converting the (Fab') 2 dimer into an Fab' monomer. The
Fab' monomer is essentially a Fab with part of the hinge region
(see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y.
(1993), for a more detailed description of other antibody
fragments). While various antibody fragments are defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that such Fab' fragments may be synthesized de novo
either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein also includes antibody
fragments either produced by the modification of whole antibodies
or synthesized de novo using recombinant DNA methodologies.
Preferred antibodies include single chain antibodies, more
preferably single chain Fv (scFv) antibodies in which a variable
heavy and a variable light chain are joined together (directly or
through a peptide linker) to form a continuous polypeptide.
[0059] A single chain Fv ("scFv" or "scFv") polypeptide is a
covalently linked VH:VL heterodimer which may be expressed from a
nucleic acid including VH- and VL-encoding sequences either joined
directly or joined by a peptide-encoding linker. Huston, et al.
(1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883. A number of
structures for converting the naturally aggregated--but chemically
separated light and heavy polypeptide chains from an antibody V
region into an scFv molecule which will fold into a three
dimensional structure substantially similar to the structure of an
antigen-binding site. See, e.g. U.S. Pat. Nos. 5,091,513 and
5,132,405 and 4,956,778.
[0060] An "antigen-binding site" or "binding portion" refers to the
part of an immunoglobulin molecule that participates in antigen
binding. The antigen binding site is formed by amino acid residues
of the N-terminal variable ("V") regions of the heavy ("H") and
light ("L") chains. Three highly divergent stretches within the V
regions of the heavy and light chains are referred to as
"hypervariable regions" which are interposed between more conserved
flanking stretches known as "framework regions" or "FRs". Thus, the
term "FR" refers to amino acid sequences that are naturally found
between and adjacent to hypervariable regions in immunoglobulins.
In an antibody molecule, the three hypervariable regions of a light
chain and the three hypervariable regions of a heavy chain are
disposed relative to each other in three dimensional space to form
an antigen binding "surface". This surface mediates recognition and
binding of the target antigen. The three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity determining regions" or "CDRs" and are
characterized, for example by Kabat et al. Sequences of proteins of
immunological interest, 4th ed. U.S. Dept. Health and Human
Services, Public Health Services, Bethesda, Md. (1987).
[0061] As used herein, the terms "immunological binding" and
"immunological binding properties" refer to the non-covalent
interactions of the type which occur between an immunoglobulin
molecule and an antigen for which the immunoglobulin is specific.
As used herein, a biological sample is a sample of biological
tissue or fluid that, in a healthy and/or pathological state, that
is to be assayed for the analyte(s) of interest. Such samples
include, but are not limited to, sputum, amniotic fluid, blood,
blood cells (e.g., white cells), tissue or fine needle biopsy
samples, urine, peritoneal fluid, and pleural fluid, or cells
therefrom. Biological samples may also include sections of tissues
such as frozen sections taken for histological purposes. Although
the sample is typically taken from a human patient, the assays can
be used to detect the analyte(s) of interest in samples from any
mammal, such as dogs, cats, sheep, cattle, and pigs. The sample may
be pretreated as necessary by dilution in an appropriate buffer
solution or concentrated, if desired. Any of a number of standard
aqueous buffer solutions, employing one of a variety of buffers,
such as phosphate, Tris, or the like, preferably at physiological
pH can be used.
[0062] As used herein, the term "receptor" or "drug receptor"
refers to a biological structure that is a target for drug therapy,
and includes proteins such as membrane-bound structures like
G-protein Coupled Receptors, nuclear receptors like hormone
receptors; proteins which modulate the expression of genes, such as
promoters and inducers; nucleic acid targets such as genes,
expressed sequences, regulatory and signaling sequences; other
proteins in biological systems which modulate or mediate
physiological activities of a given organism.
[0063] As used herein, the term "signal path" refers to a
transmission medium along or through the bio-electrical interface
which is capable of supporting an electromagnetic signal of any
useful frequency including a DC static field. A non-exhaustive list
of signal paths include conductive and dielectric waveguide
structures, conductive and dielectric transmission line structures,
multiple-conductor and multiple dielectric transmission mediums
such as transverse electromagnetic (TEM) transmission lines,
transmission lines with three or more conductive or dielectric
elements which support Transverse Electric (TE), Transverse
Magnetic (TM), or TEM modes of propagation such as quadrupolar and
octupolar lines; coupled waveguides and conductive and dielectric
resonant cavity structures which may or may not be coupled;
conductive and dielectric antenna structures such as dipole and
quadrupole antennas; evanescent wave structures such as evanescent
waveguides, both coupled and uncoupled, evanescent wave
transmission lines, and evanescent wave antennas; other non-modal
structures like wires, printed circuits, and other distributed
circuit and lumped impedance conductive structures, and the like.
In embodiments in which the signal path consists of a conductive
region or regions, the conductive region extends continuously over
that range. In embodiments in which the signal path is
non-metallic, e.g., a dielectric waveguide, antenna, or
transmission line, the signal path is defined as the path having
either the greatest conductivity at the frequency or range of
frequencies being used, or as the molecular binding region
itself.
[0064] As used herein, the term "molecular binding region" or "MBR"
refers to a surface layer or a volume element having of at least
one molecular structure (i.e., an analyte, antiligand, or a
ligand/antiligand pair, etc.) coupled to the signal path along or
between the bio-electrical interface. The molecular binding region
may consist of one or more ligands, antiligands, ligand/antiligand
complexes, linkers, matrices of polymers and other materials, or
other molecular structures described herein. Further, the molecular
binding region may be extremely diverse and may include one or more
components including matrix layers and/or insulating layers, which
may have one or more linking groups. The molecular binding region
is coupled to the signal path either via a direct or indirect
physical connection or via electromagnetic coupling when the ligand
is physically separated from the signal path. The molecular binding
region may be of a derivatized surface such as by thiol linkers,
alkanethiols, heterobifunctional alkanes, branched dextrans,
biotinylated metals and the like, all in accordance with standard
practice in the art.
[0065] As used herein, the term "binding event" refers to an
interaction or association between two or more molecular
structures, such as a ligand and an antiligand. The interaction may
occur when the two molecular structures as are in direct or
indirect physical contact or when the two structures are physically
separated but electromagnetically coupled. Examples of binding
events of interest in a biological context include, but are not
limited to, ligand/receptor, antigen/antibody, drug-receptor,
protein-protein, enzyme/substrate, DNA/DNA, DNA/RNA, RNA/RNA,
nucleic acid mismatches, complementary nucleic acids and nucleic
acid/proteins. Alternatively, the term "binding event" may refer to
a single molecule or molecular structure described herein, such as
a ligand, or an antiligand/ligand complex, which is bound to the
signal path. In this case the signal path is the second molecular
structure.
[0066] As used herein, the term "ligand/antiligand complex" refers
to the ligand bound to the antiligand. The binding may be specific
or non-specific, and the bonds are typically covalent bonds,
hydrogen bonds, immunological binding, Van der Waals forces, or
other types of binding.
[0067] As used herein, the term "coupling" refers to the transfer
of energy between two structures either through a direct or
indirect physical connection or through any form of signal
coupling, such as electrostatic or electromagnetic coupling,
matter-field interactions, and the like.
[0068] As used herein, the term "test signal" refers to a d.c,
frequency domain, or time domain signal used to probe the bio-assay
device. Frequency domain signals may propagate at any useful
frequency defined within the electromagnetic spectrum. For example,
the frequency range within which a test signal may propagate is for
example at or above 1 MHz, such as 5 MHz 10 MHz, 20 MHz, 45 MHz,
100 MHz, 500 MHz, 1 GHz, 5 GHz, 10 GHz, 30 GHz, 50 GHz, 100 GHz,
500 GHz, 1000 GHz and frequencies ranging therebetween. Time domain
test signals may be generated in square, sawtooth, triangle, or
other known waveforms and propagate at periodic or aperiodic
intervals, Time domain signals may consist of amplitudes and
rise/fall times which permit modulation which coupled to the
molecular binding region. For example, a time domain test signal
may consist of a square waveform having an amplitude between 0V and
50V, and a rise/fall time of between 0.1 pS and 1 uS, or range
anywhere therebetween.
[0069] As used herein, the term "enzyme," refers to a protein which
acts as a catalyst to reduce the activation energy of a chemical
reaction in other compounds or "substrates", but is not a final
product in the reaction.
[0070] As used herein, the term "sample" and/or "solution" includes
a material in which a ligand resides. A non-exhaustive list of
solutions includes materials in solid, liquid or gaseous states.
Solid solutions may be comprised of naturally-occurring or
synthetic molecules including carbohydrates, proteins,
oligonucleotides, or alternatively, any organic polymeric material,
such as nylon, rayon, dacryon, polypropylene, teflon, neoprene,
delrin or the like. Liquid solutions include those containing an
aqueous, organic or other primary components, gels, gases, and
emulsions. Exemplary solutions include celluloses, dextran
derivatives, aqueous solution of d-PBS, Tris buffers, deionized
water, blood, physiological buffer, cerebrospinal fluid, urine,
saliva, water, organic solvents. The solution is used herein to
refer to the material in which the ligand and/or antiligand are
applied to the binding surface. The solution contains the sample to
be analyzed.
[0071] As used herein, the term "linking group" or "linker" refers
to chemical structures which are used to attach any two components
on the bio-assay device. The linking groups thus have a first
binding portion that binds to one component, such as a conductive
surface or dielectric matrix, and have a second binding portion
that binds to another component such as the matrix or the
antiligand.
[0072] As used herein, the term "bio-assay device" refers to a
structure on which the molecular binding region is formed. The
bio-assay device may consist of a surface, recessed area, volume,
or a hermetically sealed enclosure, each of which may be any
particular size or shape.
[0073] As used herein, the "bio-assay system" refers to the
bio-assay device as described above, in connection with the
components necessary to electromagnetically probe and detect the
bio-assay device. These components include, but are not limited to,
the signal path(s), substrate(s), electronic devices such as signal
generators, oscilloscopes, network analyzers, time domain
reflectometers or other equipment necessary to probe and detect
signals from the bio-assay device, microchips and microprocessors
which can probe and detect electromagnetic signals and analyze
data, and the like.
[0074] As used herein, the term "resonant" or "resonance" refers
generally to a rapidly changing dielectric response as a function
of frequency.
[0075] As used herein, the term "dispersion" refers to the
functional dependence of the dielectric properties of a material on
the frequency of the probing radiation, and in particular is used
to distinguish regions of the electromagnetic spectrum in which the
dielectric properties of a given material has a strong functional
dependence on the frequency of the probing electromagnetic
energy.
[0076] As used herein, "bio-electrical interface" refers to an
interface region which includes the signal path for supporting test
signal propagation and the molecular binding region of a
sample.
[0077] As used herein, the term "matrix" or "binding matrix" refers
to a layer or volume of material on the bio-assay chip that is used
as a spacer or to enhance surface area or volume available for
binding or to optimize orientation of molecules for enhanced
binding, or to enhance any other property of binding so as to
optimize the bio-assay device. The matrix layer may be comprised or
carbohydrates such as dextran, poly amino acids, cross-linked and
non-cross linked proteins, and the like.
[0078] As used herein, the term "structural change" refers to any
change of position, chemical make-up, orientation, conformation,
relative orientation of sub-structures or sub-units of a molecule
or molecular system. A non-exhaustive list includes conformational
changes, dimerization and polymerization, covalent binding,
sub-unit motion, interactions with other molecules such as covalent
and non-covalent binding, hydrophobic bonding, denaturation and
re-naturation, hybridization, ionization, substitution, and the
like.
[0079] II. General Overview of the Bio-Assay System
[0080] The present invention makes use of the observation that a
vast number of molecules can be distinguished based upon the unique
dielectric properties most molecules exhibit. These distinguishing
dielectric properties can be observed by coupling an
electromagnetic signal to the bound molecular structure. The unique
dielectric properties modulate the signal, giving it a unique
signal response. The unique signal response can then be used to
detect and identify the ligands and other molecules which make up
the molecular binding region.
[0081] FIG. 1A illustrates a side view of one embodiment of a
bio-assay system 100 in accordance with the present invention. The
system 100 is illustrated in a two conductor, signal-plane
ground-plane, circuit topology which may be realized in a multitude
of architectures including lumped or distributed element circuits
in microstrip, stripline, coplanar waveguide, slotline or coaxial
systems. Moreover, those of skill in the art of electronics will
readily appreciate that the system may be easily modified to a
single conductor waveguide system, or a three or more conductor
system.
[0082] As illustrated, the system 100 includes a signal source 110,
transmission lines 120, a ground plane 130, a bio-assay device 150,
and a signal detector 160. The illustrated embodiment shows two
transmission lines 120 coupled to the bio-assay device 150,
although in an alternative embodiment, the system may consist of a
single transmission line coupled to the bio-assay device for making
a single port measurement. Further alternatively, three or more
transmission lines may be coupled to the bio-assay device 150 for
multiple port measurements.
[0083] Transmission lines 120 are formed from a material which can
support the propagation of a D.C voltage/current of an A.C. time or
frequency domain signal over the desired frequency of operation.
Transmission lines 120 may be realized as a conductive layer, such
as a center conductor in a coaxial cable or a gold transmission
line, deposited on a substrate, such as alumina, diamond, sapphire,
polyimide, or glass using conventional photolithography or
semiconductor processing techniques. Signal interconnections 122
may be made via wire/ribbon bonds, soldering, conductive epoxy,
connectors, or other conventional connection techniques appropriate
for the frequency of operation.
[0084] The system 100 further includes a bio-assay device 150 which
includes a dielectric substrate 151 and a signal path 152. The
dielectric substrate 151 may consists of any insulating material
such as glass, alumina, diamond, sapphire, silicon, gallium
arsenide or insulating materials used in semiconductor processing.
Alternatively, dielectric material such as RT/Duroid.RTM.
manufactured by the Rodgers Corporation or other similar dielectric
materials may be used.
[0085] The signal path 152 is designed to provide a low insertion
loss medium and can consist of any TE, TM, or TEM signal
architecture. In an exemplary embodiment, the signal path 152
consists of a photolithographically formed microstrip transmission
line having a sputtered gold thickness on the order of between 0.1
um to 1000 um. In this embodiment, the transmission line is
designed to provide low signal loss from D.C. to 110 GHz. Other
condutive materials such as indium tin oxide (ITO), copper, silver,
zinc, tin, antimony, gallium, cadmium, chromium, manganese, cobalt,
iridium, platinum, mercury, titanium, aluminum, lead, iron,
tungsten, nickel, tantalum, rhenium, osmium, thallium or alloys
thereof may be used to form the transmission line. In another
embodiment, the signal path 152 consists a dielectric region,
further described below.
[0086] A bio-electrical interface region 153 defines the region
over the signal path 152 and the MBR 156 of the applied sample 157
are electromagnetically coupled. In one embodiment of the
invention, the MBR 156 specifically binds to the signal path 152.
In another embodiment of the invention, the MBR 156 binds
non-specifically to the signal path 152. In still another
embodiment of the invention, the MBR is electromagnetically coupled
to, but is separate from the signal path 152. Sufficient
electromagnetic coupling may occur either through direct binding to
the signal path 152 or from the molecular structures of the MBR 156
being suspended in close proximity to the signal path 152. When
direct molecular binding to the signal path is sought, the signal
path may include linker and/or matrix layers as further described
in the commonly-owned, co-pending U.S. patent application entitled
"Method and Apparatus for Detecting Molecular Binding Events," Ser.
No. 09/243,194, filed Feb. 2, 1999 incorporated herein by
reference.
[0087] The MBR 156 is primarily composed of one or more ligands,
although other molecules and structures may also be included, as
described herein. The MBR 156 may consist of only one bound ligand
tier, for instance in the case of primary binding, or it may
consist of two, three, four, five or more bound ligand tiers, in
the instances where there are secondary or higher-order binding
events occurring. Multiple ligand tiers may occur at different
binding surfaces 155 over the same signal path. Additionally, the
MBR 156 may comprise a matrix in a volume, with ligands and
antiligands attached to structural components such as branched
dextran, polymers, amino acid chains, other linkers known in the
art, and the like.
[0088] In the illustrated embodiment, dielectric substrate 151 is
located between the signal path 151 and the ground plane 159.
However, the MBR 156 and sample 157 may be located proximate to the
ground plane 159 such that MBR 156 is electromagnetically coupled
to ground plane 159 alternatively or in addition to the MBR's
location to the signal path 152 as shown in FIG. 1A.
[0089] The system 100 includes a signal source 110 which launches a
test signal 112 onto the transmission line 120 and towards the
bio-assay device 150. A signal detector 160 is positioned along the
transmission path to receive the modulated test signal 162 (either
reflected or transmitted or both). When the test signal 120
propagates along the bio-electrical interface region 153 of the
bio-assay device 150, the dielectric properties of the MBR 156
modulate the test signal. The modulated test signal 162 is then
recovered by the detector 160 and used to detect and identify the
molecular binding events occurring within the MBR 156.
[0090] FIG. 1B illustrates a second embodiment of the bio-assay
test system in accordance with the present invention. Reference
numbers used in FIG. 1A are reused to indicate previously described
elements. The system includes the described signal source 110,
transmission lines 120, connections 122, ground plane 130,
bio-assay device 150 and signal detector 160.
[0091] The bio-assay device 170 includes a dielectric substrate 151
and ground plane 159, previously described. The signal path
includes transmission lines 172 and a dielectric region 156 formed
across the bio-electrical interface region 153 between transmission
lines 120. The dielectric region 156 is composed of the MBR and
formed from the molecular binding events of the sample 157. The
dielectric region is designed to provide a DC-blocked, low signal
loss medium between transmission lines 172. The D.C. blocking
properties of the dielectric region 156 prevents D.C. voltages and
currents from passing between the input and output which could
interfere with the operation of the test system, further described
below. Dielectric region 156 provides low signal loss over the
desired testing frequencies, some examples being 1 MHz, 5 MHz 10
MHz, 20 MHz, 45 MHz, 80 MHz, 100 MHz, 250 MHz, 500 MHz, 750 MHz, 1
GHz, 2.5 GHz, 5 GHz, 7.5 GHz, 10 GHz, 12 GHz, 18 GHz, 20 GHz, 22
GHz, 24 GHz, 26 GHz, 30 GHz, 33 GHz, 40 GHz, 44 GHz, 50 GHz, 80
GHz, 96 GHz, 100 GHz, 500 GHz, 1000 GHz, or frequencies ranging
therebetween.
[0092] As described above, the MBR operates to modulate the test
signal. The architecture of the dielectric region 156 serves to
signal support propagation through the bio-electrical interface
region without high signal loss. An insulating substrate 176 is
used as a binding surface for the MBR in order to form the
dielectric region 156 and the MBR may bind either specifically or
non-specifically to the insulating substrate 176. The insulating
substrate 151 may consist of the same or different dielectric
material as the dielectric substrate 151 and may, alternatively or
in addition, consist of linker, matrix, and/or insulating layers
further described in the incorporated patent application entitled:
"Method and Apparatus for Detecting Molecular Binding Events," Ser.
No. 09/243,194.
[0093] The length of the dielectric region (MBR) 156 is selected to
provide sufficient test signal modulation while minimizing through
loss. Typical lengths are on the order of 10.sup.-1m, 10.sup.-2m
10.sup.-3m, 10.sup.-4m, 10.sup.-5m, 10.sup.-6m, 10.sup.-7m,
10.sup.-8m, 10.sup.-9m, 10.sup.-10m 10.sup.-11m, or range anywhere
therebetween.
[0094] As indicated, detection and identification of a ligand is
also possible when the ligand is physically separated from but
electromagnetically coupled to the signal path 151. In this
instance, the coupling between the signal path 151 and the
suspended ligand will alter the response of the test signal
propagating along the signal path 151, thereby providing a means
for detecting and/or identifying it. The maximum separation between
the signal path 151 and suspended ligand is influenced by such
factors as the effective dielectric constant of the medium between
the signal path 151 and the ligand, the total coupling area, the
sensitivity of the signal detector, concentration of the ligands in
solution, and the desired detection time. Separation distances are
typically on the order of 10.sup.-1m, 10.sup.-2m 10.sup.-3m,
10.sup.-4m, 10.sup.-5m, 10.sup.-6m, 10.sup.-7m, 10.sup.-8m,
10.sup.-9m, 10.sup.-10m or range anywhere therebetween.
[0095] In some embodiments, such as cell based assays, the MBR 156
may be electromagnetically coupled to the signal path 151 through
the sample. Thus, cells, and in particular cell membranes and
membrane-based structures may couple to the signal path
indirectly.
[0096] III. Single Path Test System and Bio-assay
[0097] Molecular binding events occurring within the MBR maybe
detected and identified using various test systems which generate,
recover, and subsequently analyze changes in the generated test
signal. Test systems which are capable of use with the present
invention include those systems designed to detect changes in the
signal's voltage, current, impedance, admittance, reactance,
amplitude, phase, delay, frequency, wave shape and/or timing, and
other signal properties.
[0098] A. Test System
[0099] FIG. 2 illustrates one possible embodiment of a single path
test system 200 in accordance with the present invention. The test
system includes a test fixture 300, further described below, a
measurement system 240 and a computer 260. Measurement system 240
communicates test signals to and from test fixture 300 via test
cables 224. Computer 260 controls measurement system 240 via a
control bus 250.
[0100] In one embodiment, measurement system 240 includes an
S-Parameter Test Module model no. 8516A, a Frequency Synthesizer
(not shown) model no. 8341B, and a Vector Network Analyzer model
no. 8510B, all of which are manufactured by the Hewlett Packard
Company of Palo Alto, Calif. (www.hp.com). In this embodiment,
measurement system 240 provides a measurement capability between
the frequencies of 45 MHz and 40 GHz. In an alternative embodiment,
measurement system 240 may consist of model number HP 8751A network
analyzer which provides a measurement capability between 5 Hz and
500 MHz. In a further embodiment, measurement system may consist of
model number HP 85106D which provides a measurement capability
between 33 GHz and 110 GHz, both manufactured by the Hewlett
Packard Company. Other measurement systems such as scalar network
analyzers, Time Domain Reflectometers, an other similar measurement
systems may also be used to detect a change in the test signal
which is attributable to the dielectric properties of the MBR.
[0101] Test cables 224 support the propagation of the test signals
at the desired frequency. In one embodiment, test cables consists
of model number 6Z PhaseFlex.TM. Microwave test cables manufactured
by the W. L. Gore and Associates, Inc. of Newark Del.
(www.gore.com). Control bus 250 provides communication between the
test system and computer 260 and in the illustrated embodiment
consists of a General Purpose Instrument Bus (GPIB). In alternative
embodiments, measurement system 240 and computer 260 may be
integrated within a single automated measurement unit.
[0102] Computer 260 controls measurement system 240 to generate
test signals at one or more frequencies, output power levels,
signal shapes, phase offsets or other measurement settings. In the
preferred embodiment, computer 260 includes a +450 MHz
microprocessor, such as those manufactured by the Intel Corporation
of Santa Clara, Calif. (www.intel.com). Test system control, data
acquisition, and analysis may be performed using a graphical
programming software tool, such as LabVIEW.RTM. manufactured by the
National Instruments Corporation of Austin, Tex.
(www.natinst.com).
[0103] Alternatively or in addition, measurement system 240 may
include a Time Domain Reflectometer (TDR) system, such as those
optionally available with the above-described network analyzers or
described in the incorporated patent application entitled: "Method
and Apparatus for Detecting Molecular Binding Events," Ser. No.
09/243,194. Essentially, TDR systems transmit a signal pulse
towards a unit under test. The return signal (either reflected from
or transmitted through the unit under test) can be analyzed to
ascertain information about the unit under test. Specifically in
the present embodiment, the dielectric properties of the MBR will
modulate the signal pulse, thereby enabling detection and
identification of the molecular binding events therein.
[0104] TDR measurements may be made at the fixture level using the
aforementioned systems, or at the bio-assay device level utilizing
one or more of the standard techniques of microwave monolithic
circuit (MMIC) technologies. When a TDR measurement is made at the
device level, a time-domain test signal is generated in close
proximity to the bio-assay device. This signal is then propagated
along the signal path to the bio-assay element via standard
conductive geometries used in MMIC technologies. The molecular
binding region modulates the time-domain test signal, and the
modulated signal is then recovered to be analyzed.
[0105] B. Test Fixture
[0106] The test fixture of the present invention is designed to
provide a signal path and to secure the MBR of the applied sample
in direct contact with or in close proximity to the signal path
such that a test signal propagating therealong will
electromagnetically couple to the MBR. The test fixture may consist
of a wholely or partially enclosed, or recessed structure over or
into which the sample may be deposited, injected, or otherwise
applied.
[0107] FIG. 3A illustrates in a side view one possible embodiment
of the test fixture 300 in accordance with the present invention.
Test fixture 300 includes a top plate 302 and a bottom plate 304.
Top plate 302 includes ports 350a and 350b for injecting the sample
solution. Top plate 302 further includes the top half of a sample
cavity 340a. Bottom plate 304 includes the bottom half of the
sample cavity 340b. In the preferred embodiment, top and bottom
plates 302 and 304 are each composed of machined stainless steel
and each measures 0.0320 cm.times.1.575 cm.times.3.15 cm.
[0108] Contained with the sample cavity 340 is a reaction vessel
310, an O-ring 320, a bio-assay device 400 (further described in
FIG. 4 below), and a bottom spacer 330. Reaction vessel 310
includes ports 312a and 312b for receiving the sample. Reaction
vessel 310 further includes an O-ring cavity 318 for accommodating
the O-ring 320. O-ring 320 is positioned between the reaction
vessel 310 and the bio-assay device 400 to secure the sample along
the bio-assay device 400. Bio-assay device 400 provides the signal
path and bioelectrical interface along which the MBR will form.
Bottom spacer 330 is provided to elevate the bio-assay device 400
to the proper height so that it may couple to input and output
transmission lines (not shown) formed between the top and bottom
plates 302 and 304.
[0109] The sample is injected into sample cavity 340 via feed tubes
(not shown) coupled to ports 350a and 350b. Sample flows through
reaction vessel ports 312a and 312b into the reaction vessel 310.
In the preferred embodiment, the sample is injected by applying
positive pressure in one feed tube and negative pressure to the
other feed tube.
[0110] FIG. 3B illustrates an end view of the test fixture shown in
FIG. 3A. As illustrated, test fixture 300 includes connectors 360a
and 360b for communicating signals into and/or out of the test
fixture 300. Connectors 360a and 360b are secured to top and bottom
plates 302 and 304 via screws 361. Connectors 360 and 360 include
center conductors 362 which are coupled to the bio-assay device 400
via transmission lines (not shown) formed between the top and
bottom plates 302 and 304, respectively. In the preferred
embodiment, connectors 360 are SMA connectors such as those
manufactured by the SRI Connector Gage Company of Melbourne, Fla.
(www.sriconnectorgage.com). In alternative embodiments, connectors
360 may consist of N, 3.5 mm, 2.9 mm, 2.4 mm or other connectors
appropriate for the test frequency range.
[0111] FIG. 3C illustrates a top view of top plate 302 showing
ports 350a and 350b and top half of sample cavity 340a. In its
preferred embodiment, top half of sample cavity 340a measures 0.4
cm.times.0.4 cm.times.0.080 cm. FIG. 3D illustrates a top view of
bottom plate 304 showing the bottom half of sample cavity 340b,
also measuring 0.40 cm.times.0.40 cm.times.0.080 cm in the
preferred embodiment. FIGS. 3E and 3F illustrate side and bottom
views respectively of reaction vessel 310. In its preferred
embodiment, reaction vessel is composed of Lexan.RTM. and measures
0.4 cm.times.0.4 cm.times.0.070 cm. Ports 312a and 312b are 0.030
cm diameter. O-ring cavity 318 has an diameter of 0.240 cm.
[0112] FIGS. 3G and 3H illustrate top and side views of O-ring 320,
respectively. In the preferred embodiment, O-ring 320 is composed
of an elastomer, such as Viton.RTM. and measures 0.100
cm.times.0.240 cm with an inner diameter of 0.030 cm. FIG. 3I and
3J illustrate top and side views of bottom spacer 330. In the
preferred embodiment, bottom spacer is composed of Lexan.RTM. or
alumina and measures 0.4 cm.times.0.4 cm.times.0.025 cm.
[0113] C. Bio-Assay Device
[0114] The bio-assay device forms the bio-electrical interface of
the present detection system. The device includes a signal path
electromagnetically coupled to the MBR. One or more input/output
ports are connected to the signal path to communicate the test
signal. A single input/output port may be used, when for instance a
reflection measurement, known in the art, is sought. Alternatively,
separate input and output ports may be used when a through
measurement, also known in the art, is sought alternatively or in
addition to the reflection measurement.
[0115] The signal path is preferably formed along a direction which
is non-orthogonal to the MBR. In one embodiment, the test signal
propagates in parallel to a tangent on the surface on which the MBR
is formed. In other embodiments, the test signal may propagate at
an angle of .+-.1.degree., .+-.2.degree., .+-.3.degree.,
.+-.4.degree., .+-.5.degree., .+-.10.degree., .+-.15.degree.,
.+-.20.degree., .+-.30.degree., .+-.40.degree., .+-.45.degree.,
.+-.50.degree., .+-.60.degree., .+-.70.degree., .+-.80.degree., or
.+-.85.degree. relative to the MBR binding surface, or any ranges
therebetween. In a first embodiment, the signal path consists of a
transmission line in a two conductor structure and the direction of
the signal path is defined by the Poynting vector as known in the
art of electromagnetics. In a second embodiment, the transmission
line may consist of a conductive region or layer which extends
continuously along the bio-electrical interface region. In a third
embodiment, the signal path maybe defined as the path having the
least amount of signal loss along the bio-electrical interface over
the desired frequency range of operation. In a fourth embodiment,
the signal path maybe defined as having an a-c. conductivity of
greater than 3 mhos/m, i.e., having a conductivity greater than
that a saline solution, typically greater than 5 mhos/m, but
ideally in the range of 100 to 1000 mhos/m and greater. As
described above, the MBR may be either be in direct contact with or
physically separated from but electromagnetically coupled to the
signal path.
[0116] The signal path may be realized in a number of different
architectures, such as a conductive wire, a transmission line, a
conductive or dielectric waveguide structure, a resonant cavity, or
any other transmission medium that will support the propagation of
the test signal over the desired frequency range. At high test
frequencies (frequencies above 10 MHz, for example) the signal path
may be realized in microstrip, stripline, suspended substrate,
slotline, coplanar waveguide, conductive or dielectric waveguide,
or other high frequency signal path architectures such as those
described in R. E. Collins Foundations for Microwave Engineering,
McGraw-Hill Publishing Co., 1966; and S. March, Microwave
Transmission Lines and Their Physical Realizations, Les Besser and
Associates, Inc., 1986. The following examples are but a few of the
possible signal path embodiments within the scope of the present
invention.
[0117] Through Microstrip Transmission Line
[0118] FIG. 4A illustrates a top view of a standard microstrip
transmission line bio-assay 410 for use with the test fixture of
FIG. 3A. As illustrated, the signal path consists of a transmission
line 412 of width of 0.065 cm and length of 1.0 cm between the
input/output ports 411. Bio-assay 410 is formed using standard
photolithographic techniques and fabricated using sputtered gold
transmission lines on a 0.55 mm thick quartz glass substrate having
a dielectric constant of approx. 3. Those of skill in the art will
appreciate that other signal path architectures, conductive and
substrate materials, and photolithographic techniques may be
alternatively employed.
[0119] During a testing operation, a sample is applied over the
transmission line 412 and a MBR is formed along the exposed surface
of the transmission line 412. The MBR may be either in direct
physical contact with the transmission line 412 or separated from
but electromagnetically coupled to the line 412. In the embodiment
in which the MBR is in direct contact with the transmission line,
linker and/or matrix layers may be employed to facilitate binding
thereto as further described in the incorporated patent application
entitled: "Method and Apparatus for Detecting Molecular Binding
Events," Ser. No. 09/243,194.
[0120] Next, a test signal is launched on to the transmission line
412 through, for example, an SMA type connector 360, shown in FIG.
3B. As the test signal propagates along the transmission line
portions have a MBR attached or in close proximity thereto, the
dielectric properties of the MBR modulate the test signal. The
modulated test signal is then be recovered and used to detect and
identify the molecular binding events occurring within the MBR.
[0121] Meandered Microstrip Transmission Line
[0122] FIG. 4B illustrates a top view of a meandered transmission
line bio-assay 420 for use with the test fixture of FIG. 3A.
Bio-assay 420 includes a meandered line coupled between an
input/output ports 421. The meander line 422 is designed to
increase the MBR surface area which provides greater measurement
sensitivity, while adding minimal length and size to the detection
structure.
[0123] In the illustrated embodiment, the meandered line 422 has a
width of 0.065 cm and length of 1.0 cm between the input/output
ports 422. Transmission line corners may be mitered, 45.degree. to
minimize signal reflection and maximize signal transmission along
the line 422. Spacing 424 is designed to minimize coupling between
proximate line sections. In one embodiment, line spacing is 0.033
cm. In an alternative embodiment line spacing 424 is defined such
that coupling between proximate line sections 422a, 422b is no more
than -7 dB. Bio-assay 420 is formed using standard
photolithographic techniques and fabricated using sputtered gold
transmission lines on a 0.55 mm thick quartz glass substrate having
a dielectric constant of approx. 3. Those of skill in the art will
appreciate that other signal path architectures, conductive and
substrate materials, and photolithographic techniques may be
alternatively employed.
[0124] During a testing operation, a sample is applied over the
meandered line 422 and a MBR is formed along the exposed surface of
the meandered line 422. The MBR may be either in direct physical
contact with the meandered line 422 or separated from but
electromagnetically coupled to the line 422. Linker and/or matrix
layers may be used to facilitate binding to the meandered line
422.
[0125] Next, a test signal is launched on to the transmission line
422 through, for example, an SMA type connector 360, shown in FIG.
3B. As the test signal propagates along the transmission line
portions have a MBR attached or in close proximity thereto, the
dielectric properties of the MBR modulate the test signal. The
modulated test signal is then be recovered and used to detect and
identify the molecular binding events occurring within the MBR.
[0126] Numerous variations in the illustrated design may be
realized to increase the detection sensitivity over a minimum
detection area. For instance, when employed miters may be designed
to provide an intentional impedance mismatch between line segments,
thereby causing signal reflections between miters. When the
effective signal length of the line segment approaches 180 degrees,
the reflected signals will combine in phase with incoming signals,
thereby a larger amplitude output signal at these frequencies.
Higher output power permits greater measurement sensitivity and the
length of the line segments can be tune to detect or more closely
inspect responses occurring at specific frequencies.
[0127] Microstrip Ring Resonator
[0128] FIG. 4C illustrates a top view of a ring resonator bio-assay
430 for use with the test fixture of FIG. 3A. The bio-assay 430
includes input/output ports 431a and 431b coupled to a ring
resonator 434. Ring resonator 434 includes three concentric rings
434a-c and a solid circular ring 434d disposed therein. Each ring
434a-c has a width of 0.1 cm and is separated from proximate
ring(s) by a spacing of 0.1 cm. The solid circular element 434d is
0.050 cm in radius and is disposed at the ring center. In
alternative embodiments, spacing 434e and/or widths may vary from
ring to ring. Bio-assay 430 is formed using standard
photolithographic techniques and fabricated using sputtered gold
transmission lines on a 0.55 mm thick quartz glass substrate having
a dielectric constant of approx. 3. Those of skill in the art will
appreciate that other signal path architectures, conductive and
substrate materials, and photolithographic techniques may be
alternatively employed.
[0129] During normal operation without an applied sample, a test
signal is injected into the port 431a through, for example, an SMA
connector 360 as shown in FIG. 3B. Via electromagnetic coupling, a
portion of the test signal propagates through the ring resonator
434 and to the output port 431b. An impedance mismatch occurs at
this interface 431b, reflecting a portion of the signal back toward
the source interface 431a. The remaining portion of the signal
propagates out of the resonant circuit along the input line segment
and to the test set. At the source interface 431a, a second
impedance mismatch occurs and reflecting a portion of the reflected
signal again toward the resonator output 431. The remaining portion
of the signal is propagated out of the resonant circuit along the
output line segment toward the test set input. The signal continues
to "ping-pong" between the interfaces 431a and 431b until the
signal is dissipated or transmitted to the source or test set. The
magnitude of the reflected wave depends in part on the magnitude of
the impedance mismatch at the interfaces 431a and 431b. The larger
the impedance mismatches, the larger the reflected signal.
[0130] At one or more frequencies, the effective signal path
between interfaces 431a and 431b approaches a 180.degree. phase
shift (or a multiple thereof). When this occurs, the reflected
signal will reach input interface 431a having a phase substantially
equal to the phase of the incoming signal. In this instance, the
incoming signal and the reflected signal will recombine in-phase,
thereby producing a stronger signal. When the stronger signal
reaches the output interface 431b, a larger magnitude signal
(compared to the non-combined signal) will exit from the output
interface 431b to the test set. Thus, the resonator 434 will output
a larger magnitude signal near frequencies in which the resonator
434 has an effective signal length near 180.degree. or a multiple
thereof. This difference in output signal strength can be monitored
and detected using the measurement systems described herein.
[0131] When the sample is applied over the resonator 430, a MBR is
formed along the exposed portion of rings 434a-d. The MBR may
either be in direct physical contact with the rings or separated
from but electromagnetically coupled to the rings 434a-d. Linker
and/or matrix layers may be employed to facilitate binding to the
resonator rings 434a-d and/or input and output interfaces 431a and
431b.
[0132] Next, a test signal is injected into the input port 431a as
above. The test signal couples between rings of the resonator 434
as before, except that the dielectric properties of the MBR
operates to change the frequency(s) at which the resonator 434
approaches 180.degree.. Further, because the dielectric properties
of each different MBR are distinct, each MBR will produce a
different "frequency marker", i.e., the frequency at which the
resonator approaches a 180.degree. phase shift and produces a
larger output signal. In this manner, samples containing different
molecular structures will exhibit different frequency markers,
which can be used to detect their presence in an unknown solution.
In addition, molecular structures within a particular class,
alpha-helices, beta-sheets and other structural motifs in proteins
may exhibit "related" frequency markers, e.g., frequency markers
within close proximity to each other or frequency markers which
occur within a predictable pattern.
[0133] Those of skill in the art of Microwave engineering will
understand that other resonant structures are also possible. For
instance, the resonator 434 may alternatively consist of a
transmission line segment connected between the input and output
interfaces 431a and 431b. In this embodiment, the transmission line
segment will have the appropriate impedance relative to the input
and output ports to provide the desired input and output impedance
mismatch and the appropriate length to provide the 180.degree.
phase shift in presence of the sample. Other resonant
configurations such as a proximately placed dielectric puck as well
as others may be used with minor modifications to detect the
presence or absence of particular molecular structures.
[0134] Microstrip Capacitive Gap
[0135] FIG. 4D illustrates a top view of a capacitive gap bio-assay
440 for use with the test fixture of FIG. 3A. Bio-assay 440
includes an input port 441a coupled to an input line segment 442a
and an output port 441b coupled to an output line segment 442b.
Disposed between the input and output line segments 442a and 442b
is a gap 444 where the sample is deposited during testing. In the
illustrated embodiment, input and output line segments 442a and
442b are each 0.495 mm long and 0.250 mm wide. Capacitive gap 444
measures 0.010 mm.times.0.250 mm. Bio-assay 440 is formed using
standard photolithographic techniques and fabricated using
sputtered gold transmission lines on a 0.55 mm thick quartz glass
substrate having a dielectric constant of approx. 3. Those of skill
in the art will appreciate that other signal path architectures,
conductive and substrate materials, and photolithographic
techniques may be alternatively employed.
[0136] During normal operation without an applied sample, a test
signal is injected into the port 441a through, for example, an SMA
connector 360 as shown in FIG. 3B. Via electromagnetic coupling, a
portion of the test signal's electromagnetic field propagates
across the capacitive gap 444 between the input and output line
segments 442a and 442b. The capacitive gap 44 prevents the
transmission of D.C. voltage and current from passing between the
input and outputs. The test signal is then recovered at the output
port 441b for processing. The width and separation of the gap 444,
impedances of input and output line segments 442a and 442b, the
dielectric constant of the substrate 445, and the frequency of
operation will influence the amount of signal power transferred
between the input and output ports 441a and 441b. The capacitive
gap circuit 440 will exhibit a signal response which varies over a
test frequency range.
[0137] When the sample is applied over the gap 444, a MBR is formed
along the edges of input and output line segments 442a and 442b.
The MBR may either be in direct physical contact with the line
segment edges 442a and 442b, or separated from but
electromagnetically coupled thereto. Linker and/or matrix layers
may be used on the line segments 442a and 442b to promote molecular
binding thereto.
[0138] The formation of the MBR on gap edges effects the signal's
transmissivity from the input port 441a to the output port 441b.
Specifically, the MBR creates a gap circuit, the response of which
varies over the test frequency range. As described above, each
distinct MBR will exhibit a different dielectric property which
serves to create a distinct frequency response or "signature." The
frequency signature of a known molecular sample can stored and
later used to identify the molecular structure in an unknown
solution. Molecular structures within the same class may exhibit a
similar frequency pattern over a common test frequency range. In
this case, the tester is able to identify the class of the unknown
molecular structure if the identity of the molecular structure
itself is known.
[0139] The capacitive configuration may be used as a single
detection element or in combination with one or more of the
detection elements listed herein to enhance, tune, or detune the
frequency response at one or more frequencies.
[0140] Dielectric Signal Path
[0141] FIG. 4E illustrates a side view of a dielectric signal path
bio-assay 450 having for use with the test fixture of FIG. 3.
Bio-assay 450 includes an input line segment 451, an output line
segment 452 formed on a dielectric substrate 456, and a dielectric
region 455 disposed between the input and output line segments 451
and 452. The bottom surface of dielectric region 455 is formed by
insulating substrate 453 which is treated to promote molecular
binding thereto. The insulating substrate 453 may consist of the
same or different material as the dielectric substrate 456.
Further, the insulating substrate 453 may include of linker and/or
matrix layers, further described in the commonly owned, copending
U.S. patent application entitled "Method and Apparatus for
Detecting Molecular Binding Events," Ser. No. 09/243,194, filed
Feb. 2, 1999 incorporated herein by reference. In the exemplary
embodiment of FIG. 4E, the bio-assay 450 is fabricated using
standard microstrip photolithographic techniques on a dielectric
substrate 456 of 0.55 mm quartz glass substrate having a dielectric
constant of approximately 3. The dielectric region 455 is 100
Angstroms deep and extends 2.5 um between the input and output line
segments 451 and 452.
[0142] When a sample 456 is applied over the dielectric region 455,
a longitudinal MBR 457 is formed along the surface of the
insulating substrate 453. The formed MBR serves as a signal path
for the test signal. As described above, the MBR 457 exhibits a
dielectric property which modulates the test signal and each MBR
457 will exhibit a different dielectric property which will in turn
will modulate the test signal differently. The modulated signals or
"signatures" are largely unique and can be associated with samples
having known molecular binding events. These stored signals can
later be used to identify the molecular structure in an unknown
solution. Molecular structures within the same class may exhibit a
similar frequency pattern over a common test frequency range. In
this case, the tester is able to identify the class of the unknown
molecular structure if the identity of the molecular structure
itself.
[0143] IV. Array Test System and Bio-Assay
[0144] A multitude of bio-assay devices, some examples of which are
described in FIGS. 4A-E, may be implemented in an N.times.M array
test structure to perform high through-put analysis. In this
configuration, N.times.M different binding events may be detected,
for instance to enable fast characterization of oligonucleotides
such as single nucleotide polymorphism, individual genes, and
longer sequences of the nucleic acides. The number of inputs may be
the same as the number of outputs in which case M=N, or the number
of inputs and outputs may differ.
[0145] The array may be fabricated using conventional
photolithographic processing to form one or more biosensors on a
substrate, such as the 0.5 mm.sup.2 devices described above.
Alternatively, the array may be fabricated using semiconductor
processing techniques, such as Silicon Dioxide (SiO.sub.2)or
Gallium Arsenide (GaAs) processing. In this embodiment, the array
in wafer form may include 10.sup.1, 10.sup.2, 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10
bio-assay devices/mm or range anywhere therebetween.
[0146] A. Test System
[0147] FIG. 5 illustrates one possible embodiment of an N.times.M
array test system 500 in accordance with the present invention. The
test system includes a test fixture 600 further described below, a
1.times.N input switch 530, a measurement system 540, a M.times.1
output switch 550, and a computer 560. Measurement system 540
communicates test signals to the test fixture 600 via input test
cable 524a and 1.times.N input switch 530. The test signal is
subsequently received from the test fixture via M.times.1 output
switch 550 and output test cable 524b. Computer 560 controls
1.times.N in put switch 530, measurement system 540, and M.times.1
output switch 550 via a control bus 550.
[0148] In one embodiment, measurement system 540 may consist of the
previous described measurement system 240 or any of the alternative
embodiments described herein. Similarly, input and output test
cables 524a and 524b, control bus 550, and computer 560 may consist
of those previously described and/or their alternatives.
[0149] The 1.times.N input switch 530 routes the test signal from
the input test cable 524a to one of the N test fixture signal
inputs. The M.times.1 output switch 550 routes the test signal from
one of the M test fixture outputs to the output test cable. Input
and output switches 530 and 550 may consist of any switching or
multiplexing means which will support the propagation of the
desired test signal. For instance, input and output switches 530
and 550 may consist of low frequency switches (DC to 2 GHz), such
as those manufactured by Amplifonix, Inc. of Philadelphia, Pa.
(www.amplifonix.com). Switches for use at higher frequencies (2-18
GHz), such as those manufactured by the General Microwave
Corporation of Amityville, N.Y. (www.generalmicrowave.com) may
alternatively be employed. Connection between bio-assay device and
input and output switches 530 and 550 may be made using insulated
cables, wire bonds, or other conventional interconnection means
appropriate for the test frequency of operation.
[0150] In an alternative embodiment, input and output switches 530
and 550 and the bio-assay array form a monolithic integrated
circuit. For instance, when the bio-assay array is fabricated using
GaAs semiconductor processing techniques, input and output switches
530 and 550 may consist of integrally formed PIN diodes which are
coupled to the bio-assay array. Further alternatively, input and
output switches 530 and 550 may form an integrated assembly in
which the input and output switches 530 and 550 are discrete
components which are connected (via wire or ribbon bonds) to the
bio-assay array. Both alternative embodiments provide advantages in
that the interconnecting structures are miniaturized or eliminated,
thereby reducing or eliminating the signal loss associated
therewith.
[0151] As explained, the bio-assay array may be fabricated in wafer
form using semiconductor processing techniques. In this embodiment,
the array test system 500 may consist of a wafer probe test
station, such as those manufactured by Cascade Microtech, Inc. of
Beaverton, Oreg. (www.cascademicrotech.com) which includes or is
coupled to the aforementioned input and output switches 530 and
550, and computer 560. The wafer probe station utilizes one or more
probe cards, each of which is capable of providing a large number
of low loss, low VSWR signal interconnections to the bio-assay
array.
[0152] The probe card(s) may be used to provide N and/or M signal
interconnections to the remotely located input and/or output
switches 530 and 550, respectively. Alternatively, input and/or
output switches 530 and 550 may be monolithically fabricated with
the bio-assay array, in which case the probe card(s) provides a
single input and/output signal transition to the measurement system
540. In this latter embodiment, the probe card(s) includes probes
for providing switch control voltages to the monolithically formed
switches.
[0153] Alternatively or in addition, measurement system 540 may
include a Time Domain Reflectometer (TDR) system, such as those
optionally available with the aforementioned network analyzers or
described in the incorporated patent application entitled: "Method
and Apparatus for Detecting Molecular Binding Events," Ser. No.
09/243,194.
[0154] B. Array Test Fixture
[0155] FIG. 6A illustrates a side view of one possible embodiment
of the N.times.M array test fixture 600 in accordance with the
present invention. Similar in construction to the single path test
fixture 300 shown in FIG. 3, test fixture 600 includes a top plate
602, bottom plate 604, and a sample cavity 640 which holds the
aforementioned reaction vessel 610, bio-assay device 700 (further
described in FIG. 7 below), and bottom spacer 630 elements. In the
NxM array test fixture embodiment, the dimensions of sample cavity
640 and correspondingly reaction vessel 610 and bottom spacer 630
are designed to accommodate the bio-assay device 700 which may be
larger or smaller than the bio-assay device 300 shown in FIG. 3.
Each array element includes a small, monolithically deposited
structure to form a recessed area over the signal path in order to
hold a portion of the applied sample in electromagnetic
communication with the signal path of each array element. In
another embodiment, MEMS (micro-electronic machining systems)
technology may be used to fabricate the sample cavity at the
bio-assay device level.
[0156] FIG. 6B illustrates an end view of the N.times.M array test
fixture 600. Test fixture 600 includes N input connectors
660a.sub.1 to 660a.sub.n and M output connectors 660b.sub.1 to
660b.sub.m. Test fixture 600 also includes N input transmission
lines (not shown) which provide a signal transition between the
fixture's N connectors 660a.sub.1 to 660a.sub.n and the bioassay's
N inputs. Test fixture 600 further includes M output transmission
lines (not shown) which transition between the bio-assay's M
outputs and the fixture's M output connectors 660b.sub.1 to
660b.sub.m. The input and output transmission lines may be realized
as insulated conductive wires, microstrip, stripline, coplanar
waveguide transmission lines deposited on a dielectric substrate,
or other conventionally known signal path architectures. The choice
of the transmission line's architecture will be influenced by the
test frequency band and the bio-assay device's input and output
port density.
[0157] C. Bio-Assay Array
[0158] Any or all of the structures shown in FIGS. 4A-4E can be
used to form a bio-assay array in accordance with the present
invention. The array may be fabricated on a discrete piece of
dielectric substrate or in wafer form using semiconductor
processing techniques. The array may include two or more of the
above-mentioned structures on a single device, and coupled to
diagnostic apparati via any of the standard switching techniques.
Further active elements such as transistors may also be used as
array elements, as will be further described below.
[0159] One, two, and three dimensional addressing may be used, with
any number of addresses on the device itself. Each address may be
designed to act as a logic gate in which a binary decision is made
regarding binding or some other change in the MBR; to make
decisions about three or more states, such as the shift in
frequencies in a band limited system of resonators; or to measure a
continuum of properties such as voltage, phase, frequency, or any
of the other parameters as discussed above.
[0160] FIG. 7A illustrates one embodiment of an integrated
bio-assay array 700 in accordance with the present invention. The
integrated array 700 is supplied with a test signal via the signal
source of measurement system 540. The array 700 includes an
integrated 1.times.N input switch and M.times.1 output switch which
are monolithically formed during the semiconductor fabrication
process. The number of inputs may be the same as the number of
outputs in which case M=N, the number of inputs and outputs may
differ.
[0161] The 1.times.N input switch routes the incoming test signal
to the desired array element. The MBR in the array element
modulates the test signal according to the dielectric properties of
the molecular binding events which make up the MBR. An M.times.1
output switch 550 routes the modulated test signal to a detector of
the measurement system 540. An analyzer of the test system 540
compares the input and modulated test signals to determine the
measured signal response. While each array element is illustrated
as a two-port device, those of skilled in the art will appreciate
that one-port or multiple port array elements may be used
alternatively.
[0162] As explained above, the array 700 and the input and output
switches may be fabricated either as discrete components or in
wafer form and integrated in varying degrees depending upon the
application. In the illustrated embodiment, the array 700 and input
and output switches are monolithically formed on a semiconductor
wafer. In another embodiment, the input and output switches are
monolithically formed separately from the array 700 and connected
via wire or ribbon bonds. In a further embodiment, input and output
switches 530 and 550 and array 700 are each discrete units. Those
skilled in the art will appreciate that other arrangements are also
possible.
[0163] FIG. 7B illustrates one embodiment of an array element,
shown as a series connected, electronically switched Field Effect
Transistor (FET) 710. FET 710 may be a Metal Semiconductor Field
Effect Transistor (MESFET) fabricated using GaAs processing. Other
transistor configurations are also possible for instance, High
Electron Mobility Transistors (HEMT), heterostructure FETs,
homogenous or heterojunction bipolar transistors, or PN junctions
devices such as PIN diodes to name a few. Other active or passive
array elements may be used alternatively or addition to these as
well.
[0164] In the embodiment of FIG. 7B, the source and drain terminals
712 and 714 of FET 710 are employed as the input and output ports,
711 and 715 respectively. The sample is applied over FET 710 such
that the MBR 716 provides a parallel path between the source and
drain terminals 712 and 714. FET 710 is designed such that when
turned off, it presents a drain to source resistance (R.sub.ds)
which is much higher than resistance through the MBR 716. In this
instance, the signal path propagates through the MBR 716 which
modulates the test signal. The modulated test signal is recovered
(through a DC blocking capacitor to remove the DC bias) and
compared to the input test signal to detect and/or identify the
molecular binding events occurring within the MBR 716. When the FET
710 is activated, it provides a much lower R.sub.ds compared to the
resistance of the MBR 716. In this instance, the MBR 716 is
effectively switched out of the signal path and the signal
propagates largely unaffected by it. Thus by simply opening or
closing a switch, an array element may be addressed.
[0165] FIG. 7C illustrates a further embodiment of a FET used as an
array element which is optically switched. FET 720 is connected
similarly to FET 710 described in FIG. 7B and may consist of a
photosensitive transistor, diode or other photosensitive device.
The gate junction 722 may be illuminated, for instance, with normal
sunlight, a laser, a Light Emitting Diode (LED), or other source
having a wavelength to which FET 720 has a high sensitivity. The
incident light activates FET 720 to switch out the MBR 722. When
the FET 720 is deactivated, the test signal propagates through the
MBR 722 and is modulated thereby. The modulated test signal is
recovered (through a DC blocking capacitor not shown) and analyzed
to determine the presence and/or identity of molecular binding
events within the MBR 722.
[0166] FIG. 7D illustrates an extension of FIG. 7B and 7C in which
two or more FETs are serially-connected. Array 750 includes a first
test path 753 along which addressable switches 753a and 753c are
coupled. In one embodiment, addressable switches are electronically
or optically controlled MESFETs, described above. Array path 753
further includes sample regions 753b and 753d, each of which
provides a parallel signal paths to the corresponding addressable
switches 753a and 753c.
[0167] As described above, addressable switches 753a and 753c
operate to switch in and out the sample regions 753b and 753d.
Thus, a particular row is made into a transmission path in which a
single assay site appears as an impedance mismatch. Each assay site
can be either switched into the circuit, or switched out of the
circuit, as desired. The nature of the impedance mismatch is a
function of binding and other changes in the MBR. Additional signal
paths such as signal path 754 may be included in the array and
cross-strapped to the other paths using other low loss switches
(not shown) to allow the test signal to propagate between signal
paths 753 and 754. Input and output switches 752 and 755 are used
to inject and recover the test signal to/from the array 750. As
those of skill in the art will appreciate, the described array may
be extended to any number of N.times.M elements to provide a two
dimensional array device.
[0168] FIG. 7E illustrates the circuit equivalent model of the
array shown in FIG. 7D. The switch impedance Zs is designed to be a
close match with the reference impedance of the signal path Zo, and
the assay impedance Z.sup.I,J is designed to be much different than
either the switch or reference impedance. Thus, small changes in
the assay impedance will dominate the electrical properties of any
given row, and will therefore be easily detectable. The exact
values for the impedances will depend on the design criteria for
the particular array, but certain general principles of engineering
apply, such as the greatest efficiency in terms of delivering power
to the load (detector) is obtained with matched-impedance design,
and reference impedances are frequently taken to be 50.OMEGA..
[0169] In an alternative embodiment, each array element may consist
of a logic gate which is capable of occupying one of two possible
states, depending on the conditions of gating. As an example, the
conditions of gating may be whether or not a particular binding
event has occurred. Such a condition may be the hybridization of
nucleic acid material to specific capture probes on the surface of
the device, or a particular drug-receptor interaction. In any case,
the device is engineered so that a binding event or structural
change in the MBR triggers the gating. Essentially the modulation
of any circuit parameter may trigger the gating; all that is
required is to have the necessary hardware and software in place to
make the decision as to whether or not the circuit parameter has
been modulated.
[0170] As an example, one may monitor a characteristic frequency of
a given system such as a resonant structure. The shift in this
frequency as a result of a particular binding event may serve as
the modulation which signals the logic state. Any parameter which
changes as a function of binding may be used to trigger logic gate.
Such parameters include, but are not limited to: frequency,
voltage, current, power, phase, delay, impedance, reactance,
admittance, conductance, resistance, capacitance, inductance, or
other parameters.
[0171] FIG. 7F illustrates one embodiment of a two-dimensional
bio-assay array 770. As shown, the array 770 includes a first
input/output (I/O) axis 772 and a second I/O axis 774 for
inputting/outputting test signals.
[0172] The array is interfaced with conventional external
diagnostic hardware which is capable of generating and detecting
the appropriate frequency or frequencies, then communicating it to
and from the assay array via a multiplexer, through the ports as
illustrated above. Such an externally supported system may be
comprised of any number of electromagnetic sources such as vector
and scalar network analyzers, time-domain devices like TDR
analyzers and other pulsed techniques; utilize any of the detection
schemes mentioned herein, including vector and network analyzers;
and use any number of well-known techniques to deliver the signals
to and from the assay array via standard and non-standard
multiplexing techniques.
[0173] Generically, such a chip may be fabricated using standard
semiconductor chip approaches. Those of skill in the art will
readily appreciate that such a configuration may be used in a
one-port format, a two port format, or utilize more than two
ports.
[0174] V. Applications
[0175] The above described bio-assay, test fixture, and test system
may be used in a number of applications to detect and/or identify
particular molecular binding events occurring within the sample. A
few of the possible applications are described in general
below.
[0176] Nucleic Acid Chemistry Application
[0177] The bio-sensors and test systems of the present application
may be used to analyze binding complexes, such as the hybridization
complexes formed between a nucleic acid probe and a nucleic acid
target. For instance, the bio-assay sensors and test system may be
used in diagnostic methods which involve detecting the presence of
one or more target nucleic acids in a sample, quantitative methods,
kinetic methods, and a variety of other types of analysis such as
sequence checking, expression analysis and de novo sequencing. One
or more of these methods may also detect binding between nucleic
acids without the use of labels. Certain methods will benefit from
utilizing the described bio-assay arrays and test systems which
allows for high throughput. Other methods will benefit from the use
of spectral profiles which makes it possible to distinguish between
different types of hybridization complexes. These methods are
further described in the incorporated, concurrently filed patent
application entitled "Methods of Nucleic Acid Analysis," Atty
Docket 019501-000600.
[0178] Drug Discovery Application
[0179] The bio-sensors and test systems of the present application
may be used to detect binding events between proteins and a variety
of different types of ligands. The bio-assay sensors and test
systems of the present invention may be used to screen libraries of
ligands to identify those ligands which bind to a protein of
interest, such methods have particular utility in drug screening
programs, for example. Additionally, the bio-assay sensors and test
system may be similarly employed with diagnostic methods to detect
the presence of a particular ligand that binds to a known protein,
or of a particular protein that binds to a known ligand. These
methods are further described in the incorporated, concurrently
filed patent application entitled "Methods for Analyzing Protein
Binding Events," Atty Docket 019501-000700.
[0180] FIG. 8 is an example of the effects of a protein binding
non-specifically to the dielectric signal path of the bio-assay
device 450 illustrated in FIG. 4E. A buffer (d-PBS) was initially
placed in the dielectric gap region 455 (FIG. 4E) and a baseline
insertion loss measurement over the frequency range 45 MHz to 40
GHz was taken. Next, a sample solution containing urease at high
concentration was added and the urease was allowed to bind to the
quartz in the dielectric gap region 455. The dielectric region 455
was then flushed with d-PBS and a second insertion loss measurement
over the same frequency range was taken. The second measurement was
compared to the first resulting in the changes in the signal's
frequency response, shown in FIG. 8.
[0181] While the above is a complete description of possible
embodiments of the invention, various alternatives, modification
and equivalents may be used to which the invention is equally
applicable. Therefore, the above description should be viewed as
only a few possible embodiments of the present invention, the
boundaries of which is appropriately defined by the metes and
bounds of the following claims.
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