U.S. patent application number 09/243196 was filed with the patent office on 2002-06-13 for method and apparatus for detecting molecular binding events.
Invention is credited to HEFTI, JOHN.
Application Number | 20020072857 09/243196 |
Document ID | / |
Family ID | 22917726 |
Filed Date | 2002-06-13 |
United States Patent
Application |
20020072857 |
Kind Code |
A1 |
HEFTI, JOHN |
June 13, 2002 |
METHOD AND APPARATUS FOR DETECTING MOLECULAR BINDING EVENTS
Abstract
Systems and methods for detecting molecular binding events and
other environmental effects using the unique dielectric properties
of the bound molecular structure or structures are presented. A
molecular binding layer is coupled along the surface of a signal
path. A test signal is propagated along the signal path, whereby
the test signal couples to the molecular binding layer, and in
response, exhibits a signal response.
Inventors: |
HEFTI, JOHN; (SAN FRANCISCO,
CA) |
Correspondence
Address: |
SIGNATURE BIOSCIENCE, INC.
21124 CABOT BLVD.
HAYWARD
CA
94545-1130
US
|
Family ID: |
22917726 |
Appl. No.: |
09/243196 |
Filed: |
February 1, 1999 |
Current U.S.
Class: |
702/19 ; 435/7.1;
436/501 |
Current CPC
Class: |
G01N 33/54373
20130101 |
Class at
Publication: |
702/19 ; 436/501;
435/7.1 |
International
Class: |
G01N 033/48; G01N
033/53 |
Claims
What is claimed is:
1. A method for detecting one or more properties associated with a
molecular binding layer, the method comprising the steps of:
providing a molecular binding layer coupled along the surface of a
signal path; and propagating a test signal along said signal path,
wherein said test signal couples to said molecular binding layer,
and in response, exhibits a signal response.
2. The method of claim 1, further comprising the step of measuring
said signal response.
3. The method of claim 1, wherein said surface of said signal path
is derivatized.
4. The method of claim 1, wherein said molecular binding layer
comprises an antiligand bound to a ligand.
5. The method of claim 1, further comprising the step of adding an
analyte to said molecular binding layer, wherein said added analyte
interacts with said molecular binding layer.
6. The method of claim 1, wherein said step of adding occurs prior
to said step of propagating said test signal.
7. The method of claim 6, wherein the analyte is in a solution.
8. The method of claim 7, wherein the solution comprises a sample
of body fluid.
9. The method of claim 1, wherein the signal path comprises a
second electromagnetically coupled original path.
10. A method for detecting one or more molecular binding events
between a ligand and an antiligand, the method comprising the steps
of: exposing a portion of a signal path to a first solution
containing a first ligand, said exposed signal path portion
comprising a first molecular binding layer, wherein said exposed
signal path portion comprises a continuous transmission line; and
propagating a first test signal along said signal path, wherein
said test signal couples to said molecular binding layer and
exhibits a first signal response indicating detection of said
binding event between said first ligand and said antiligand.
11. The method of claim 10, further comprising the steps of:
exposing said portion of a signal path to a second solution
containing a second ligand; and propagating a second test signal
along said signal path, wherein said test signal couples to said
molecular binding layer and exhibits a second signal response
indicating detection of said binding event between said second
ligand and said antiligand.
12. The method of claim 10, wherein the antiligand is an
antibody.
13. A method for detecting one or more properties associated with a
molecular binding layer, the method comprising the steps of:
providing a molecular binding layer coupled along the surface of a
signal path; and propagating a test signal along said signal path,
wherein the tangent of said surface of said signal path is
non-orthogonal to the direction of signal propagation of said test
signal, wherein said test signal couples to said molecular binding
layer, and in response, exhibits a signal response.
14. An apparatus for detecting one or more properties associated
with a molecular binding layer, the apparatus comprising: a signal
path having an input signal port, an output signal port, and a
continuous conductive region therebetween; and said molecular
binding layer coupled to said signal path.
15. The apparatus of claim 14, wherein said input signal port and
said output signal port comprises the same physical port.
16. The apparatus of claim 14, wherein said input signal port and
said output signal port comprise two physically separated
ports.
17. The apparatus of claim 14, wherein said molecular binding layer
comprises a ligand/antiligand complex.
18. The apparatus of claim 14, wherein said molecular binding layer
comprises a derivatized conductive layer.
19. The apparatus of claim 14, further comprising a retaining
structure for retaining a solution along said signal path.
20. The apparatus of claim 14, wherein said test signal propagates
at a frequency of greater than 1 MHz.
21. The apparatus of claim 14, wherein signal path comprises a
transmission line structure.
22. The apparatus of claim 14, wherein the signal path comprises a
resonant cavity.
23. A method for detecting one or more molecular binding events
between a ligand and an antiligand, the method comprising the steps
of: applying a first solution and a first ligand over a portion of
a signal path, wherein a first molecular binding layer comprising
said antiligand has previously formed along the surface of said
portion of said signal path, said molecular binding layer being
positioned more proximal to said signal path then said solution;
and propagating a test signal along said signal path, said signal
path comprising a path which is continuous along said surface,
wherein said test signal couples to said molecular binding layer
comprising a ligand/antiligand complex and exhibits a first signal
response.
24. A method for detecting one or more molecular binding events
between a ligand and an antiligand, the method comprising the steps
of: applying a solution and a ligand over a signal path, wherein a
molecular binding layer comprising said antiligand has previously
formed along at least the surface of at least a portion said signal
path; and propagating a test signal along said signal path, said
signal path comprising a non-orthogonal path relative to said
surface, wherein said test signal couples to said molecular binding
layer comprising a ligand/antiligand complex and exhibits a first
signal response.
25. The method of claim 24, further comprising the steps storing
said signal first signal response; applying a second solution
containing a ligand or antiligand over said portion of said signal
path; forming a second molecular binding layer along said signal
path, said second molecular layer comprising said ligand and said
antiligand, propagating a second test signal along said conductive
surface, wherein said second test signal couples to said molecular
binding layer and in response exhibits a second signal response,
said second signal response being uncorrelated with said first
signal response.
26. The method of claim 24, further comprising the steps:
propagating a second test signal along said signal path to obtain a
second signal response; comparing said first and second signal
responses; and determining said dielectric properties of said
solution have changed if said second response does not correlate
with said first response within a predefined range.
27. The method of claim 26, further comprising the steps of:
correlating said first signal response to a first known dielectric
property; correlating said second signal response to a second known
dielectric property; and removing the quantity of said first
dielectric property from the quantity of said second known
dielectric property.
28. A method for determining the classification of an unknown
ligand, the method comprising the steps of: providing a signal path
coupled to a first molecular binding layer, said molecular binding
layer comprising N respective antiligands for binding to N
respective ligand sub-structures; applying a solution containing a
plurality of unknown ligands over said molecular binding layer.
forming, in response, a second molecular binding layer along said
signal path, said second molecular binding layer comprising said N
antiligands; propagating N test signals to said N antiligands;
providing N known signal responses, said N known responses defining
a known classification of ligands; wherein each of said N test
signals couples to said N antiligands, and in response exhibits N
respective measured responses indicative of the presence of each of
said N sub-structures; wherein if a predetermined number of said N
known signal responses correlates within a predefined range with
said N measured responses, determining said unknown ligand s within
said known classification.
29. A method for identifying an unknown molecular binding event,
the method comprising the steps of: providing a signal path;
applying a first solution containing a first ligand over said
signal path; forming, in response, a first molecular binding layer
along said signal path, said first molecular layer comprising said
first ligand, wherein said first molecular binding layer is coupled
along the surface of the signal path; propagating a first test
signal along said signal path, wherein said first test signal
couples to said molecular binding layer and in response exhibits a
first signal response; providing a known signal response
corresponding to a known molecular binding event; comparing said
first signal response with said known signal response, wherein if
said first signal response correlates to said known signal response
within a predefined range, said unknown molecular binding event
comprises said known molecular binding event.
30. A method for quantitating an unknown concentration of ligands
in a solution comprising the steps of: providing a signal path
coupled to a first molecular binding layer, said molecular binding
layer comprising at least one antiligand; applying a solution
having a known concentration of ligands over said molecular binding
layer to obtain a first signal response from a propagated test
signal; repeating said applying step in one or more different known
concentrations; correlating the signals with the known
concentrations; measuring a second signal response to a propagated
test signal; and correlating the second signal response to said
algorithm.
31. An apparatus for detecting the presence of a ligand or
antiligand, comprising: a signal path comprising a continuous
conductive region; a molecular binding layer coupled to at least a
portion of said continuous conductive region, said molecular
binding layer comprising said ligand or antiligand; and a solution
coupled to said molecular binding layer.
32. A bio-electrical interface for detecting the presence of a
ligand in a solution, com sing: a signal path comprising a
continuous conductive region; a solution for providing said ligand;
and a molecular binding layer coupled along said signal path and
said solution, said molecular binding layer comprising said
ligand.
33. The bio-electrical interface of claim 32, further comprising: a
ground plane; and a dielectric layer coupled between said ground
plane and said solution.
34. The bio-electrical interface of claim 32, wherein said
molecular binding layer operates as a shunt circuit coupled between
said transmission path and said solution.
35. The bio-electrical interface of claim 32, wherein said solution
operates a shunt circuit coupled between said shunt MBL circuit and
said ground plane.
36. The bio-electrical interface of claim 35, wherein said
molecular binding layer comprises the electrical characteristics of
a series R-L circuit coupled along said signal path.
37. The bio-electrical interface of claim 36, wherein said solution
comprises the electrical characteristics of a series R-L circuit
coupled along said signal path.
38. The bio-electrical interface of claim 32, further comprising: a
ground plane; and a dielectric layer coupled between said signal
path and said ground plane.
39. The bio-electrical interface of claim 38, wherein said
molecular binding layer operates as a shunt circuit coupled to said
signal path.
40. The bio-electrical interface of claim 39, wherein said solution
comprises the electrical characteristics of a parallel L-R-C
circuit coupled to said molecular binding layer.
41. The bio-electrical of claim 32, wherein the molecular binding
layer is a proximal to a ground plane.
42. The bio-electrical of claim 32, wherein the molecular binding
layer is proximal to both signal and ground planes.
43. The bio-electrical interface of claim 32, wherein the molecular
binding layer is proximal to a portion of a wave guide.
44. The bio-electrical interface of claim 32, wherein the molecular
binding layer is proximal to a portion of a micro strip.
45. The bio-electrical interface of claim 32, wherein the molecular
binding is proximal to and incorporated in a portion of a resonant
cavity.
46. A bio-electrical interface of claim 32, wherein the signal path
comprises an input original path and an output signal path.
47. An apparatus for detecting the presence of a ligand using a
test signal, the apparatus comprising: a signal path comprising a
continuous conductive region and having a first port and a second
port for communicating said test signal therebetween; a molecular
binding layer coupled to said signal path, said molecular binding
layer comprising said ligand; and a solution coupled to said
molecular binding layer for transporting said ligand to said
molecular binding layer.
48. The apparatus of claim 47, wherein the solution is a body
fluid.
49. The apparatus of claim 48, wherein the body fluid is blood.
50. A system for detecting a molecular binding event, comprising: a
signal source for launching a test signal; a bio-assay device
coupled to said signal source, comprising: a signal path comprising
a continuous conductive region; a solution contain a ligand for
producing said molecular binding event; and a first molecular
binding layer comprising said ligand; and a signal detector coupled
to said signal path, wherein said test signal propagates along said
signal path and couples to said molecular binding layer comprising
said ligand, and in response exhibits a signal response, said
signal response indicating the presence of said molecular binding
event.
51. The test system of claim 50, wherein said test signal comprises
a frequency-varying signal and wherein said signal response
comprises a transmission loss S.sub.21 frequency response of said
test signal.
52. The test system of claim 50, wherein said test signal comprises
a frequency-varying signal and wherein said signal response
comprises a return loss S.sub.11 frequency response of said test
signal.
53. The test system of claim 51 or 52, wherein said test signal
comprises a frequency, varying signal which is a resonant
response.
54. The test system of claim 51 or 52, wherein said test signal
comprises a frequency, varying signal which is a non-resonant
response.
55 The test system of claim 51 or 52, wherein said test signal
comprises a pure frequency or a frequency varying signals and
wherein said signal response comprises a shift in one or more of
said frequencies.
56. The test system of claim 50, wherein said test signal comprises
a time domain waveform and said signal response comprises a
transmitted time domain response.
57. The test system of claim 50, wherein said test signal comprises
a time domain waveform and said signal response comprises a
reflected time domain waveform.
58. The test system of claim 50, wherein said test signal comprises
a time domain waveform of varying pulse intervals and said signal
response comprises a reflected time domain waveform.
59. In a computer-controlled molecular binding event detection
system for use with bio-assay device having a molecular binding
layer coupled along a signal, a computer program product for
detecting one or more properties associated with the molecular
binding layer, the computer program product comprising: code that
directs said processor to instruct said system to propagate a test
signal along said signal path, wherein said test signal couples to
said molecular binding layer, and in response, exhibits a signal
response; and a computer readable storage medium for storing said
code.
60. The compute r program product of claim 59 further comprising
code that directs said processor to instruct said system to measure
said signal response.
61. In a computer-controlled molecular binding event detection
system for use with a bio-assay device having a molecular binding
layer coupled along a signal, a computer program product for
detecting one or more molecular binding events between a ligand and
antiligand, the computer program product comprising: code that
directs said processor to instruct said system to apply a first
solution to a portion of said signal path; and code that directs
said processor to instruct said system to propagate a first test
signal along said signal path, wherein said test signal couples to
said molecular binding layer and exhibits a first signal response
indicating detection of said binding event between said first
ligand and said antiligand; and a computer readable storage medium
for storing said code.
62. The computer program product of claim 61, further comprising:
code that directs said processor to instruct said system to expose
said portion of a signal path to a second solution containing a
second ligand; and code that directs said processor to instruct
said system to propagate a second test signal along said signal
path, wherein said test signal couples to said molecular binding
layer and exhibits a second signal response indicating detection of
said binding event between said second ligand and said
antiligand.
63. In a computer-controlled molecular binding event detection
system for use with bio-assay device having a signal path
comprising a continuous conductive region coupled to molecular
binding layer comprising N antiligands for binding to N respective
ligand substructures, a computer program product for determining
the classification of an unknown ligand contained in a solution,
the computer program product comprising: code that directs said
processor to instruct said system to apply said solution containing
a plurality of said unknown ligands over said molecular binding
layer, wherein a second molecular binding layer forms along said
signal path, said second molecular binding layer comprising said N
anti ligands; code that directs said processor to instruct said
system to propagate N test signals to said N antiligands; code that
directs said processor to instruct said system to provide N known
signal responses, said N known responses defining a known
classification of ligands, wherein each of said N test signals
couples to said N antiligands, and in response exhibits N
respective measured responses indicative of the presence of each of
said N sub-structures; code that directs said processor to instruct
said system to determine if a predetermined number of said N known
signal responses correlates within a predefined range with said N
measured responses; and a computer readable storage medium for
storing said code
64. In a computer-controlled molecular binding event detection
system for use with bio-assay device having a molecular binding
layer coupled along a signal, a computer program product for
quantitating an unknown concentration of ligands in a solution, the
computer program product comprising: code that directs said
processor to instruct said system to apply a solution having a
known concentration of ligands over said molecular binding layer to
obtain a first signal response from a propagated test signal; code
that directs said processor to instruct said system to repeat said
applying step in one or more different known concentrations; code
that directs said processor to instruct said system to correlate
the signals with the known concentrations; code that directs said
processor to instruct said system to measure a second signal
response to a propagated test signal; code that directs said
processor to instruct said system to correlate the second signal
response to said algorithm; and a computer readable storage medium
for storing said code.
Description
BACKGROUND OF THE INVENTION
[0001] Virtually every area of 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, military applications, 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.
[0002] For analysis in the fields of chemistry, biochemistry,
biotechnology, molecular biology and numerous others, it is often
useful to detect the presence of one or more molecular structures
and measure binding between structures. The molecular structures of
interest typically include, but are not limited to, cells,
antibodies, antigens, metabolites, proteins, drugs, small
molecules, proteins, 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. Additionally,
DNA and RNA 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.
[0003] 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, calorimetric 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.
[0004] 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. Typically these use the biological sample-be it tissues,
cellular systems, or molecular systems-as a shunt or series element
in the electrical circuit topology. This configuration has 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.
[0005] 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 that are actually taking place in a given
system would represent a significant breakthrough.
[0006] 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 bioassays that can be used
for an assay and then thrown away, and be highly adaptable to a
wide range of assay applications.
[0007] 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.
[0008] The present invention fulfills many of the needs discussed
above and other needs as well.
SUMMARY OF THE INVENTION
[0009] The present invention provides systems and methods for
detecting molecular binding events and other environmental effects
using the unique dielectric properties of the bound molecular
structure or structures, and the local environment, and also
identifying the presence and concentrations of molecular species,
as well as physical properties of the local environment, in a
particular biological system.
[0010] In a first embodiment of the invention, a method for
detecting a molecular binding event includes the steps of providing
a signal path and a molecular binding layer, which is formed along
the signal path. A test signal is propagated along the signal path
and couples to the molecular binding layer. In response to the
coupling, the signal exhibits a response which is indicative of
both the molecular binding event and the molecular binding layer
itself.
[0011] In a second embodiment of the invention, a method for
determining the classification of an unknown ligand is presented.
The method comprises the steps of providing a signal path coupled
to a first molecular binding layer having N respective antiligands
for binding to N respective ligand sub-structures. Next a solution
containing a number of unknown ligands is applied to the said
molecular binding layer. In response, a second molecular binding
layer is formed along the signal path, the second molecular binding
layer having N ligands. N respective test signals are propagated to
the N respective ligands. N known signal responses defining a known
ligand classification are provided. Finally, each of the test
signals couples to the N ligand/antiligand complexes, and in
response exhibits N respective measured responses indicative of the
presence of each of said N sub-structures, so that if a
predetermined number of said N known signal responses correlates
within a predefined range with the N measured responses, the ligand
is determined to be within the known classification.
[0012] In a third embodiment of the invention, a method for
identifying an unknown molecular binding event is presented. The
method includes the steps of providing a signal path, applying a
first solution containing a first ligand over the signal path, and
forming, in response, a first molecular binding layer along the
signal path, whereby the first molecular layer includes the first
ligand and is positioned along the signal path and the first
solution. A first test signal is propagated along the signal path,
the portion of which includes the molecular binding layer comprises
a continuous transmission line, whereby the signal couples to the
molecular binding layer and in response exhibits a first signal
response. A known signal response corresponding to a known
molecular binding event is provided and the first signal response
is then compared to the known signal response, wherein if the first
signal response correlates to the known signal response within a
predefined range, the unknown molecular binding event comprises the
known molecular binding event.
[0013] In a fourth embodiment of the invention, a method for
quantitating an unknown concentration of ligands in solution is
presented. The method includes the steps of providing a signal path
which is coupled to a first molecular binding layer having at least
one antiligand, applying a solution having a known concentration of
ligands over the molecular binding layer, and propagating a test
signal along the signal path. Next a first signal response is
measured and an extrapolation algorithm is generated. A second test
signal is subsequently propagated and a second signal response is
measured. The second signal response is then correlated to the
algorithm.
[0014] In a fifth embodiment of the invention, a bio-electrical
interface is provided for detecting the presence of a ligand in a
solution. The bio-electrical interface includes a signal path, a
solution for providing the ligand and a molecular binding layer.
The molecular binding layer includes the ligand and is coupled
along the signal path and the solution.
[0015] In a sixth embodiment of the invention, a bio-assay device
is provided for detecting one or more properties associated with a
molecular binding layer, such as the presence of a ligand, using a
test signal. The apparatus includes a signal path having a first
port and a second port for communicating the test signal, and a
continuous conductive region therebetween. The bio-assay device
further includes a molecular binding layer, which may have a
ligand, and which is coupled to the signal path. The bio-assay
device may further include a solution coupled to said molecular
binding layer, which may transport the ligand to the molecular
binding layer.
[0016] In a seventh embodiment of the invention, a system for
detecting a molecular binding event is presented. The system
includes a signal source for launching a test signal, a bio-assay
device coupled to said signal source and a second detector coupled
to the bio-assay device. The bio-assay device includes a signal
path and a first molecular binding layer, which may include a
ligand or antiligand, and which may be coupled to a solution and
the signal path. The test signal propagates along the signal path,
which is continuous throughout the region of the molecular binding
layer, and couples to the molecular binding layer, and in response
exhibits a signal response which indicates the presence of said
molecular binding event.
[0017] In one aspect, the present invention is the use of the
interaction of electromagnetic radiation, typically between about 1
MHz and 1000 GHz, with molecular structures in a molecular binding
layer to determine properties of the structures, such as dielectric
properties, structural properties, binding events and the like.
Also, the present invention uses a test signal on a bio-electrical
interface having a signal path along which the molecular binding
layer is coupled to detect analytes therein.
[0018] The nature and advantages of the present invention will be
better understood with reference to the following drawings and
detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A illustrates one embodiment of the bio-assay system
in accordance with the present invention.
[0020] FIG. 1B illustrates a second embodiment of the bio-assay
system in accordance with the present invention.
[0021] FIG. 1C illustrates a cross-section view of the bio-assay
system shown in FIG. 1B.
[0022] FIG. 1D illustrates one embodiment of a molecular binding
layer in accordance with the present invention.
[0023] FIG. 1E illustrates one embodiment of a molecular binding
layer having multiple antiligands which are spatially separated in
accordance with the present invention.
[0024] FIG. 1F illustrates one embodiment of a molecular binding
layer having multiple classes of anitligands in accordance with the
present invention.
[0025] FIG. 1G illustrates a molecular binding layer comprising one
or more cells in accordance with the present invention.
[0026] FIG. 1H illustrates a molecular binding layer comprising
cell membranes and membrane associated structures in accordance
with the present invention.
[0027] FIG. 2A illustrates one embodiment of the bio-assay device
in accordance with the present invention.
[0028] FIG. 2B illustrates a second embodiment of the bio-assay
device in accordance with the present invention.
[0029] FIG. 3 illustrates one embodiment of the binding surface
chemistry which occurs along the conductive layer of the
bio-electrical interface.
[0030] FIG. 4A illustrates one embodiment of an equivalent circuit
model for the bio-electrical interface structure shown in FIG.
2A.
[0031] FIG. 4B illustrates one embodiment of a circuit
corresponding to the equivalent circuit model shown in FIG. 4A.
[0032] FIG. 4C illustrates one embodiment of an equivalent circuit
model for the bio-electrical interface structure shown in FIG.
2B.
[0033] FIG. 4D illustrates one embodiment of a circuit
corresponding to the equivalent circuit model shown in FIG. 4C.
[0034] FIGS. 5A-5G illustrate specific embodiments of the
bio-electrical interface implemented in a two conductor circuit
topology in accordance with the present invention.
[0035] FIG. 6A illustrates one embodiment of a method for detecting
molecular binding events in accordance with the present
invention.
[0036] FIG. 6B illustrates one embodiment of a method for detecting
secondary and higher-order binding events in accordance with the
present invention.
[0037] FIG. 6C illustrates one embodiment of a method for measuring
dielectric changes of the molecular binding layer in accordance
with the present invention.
[0038] FIG. 6D illustrates one embodiment of a method for
identifying a ligand in an unknown solution in accordance with the
present invention.
[0039] FIG. 6E illustrates one embodiment of a method for
identifying the class of a ligand in accordance with the present
invention.
[0040] FIG. 6F illustrates one embodiment of a method for
quantitating the ligand concentration of a solution in accordance
with the present invention.
[0041] FIG. 6G illustrates one embodiment of a method for providing
a self-diagnostic capability of the bio-assay device in accordance
with the present invention.
[0042] FIG. 7A illustrates one embodiment of a computer system for
executing a software program designed to perform each of the
methods shown in FIGS. 6A-G.
[0043] FIG. 7B illustrates a simplified system block diagram of a
typical computer system used to execute a software program
incorporating the described method.
[0044] FIG. 8A illustrates one embodiment of a frequency
measurement system in accordance with the present invention.
[0045] FIG. 8B illustrates a first frequency response measured
which can be used to detect or identify a molecular structure in
accordance with the present invention.
[0046] FIG. 8C illustrates a second frequency response which can be
used to detect or identify a molecular structure in accordance with
the present invention.
[0047] FIG. 9 illustrates a second embodiment of a frequency
measurement system in accordance with the present invention.
[0048] FIG. 10 illustrates one embodiment of a time domain
measurement system in accordance with the present invention.
[0049] FIG. 11 illustrates one embodiment of a dielectric
relaxation measurement system in accordance with the present
invention.
[0050] FIGS. 12A-B illustrate the return loss and transmission loss
measurements, respectively, of the primary binding of urease to an
ITO surface.
[0051] FIGS. 12C and 12D illustrate the transmission loss
measurements of the primary binding effects of collagenase and
lysozyme.
[0052] FIG. 12E illustrates the transmission loss response of bound
and unbound dextran.
[0053] FIG. 12F illustrates the response of con-A unbound and bound
to glucose.
[0054] FIG. 12G illustrates the transmission loss of biotin/Avidin
relative to the Avidin response.
[0055] FIG. 12H illustrates the results of a competition titration
between dextran and glucose.
[0056] FIG. 12I illustrates the return loss of con-A as a function
of glucose concentration at resonance.
[0057] FIG. 12J illustrates the transmission loss of DNA/Polylysine
complexes relative to the Polylysine response.
[0058] FIG. 12K illustrates the change in the transmission loss
response as a function of pH for a series of buffers at 100 MHz, 1
GHz, and 10 GHz.
[0059] FIG. 12L illustrates the change in the transmission loss
response as a function of ionic concentration for a series of
buffers at 100 MHz, 1 GHz, and 10 GHz.
[0060] FIG. 12M illustrates the transmission loss response for 10
samples of whole blood probed at 1 GHz indicating detection
capability in a complex environment.
[0061] FIG. 12N illustrates the result of avidin binding indicating
quadrapole moment detection.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0062] Table of Contents
[0063] I. Definition of Terms
[0064] II. Introduction
[0065] A. Bio-Assay System
[0066] B. Chemistry of the System
[0067] III. The Bio-Assay Device
[0068] A. Device Structure
[0069] B. Binding Surface Chemistry
[0070] C. Bio-Electrical Interface
[0071] D. Specific Embodiments
[0072] IV. Measurement Methodology
[0073] A. General Overview
[0074] B. Detecting Molecular Binding Events
[0075] C. Detecting Changes in the Dielectric Properties
[0076] D. Identifying Molecular Binding Events
[0077] E. Identifying Classes of Bound Molecular Structures
[0078] F. Quantitating Concentrations
[0079] G. Bio-Assay Device Self-Calibration
[0080] V. Measurement Systems
[0081] A. Frequency Measurement System
[0082] B. Time Domain Measurement System
[0083] C. Dielectric Relaxation Measurement System
[0084] VI. Examples
[0085] VII. Applications
[0086] I. Definition of Terms
[0087] As used herein, the terms biological "binding partners" or
"ligand/antiligand" or "ligand/antiligand complex" refers to
molecules that specifically recognize (e.g. bind) other molecules
to form a binding complex such as antibody-antigen,
lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, 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".
[0088] 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 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 cells, cell
membranes, organelles and synthetic analogues thereof.
[0089] 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.
[0090] 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 or conductive surface. 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.
[0091] 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
heterogenous population of proteins and/or other biologics. Thus,
under designated conditions (e.g. immunoassay conditions in the
case of an antibody), the specified ligand or antibody binds to its
particular "target" (e.g. a hormone specifically binds to its
receptor) and does not bind in a significant amount to other
proteins present in the sample or to other proteins to which the
ligand or antibody may come in contact in an organism or in a
sample derived from an organism. Similarly, nucleic acids may
hybridize to one another under preselected conditions.
[0092] 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.
[0093] 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.
[0094] As used herein, the terms "polypeptide", "peptide" and
"protein" are used interchangeably to refer to a 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.
[0095] 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.
[0096] 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.
[0097] 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').sub.2 dimer into an Fab' monomer. The
Fab' monomer is essentially an 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.
[0098] 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.
[0099] 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).
[0100] 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.
[0101] As used herein, the term "signal path" refers to a
transmission medium along 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, multiple-conductor transmission mediums such as
transverse electromagnetic (TEM) transmission lines, transmission
lines with three or more conductive elements which support TE, TM
or TEM mode propagation such as quadrupolar and octupolar lines,
coupled waveguides, resonant cavity structures which may or may not
be coupled, other non-modal structures like wires, printed
circuits, and other distributed circuit and lumped impedance
conductive structures, and the like. The signal path may
structurally comprise the signal plane, the ground plane, or a
combination of both structures. Typically, the signal path is
formed along a direction which is non-orthogonal to the surface of
the MBL. In embodiments in which the signal path consists of a
conductive layer or region, the conductive region extends
continuously over that range. In embodiments in which the signal
path is non-metallic, i.e., a dielectric waveguide, the signal path
is defined as the path having the least amount of signal loss or as
having a conductivity of greater than 3 mhos/m.
[0102] As used herein, the terms "molecular binding layer" or "MBL"
refers to a layer having of at least one molecular structure (i.e.,
an analyte, antiligand, or a ligand/antiligand pair, etc.) coupled
to the signal path along the bio-electrical interface. The
molecular binding layer 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 layer 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 MBL 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 MBL may be of a derivatized surface such as by thiol
linkers biotinylated metals and the like, all in accordance with
standard practice in the art.
[0103] As used herein, the term "binding event" refers to an
interaction or association between a minimum of two 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 therebetween. Examples of
binding events of interest in a medical context include, but are
not limited to, ligand/receptor, antigen/antibody,
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.
[0104] 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.
[0105] 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.
[0106] As used herein, the term "test signal" refers to a signal
propagating at any useful frequency defined within the
electromagnetic spectrum. For examples, the test signal frequency
is 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.
[0107] 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.
[0108] As used herein, the term "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.
[0109] 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 the conductive
surface, and have a second binding portion that binds to another
component such as the matrix or the antiligand.
[0110] As used herein, the term "bio-assay device" refers to a
structure in which the molecular binding layer is formed. The
bio-assay device may consist of a surface, recessed area, or a
hermetically sealed enclosure, all of which may be any particular
size or shape.
[0111] 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, and vector analyzers necessary to probe
to and detect signals from the bio-assay device, microchips and
microprocessors which can probe and detect electromagnetic signals
and analyze data, and the like.
[0112] As used herein, the term "resonant" or "resonance" refers
generally to a rapidly changing dielectric response as a function
of frequency.
[0113] As used herein, "bio-electrical interface" refers to an
interface structure between a signal path for supporting the
propagation of a test signal and a molecular binding layer.
[0114] As used herein, the term "matrix" or "binding matrix" refers
to a layer of material on the bioassay chip that is used as a
spacer or to enhance surface area 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.
[0115] II. Introduction
[0116] A. The Bio-assay System
[0117] 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 a 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 layer.
[0118] FIG. 1A illustrates 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.
[0119] 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 alternative embodiments a single transmission line may
be coupled to the bio-assay device or further alternatively, three
or more transmission lines may coupled to the bio-assay device 150.
Transmission lines 120 are formed from a material which can support
the propagation of a signal over the desired frequency of
operation. Transmission lines 120 are realized as a conductive
layer, such as gold, deposited on a substrate, such as alumina,
diamond, sapphire, polyimide, or glass using conventional
photolithography or semiconductor processing techniques.
[0120] The system 100 further includes a bio-assay device 150
coupled to the transmission lines 120. The bio-assay device 150
contains a supporting substrate 151 onto which a conductive layer
153 is disposed. The conductive layer 153 forms an interface for
supporting the propagation of a test signal. The supporting
substrate 151 may consists of any insulating material such as
glass, alumina, diamond, sapphire, silicon, gallium arsenide or
other insulating materials used in semiconductor processing.
[0121] A molecular binding layer (MBL) 156 is coupled to one or
more areas of the interface transmission line 153. As those of
skill in the art of electronics will appreciate, coupling may occur
either through a direct connection between the interface
transmission line 153 and MBL 156 as illustrated, or alternatively
through signal coupling, further described below.
[0122] The MBL 156 is primarily composed of one or more ligands,
although other molecules and structures may also be included, as
described herein. The MBL 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 interface transmission line
153.
[0123] In the illustrated embodiment, dielectric substrate 158 is
located between solution 157 and ground plane 159. In the
illustrated embodiment, dielectric layer 158 and ground plane 159
are located within the bio-assay device 150, although in
alternative embodiments, one or both may be located externally.
Furthermore, the MBL 156 and solution 157 arrangement may be
switched and moved towards the ground plane alternatively, or in
addition to these layers' proximity to the interface transmission
line 153.
[0124] The system 100 includes a signal source 110 which launches
the test signal onto the transmission line 120 and towards the
bio-assay device 150. A signal detector 160 is positioned along the
transmission path to detect the resulting signal (either reflected
or transmitted or both). When the signal propagates along the
interface transmission line 153 of the bio-assay device 150, the
dielectric properties of the MBL 156 modulates the test signal. The
modulated signal can then be recovered and used to detect and
identify the molecular binding events occurring within the
bio-assay device, further described below.
[0125] In an alternative embodiment of the invention, detection and
identification of a ligand, antiligand/ligand complex or other
molecular structure described herein is possible when it is
physically separated from the interface transmission line 153. In
this embodiment, the ligand is separated from but electrically or
electromagnetically coupled to the interface transmission line 153.
The coupling between the interface transmission line 153 and the
suspended ligand will alter the response of the test signal
propagating along the interface transmission line 153, thereby
providing a means for detecting and/or identifying it. The maximum
separation between the interface transmission line 153 and
suspended ligand is determined by such factors as the effective
dielectric constant of the medium between the interface
transmission line 153 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.-1 m, 10.sup.-2 m 10.sup.-3 m,
10.sup.-4 m, 10.sup.-5 m, 10.sup.-6 m, 10.sup.-7 m, 10.sup.-8 m,
10.sup.-9 m, 10.sup.-10 m or range anywhere therebetween.
[0126] In some embodiment, such as cell based assays, the MBL may
be electromagnetically coupled to the signed path through the
solution. Thus, cells, and in particular cell membranes and
membrane-based structures may couple to the signal.
[0127] FIG. 1B illustrates a second embodiment of the bio-assay
system comprising an array of resonant microstrip circuits 170.
Each resonant circuit 170 consists of a transmission line 172
terminating in an open-circuited stub 176. Those skilled in the art
of circuit design will appreciate other resonant structures may be
employed in lumped element, distributed, or a combination of both
circuit topologies.
[0128] FIG. 1C illustrates a cross-section view of one resonant
circuit 170. The open-circuited stub 176 forms the bio-electrical
interface of the resonant circuit 170 and closely parallels the
bio-electrical interface shown in FIG. 1A. In particular, the
open-circuited stub 176 consists of an interface transmission line
176a deposited on a dielectric layer 176b, and is positioned above
ground plane 176c.
[0129] In this embodiment, the MBL 176d is coupled via a direct
connection to transmission line 176a. The MBL 176d can bind along
the interface transmission line in a specific or non-specific
manner. As above, the subject molecular structure may be suspended
from but electrically coupled or electromagnetically coupled to the
interface transmission line 176a to provide binding event detection
and identification information.
[0130] The dimensions of the interface transmission line 176a are
influenced by considerations such as the desired measurement time
(a larger area resulting in faster detection time), the desired
resonant frequency f.sub.res, certain impedance matching conditions
to achieve higher efficiency or cause discontinuities to highlight
binding events, and the process by which the entire array is
formed. For instance, if conventional microwave photolithography is
used, the binding surface area may range from 10.sup.-1 m.sup.2 to
10.sup.-6 m.sup.2 using a relatively thick dielectric layer such as
alumina, diamond, sapphire, duriod or other conventional substrate
materials. Alternatively, if semiconductor processing is used, the
binding surface area may range from 10.sup.-6 m.sup.2 to 10.sup.-12
m.sup.2 using a relatively thin dielectric layer of silicon or
gallium arsenide.
[0131] Using conventional microwave design techniques or CAD tools
such as Microwave Spice.TM., EEsof Touchstone.TM. and Libra.TM.,
the length and impedance of the transmission line 172, the
dimensions of the interface transmission line 176a, and the
thickness and dielectric constant of the dielectric layer 176b can
be selected such that the resonant structure exhibits a resonant
signal response at a desired resonant frequency point f.sub.res.
The desired resonant frequency f.sub.res point is typically the
frequency range over which the molecules of interest exhibit a
dramatic change in their dielectric properties, the measurement of
which will enable their detection. Alternatively, the resonant
frequency point f.sub.res can be defined as the center of the
desired test frequency range to allow for the widest range of
signal detection. In the illustrated embodiment, the resonant
frequency f.sub.res includes 10 MHz, 20 MHz, 45 MHz, 100 MHz, 500
MHz, 1 GHz, 5 GHz, 10 GHz, 30 GHz, 50 GHz, 100 GHz, 500 GHz, 1,000
GHz or frequencies ranging therebetween.
[0132] During measurement, the solution 176e is applied over one or
more of the open-circuited stubs 172. A MBL 176d is formed when one
or molecules within the solution bind to the interface transmission
line 176a. In this instance, the MBL 176d and the solution
electrically behave as a parasitic circuit, further described
below, which causes the resonant frequency point f.sub.res to shift
above or below its original resonant frequency point. This shift in
frequency can be detected, and is used to indicate the occurrence
of a molecular binding event. The signal response may also be
interrogated over a wide spectrum to ascertain the identity of the
bound molecular structure, as described below. Each resonant
circuit 170 may be fabricated to bind different molecular
structures and each resonant circuit 170 be made addressable,
thereby permitting simultaneous detection and identification of a
large numbers of molecular structures within the same solution. In
an alternative embodiment, each resonant circuit 170 may be
designed to exhibit a distinct resonant frequency, in which case
all of the resonant circuits 170 may be interrogated over a
continuous frequency spectrum to determine molecular binding.
[0133] B. Chemistry of the System
[0134] The chemistry of the system generally occurs within the
bio-assay device, and in particular along the conductive layer
(interface transmission line in FIGS. 1A-1C). The conductive layer
is fabricated from materials and having a morphology which is
conducive to support the propagation of the high frequency test
signal. The conductive surface is constructed from materials
exhibiting appropriate conductivity over the desired test frequency
range as well as possessing good molecular binding qualities as
described above. Such materials include, but are not limited to
gold, 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. The
conductive layer may also be formed from semiconducting materials
which may be either crystalline or amorphous in structure,
including chemically doped or pure carbon, silicon, germanium,
gallium-arsenide, idium-gallium arsenide, or the like. The
conductive material may also be formed from polymers especially
those that are conductive such as polyacetylene, polythiophene and
the like. The conductive layer may be thick or only several
molecular layers in depth as the application requires. The
conductive layer may be comprised of an evaporated thin metal layer
or an epitaxial layer of gallium arsenide or other semiconductor
materials rendered conductive through known semiconductor
processing techniques. In addition, the conductive layer may be
derivatized, the process by which is well known, e.g., see Kumar et
al., "Patterned Self-Assembled Monolayer and Mesoscale Phenomena,"
Accounts of Cemical Research, 28:219-226 (1995).
[0135] The conductive layer is additionally fabricated from
materials and having a morphology which is conducive to facilitate
molecular binding. Ligands may bind directly, indirectly through
other molecular structures, or through both configurations to bind
to the conductive layer. The range of molecules that may bind to
the conductive layer include but are not limited to proteins,
nucleic acids, small molecules, saccharides, lipids, and any other
molecule of interest. The chemistry may involve only a single
species of molecules attached to the surface, a whole array of
different species attached to the surface, or multiple binding
events between species directly attached to the surface and ligands
of interest in the solution.
[0136] The typical chemistry involved in attaching a ligand to the
conductive layer will in general depend on the nature of the ligand
and any antiligand to which it binds, and their functions in the
assay. A list of possible types of interactions that may occur on
the surface include but are not limited to: Protein/protein
interactions, DNA/protein interactions, RNA/protein interactions,
nucleic acid hybridization, including base pair mismatch analysis,
RNA/RNA interactions, tRNA interactions, Enzyme/substrate systems,
antigen/antibody interactions, small molecule/protein interactions,
drug/receptor interactions, membrane/receptor interactions,
conformational changes in solid phase ligands, protein/saccharide
interactions, and lipid/protein interactions.
[0137] The actual surface chemistry may be described in one
embodiment as primary binding and secondary binding. Additional
layers of molecular binding may also occur. Primary binding refers
to the attachment of an antiligand to the conductive surface, which
can be done through the assistance of a linker molecule. Secondary
binding refers to the binding of a ligand to the antiligand, which
may be another molecule in the MBL or directly to the conductive
surface itself. Typically, the binding involves a liquid phase
ligand binding to an immobilized solid phase antiligand. For
example, primary binding could be the attachment of a specific
antibody to the conductive layer of the bioassay device and
secondary binding would involve the binding of a specific antigen
in a sample solution to the antibody. Alternatively, secondary
binding may be the direct attachment of a protein to the conductive
surface, such as the amine terminus of a protein attaching directly
to a gold conductive layer.
[0138] The aforementioned binding results in the formation of a
molecular binding layer (MBL) 180 along one or more areas of the
conductive layer, one embodiment of which is illustrated in FIG.
1D. In this embodiment, the MBL 180 optionally consists of a first
linker 181, an insulator 182, a second linker 183, a matrix 184, a
third linker 185, an antiligand layer 186, and a ligand layer
187.
[0139] First linker 181 provides attachment between insulating
layer 182 and conductive layer (not shown). First linker 181
consists of molecule such as thiols, amines, amides, or metals such
as chromium or titanium. Insulating layer 182 provides a barrier
between the conductive layer and the MBL 180 and solution (not
shown). Insulating layer 182 may provide a hermetic barrier to
prevent structural deterioration of conductive layer due to
exposure to the MBL and/or solution. Alternatively, or in addition,
insulating layer 182 may consist of an electrically non-conductive
material to prevent the flow of DC or low frequency energy from the
conductive layer to the MBL and/or solution which could interfere
with the measurement. The insulating layer may include polyimide,
alumina, diamond, sapphire, non-conductive polymers, semiconductor
insulating material such as silicon dioxide or gallium arsenide or
other materials which provide hermetic and/or electrically
insulating characteristics. The insulating layer may also consist
of air, or another gaseous substance, in which case linker 181 may
be deleted.
[0140] Second linker 183 provides attachment between the insulating
layer 182 and matrix 184 and consists of the same or similar
molecules as first linkers 181. Matrix layer 184 may consist of a
polymer layer, but is also optionally a carbohydrate, protein,
poly-amino acid layer or the like. Third linker 185 consists of
molecules suitable for attaching the matrix layer to the antiligand
186 and may consist of the same or similar molecules as either
first and/or second linkers 181 and 183.
[0141] Antiligand 186 is used to specifically or non-specifically
bind the ligand 187 within solution and/or to measure physical
properties of the solution, some examples of which are temperature,
pH, ionic strength, and the like. Antiligand consists of a molecule
or molecular structure which specifically or nonspecifically binds
to ligand 187. For instance, in the case in which the ligand
consists of an antigen, antiligand 186 will consist of an antibody.
Ligand 187 consists of a molecule or structure which specifically
or nonspecifically binds to the antiligand 186.
[0142] Generally, the MBL will be sufficient to interact measurably
as described herein with an electromagnetic test signal along the
associated signal path. Thus, essentially any MBL composition that
exhibits varying dielectric properties can be analyzed. In most
embodiments, the MBL will range in thickness between about 1-5
.ANG. to 1 cm. For simple molecular binding events, the range will
be usually between about 10 .ANG. to 10,000 .ANG., typically
between 100 .ANG. and 5,000 .ANG., or 500 .ANG. to 1,000 .ANG.. In
larger interactions (e.g. cellular) the MBL will range between 1
.mu.m and 100 .mu.m, preferably 5 .mu.m to 50 .mu.m. With
insulators, matrices and the like, the size will range
significantly higher.
[0143] The embodiment of FIG. 1D is not intended to be exhaustive
of all possible MBL configurations. Those of skill in the art will
appreciate that a vast multiplicity of combinations making up the
MBL can be designed, as dictated by the specific applications. For
instance, first, second and third linkers 181, 183, 185, insulating
layer 182, and matrix layer 184 are not implemented and the MBL
consists of antiligand 186 and ligand 187. Further alternatively,
first linker 181 and insulating layer 182 may be deleted. Other
alternative embodiments in which one or more of the described
layers are deleted, or additional layers added, will be apparent to
one skilled in the art.
[0144] Further, the MBL may be composed of heterogeneous molecules
which may be spatially grouped or randomly layered or distributed
depending upon the particular array format. For example, FIG. 1E
illustrates a top view of an MBL 180 having four different
antiligands 190, 191, 192 and 193, which are spatially separated.
FIG. 1F illustrates an MBL 180 in which four different antiligands
190, 191, 192 and 193 are randomly distributed throughout. In
another embodiment, FIG. 1G illustrates a cross-sectional view in
which the MBL 180 contains cells 194 in solution 157 coupled to
signal path 153. In another embodiment, a cell membrane 195, with
membrane bound structures (not shown), is in solution 157 coupled
to signal path 153. The layers may include for example, linkers,
matrices, antiligands, ligands and one or more insulating layers.
In some embodiments, one or more membranes may be employed, such as
those controlling ion transport, size or charge selection or
supporting the attachment of antiligand or other molecular
structures.
[0145] Electrically, the MBL exhibits unique dielectric properties
which are in part attributable to the structural and conformational
properties, and changes therein, of bound molecules, both isolated
and in the presence of environmental changes such as binding
events, pH changes, temperature, ionic strength and the like. The
dielectric properties of the bound molecular structures, along with
the local structures of the solvating medium (the solution) may
also be attributable to changes in the intramolecular and
intermolecular bonds caused by primary or other higher-order
binding, and the displacement of the solvating medium near the
conductive layer.
[0146] Once a conductive layer is provided, one of skill in the art
will be generally familiar with the biological and chemical
literature for purposes of selecting a system with which to work.
For a general introduction to biological systems, see, Current
Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc, and John Wiley & Sons, Inc. (through 1997 Supplement)
(Ausubel); Watson et al. (1987) Molecular Biology of the Gene,
Fourth Edition, The Benjamin/Cummings Publishing CO., Menlo Park,
Calif.; Alberts et al. (1989) Molecular Biology of the Cell, Second
Edition Garland Publishing, NY; The Merck Manual of Diagnosis and
Therapy, Merck & Co., Rathway, N.J. Product information from
manufacturers of biological reagents and experimental equipment
also provide information useful in assaying biological systems.
Such manufacturers include, e.g., the SIGMA chemical company (Saint
Louis, Mo.), R&D systems (Minneapolis, MN), Pharmacia LKB
Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo
Alto, Calif.), Aldrich Chemical Company (Milwaukee, Wis.), GIBCO
BRL Life Technologies, Inc, (Gaithersberg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland,
Applied Biosytems (Foster City, Calif.), as well as many other
commercial sources known to one skilled in the art.
[0147] Biological samples can be derived from patients using well
known techniques such as venipuncture, lumbar puncture, fluid
sample such as saliva or urine, or tissue biopsy and the like. When
the biological material is derived from non-humans, such as
commercially relevant livestock, blood and tissue samples are
conveniently obtained from livestock processing plants. Similarly,
plant material used in the invention may be conveniently derived
from agriculture or horticultural sources, and other sources of
natural products. Alternatively a biological sample may be obtained
from a cell or blood bank where tissue and/or blood are stored, or
from an in vitro source, such as a culture of cells. Techniques for
establishing a culture of cells for use as a source for biological
materials are well known to those of skill in the art. Freshney,
Culture of Animal Cells a Manual of Basic Technique Third Edition,
Wiley-Liss, NY (1994) provides a general introduction to cell
culture.
[0148] The present invention can be practiced in a number of
embodiments. Some are detailed below, additional embodiments and
applications are detailed in the applications section.
[0149] In one embodiment, the invention is used to detect binding
of a molecular structure to the signal path. In this embodiment, a
signal is propagated along the signal path. As it propagates, it
couples to the bound structure and is modulated. Analysis of the
modulated response indicates binding.
[0150] In another embodiment, the invention may be used to identify
secondary binding. For example, primary binding may be the
attachment of an antibody to the conductive surface. Secondary
binding might involve the measurement of binding between the
immobilized antibody and its antigen in solution. After primary
binding has been detected as described in the previous paragraph,
the solution containing the antibody is added to the bio-assay
device and the response measured again. The response is compared to
the primary binding response. A change would indicate that a
binding event has occurred.
[0151] In one aspect, the present invention may be used to identify
ligands, for example proteins, in the primary binding stage. In the
calibration phase the responses of a large number of known proteins
can be determined and stored. After attaching an unknown protein to
the assay surface, the dielectric properties of the system could be
measured and the dielectric properties of the signal used to
identify the protein on the surface. Because each protein's
fingerprint response is stored, the unknown response can be
compared with the stored responses and pattern recognition may be
used to identify the unknown protein.
[0152] In another embodiment, the invention may be used in an array
format. The device will have multiple addressable sites, each of
which has bound to it a specific antiligand. After delivering
solution to the device, binding responses at each site will be
measured and characterized. A device of this type may be used to
measure and/or identify the presence of specific nucleic acid
sequences in a sample. At each of the addressable sites a unique
nucleic sequence is attached as the antiligand. Upon exposure to
the sample, complementary sequences will bind to appropriate sites.
The response at each site will indicate whether a sequence has
bound. Such measurement will also indicate whether the bound
sequence is a perfect match with the antiligand sequence or if
there are one or multiple mismatches. This embodiment may also be
used to identify proteins and classes of proteins.
[0153] In another embodiment, this invention may be used to
generate a standard curve or titration curve that would be used
subsequently to determine the unknown concentration of a particular
analyte or ligand. For example, an antibody could be attached to
the device. The device could be exposed to several different
concentrations of the ligand and the response for each
concentration measured. Such a curve is also known to those skilled
in the art as a dose-response curve. An unknown sample can be
exposed to the device and the response measured. Its response can
be compared with the standard curve to determine the concentration
of the ligand in the unknown sample.
[0154] In another embodiment, this invention may be used to
internally self-calibrate for losses due to aging and other
stability issues. For example with antibody-antigen systems, this
invention allows one to measure the amount of active antibody on
the surface by measuring a primary response before exposure the
unknown. The value of the primary response is used to adjust the
secondary response, antigen binding, by a constant that reflects
the amount of active antibody that remains on the device.
[0155] III. The Bio-assay Device
[0156] A. Device Structure
[0157] Structurally, the bio-assay device includes a signal path
and a bio-electrical interface. The signal path may consist of a
single input/output signal port; one input signal port path and one
output port path, or multiple input and/or output signal port
paths. The signal path(s) may be realized in a number of different
architectures, such as a conductive wire, a transmission line, a
waveguide structure, resonant cavity, or any other transmission
medium that will support the propagation of the test signal over
the desired frequency range. For possible embodiments, see 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. Further, the bio-assay device may also be realized in a
variety of different configurations. Non-exhaustive configurations
include large to miniaturized structures using conventional
manufacturing techniques, conventional etching and
photolithography, or semiconductor processing techniques.
[0158] FIG. 2A illustrates one embodiment of the bio-assay device
as shown in cross-sectional view. The bio-assay device 230 consists
of a top plate 231, contact terminals 237, and a bottom plate 239.
Top plate 231 includes a bottom surface having an interface
transmission line 233 disposed thereon. The dielectric substrate
240 and the ground plane 250 are located external to the bio-assay
device. Top plate 231 and/or dielectric substrate 240 are formed
from an insulating material, such as glass, which are preferably
compatible with conventional photolithography or gold sputtering,
etching or chemical vapor deposition (CVD) processing. Other
materials such as alumina, silicon, gallium arsenide or other
insulating materials, may alternatively be used.
[0159] As illustrated in FIG. 2A, the bottom surface of the
interface transmission line 233 is in contact with the molecular
binding layer (MBL) 234. As illustrated, the MBL may consist of
bound molecular structures of different layers or types as well as
molecular structures occurring within the solution. In alternative
embodiments, the MBL 234 may extend over small or large portions of
the interface transmission line 233 and may consist of different
bound molecular structures as shown. The MBL may consist solely of
antiligand/ligand structures, or a variety intermediate of linker,
matrix, and insulating layers, as shown in FIG. 1D. When
implemented, the insulating layer 182 (FIG. 1D) may consist of air,
polyimide, alumina, diamond, sapphire, or semiconductor insulating
material such as silicon dioxide or gallium arsenide or a
non-conductive material in addition to other conventional
insulating materials. The thickness and dielectric constant of the
insulating layer are such that the MBL 234 and the interface
transmission line 233 are tightly coupled together during signal
transmission. The thickness of the insulating layer 182 maybe
10.sup.-1 m, 10.sup.-2 m, 10.sup.-3 m, 10.sup.-4, 10.sup.-5 m,
10.sup.-6 m, 10.sup.-7 m, 10.sup.-8 m, 10.sup.-9 m, 10.sup.-10 m or
less in thickness, or values ranging therebetween, depending the
amount of coupling required, the dielectric constant of the
insulating layer, and the total coupling area. Coupling may be
accomplished through a number of different configurations,
including broadside and offset coupled configurations in
multi-layer, coplanar, or waveguide circuit topologies.
Implementing an insulating layer may be advantageous for
hermetically sealing the interface transmission line from the
solution medium and/or for preventing DC or low frequency current
from flowing into the solution which could possibly disrupt
molecular binding events occurring therein.
[0160] The interface transmission line 233 consists of a material
which is capable of supporting signal propagation and which is
capable of binding the MBL 234. The material will vary depending
upon the makeup of the MBL, but some will include gold, 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. Alternatively, the interface
transmission line 233 may include one or more molecular structures
(antiligands) (which forms a part of the MBL 234) for forming bonds
with one or more targeted molecules (ligands). The material
comprising the interface transmission line may also be chosen to
promote the attachment of linkers as well as to support signal
propagation. Other materials that can be used to form the interface
transmission line 233 will be readily apparent to those of skill in
the art.
[0161] The ligands may be transported to the MBL 234 using a
solution 260, such as Dulbecco's phosphate-buffered saline (d-PBS)
for example. The protein, nucleic acid, or other ligand of interest
can be applied to the binding surface using a variety of techniques
such as wicking, pipeting, dipping, dropping, direct contact, or
through capillary action.
[0162] In a specific embodiment, the interface transmission line
233 is designed to provide low signal loss and close impedance
matching to the external transmission lines 270. Low signal loss is
achieved by fabricating the interface transmission line 233 from a
conductive material, some examples being gold, copper, aluminum,
indium tin oxide (ITO) or other conductive materials described
above. Close impedance matching is achieved by defining the width
of the interface transmission line 233 at approximately the width
of external transmission lines 270, depending on the relative
dielectric properties of the substrate, the solution, and the MBL.
Signal continuity between the interface transmission line 232 and
the external transmission lines 270 is provided via contact
terminals 237. As explained above, the MBL 234 and solution medium
260 may be located proximate to the ground plane 250 alternatively,
or in addition to these layer's location proximate to the interface
transmission line 232.
[0163] Additional analog and/or digital circuitry in lumped element
form, distributed form, or a combination of both may be included at
the input and/or output ports of the bio-assay device. For
instance, impedance matching circuits and/or buffer amplifier
circuits may be employed at the input port. Alternatively, or in
addition, impedance matching circuitry and one or more output
amplifiers may be implemented to further enhance the output signal.
Those of skill in the art of electronics will appreciate that other
types of conditioning circuitry may be used in alternative
embodiments as well.
[0164] FIG. 2B illustrates a second embodiment of the bio-assay
device. In this embodiment, the solution occupies a space above the
interface transmission line 233 which is formed on the top surface
of bottom plate 239. The top side of the interface transmission
line 233 forms the binding surface to which the MBL 234 adheres.
Dielectric layer 240 is positioned between interface transmission
line 233 and the ground plane 250. Contact terminals 237 provide a
signal path to the external transmission lines 270. The interface
transmission line, top plate, bottom plate, contact terminals, and
dielectric layer may be formed from the materials and the processes
as described above. The MBL may also be configured as described
above in FIG. 1D, or variations thereof. Further, the MBL 234 and
solution medium 260 may be located proximate to the ground plane
250 alternatively, or in addition to these layer's location
proximate to the interface transmission line 233.
[0165] Additional structural embodiments include bio-assay devices
having multi-element transmission lines, waveguides, and resonant
cavities, in which the MBL may be attached to one or more of the
line or cavity elements in such a way as to enhance detection
specificity and sensitivity. Examples of such structures include
parallel arranged signal combiners, resonant cavities, or
waveguides along which the bound MBL on one element alters the
signal propagation properties as compared to another parallel
element without the bound structure, and thus serve to change the
mode properties of the combined signal, resulting in readily
detectable output signal properties. These latter effects make use
of well-known techniques to measure frequency, frequency stability,
and very small changes in the frequency with ultra-high
precision.
[0166] B. Binding Surface Chemistry
[0167] FIG. 3 illustrates one embodiment of the binding surface
chemistry which occurs along the conductive layer of the
bio-electrical interface. The bio-electrical interface includes a
substrate 320, a conductive layer 330, a MBL 340, and solution 350.
The substrate 320 may be any of the dielectric layer or substrate
materials described herein including alumina, diamond, sapphire,
plastic, glass and the like and may provide structural support to
the conductive layer 320. In an alternative embodiment, substrate
320 is removed and structural support is provided via insulating
layer 342.
[0168] The conductive layer 330 consists of a material and
morphology which will promote signal propagation over the desired
frequencies and which will promote binding of the MBL 340, as
described above. In a two-conductor circuit topology, conductive
layer 330 may comprise the signal plane or the ground plane. In
either case however, a second conductive layer (either the signal
plane or the ground plane, not shown) is located either below the
substrate 320 (the arrangement of FIG. 2B) or at least one
substrate layer removed from the solution 350 (an inverted
arrangement of FIG. 2A). Alternatively, conductive layers may be
positioned at both of these levels.
[0169] Solution 350 is coupled to the MBL 340 for permitting the
flow of ligands to the MBL 340. Ligand flow from solution 350 to
MBL 340 may directionally or non-directional. Solution consists of
any transporting medium such as gases, ligius, or solid phase
materials, some examples being aqueous d-PBS, Tris buffer,
phosphate buffers, and the like.
[0170] Along the bio-electrical interface, the MBL is positioned
between at least a portion of the solution and the signal path,
such that the MBL is more proximate to the signal path than the
solution along that portion. In the embodiment of FIG. 3, the MBL
340 is positioned between the solution 350 and the conductive layer
330, closer in proximity to the latter. In one embodiment (shown in
FIG. 2A), the solution is positioned between the signal and ground
planes. In a second embodiment (shown in FIG. 2B), the solution is
positioned outside of the signal-ground plane region.
[0171] The typical chemistry involved in binding the MBL to the
conductive surface will in general depend on the nature and content
of the MBL its function in the assay. The MBL may consist of a
ligand, ligand/antiligand complex, or other molecular structures as
described herein. Typically, the ligand will be functionally
intact, as close to the surface as possible, and the surface
density of the antiligand will be high enough to provide the
greatest dielectric effect, but not so high as to impair the
function of binding, such as by steric hindrance or physically
blocking the active binding site of the immobilized antiligand by
neighboring molecules.
[0172] Ligands may bind specifically or non-specifically either
directly to the conductive layer 320 or intermediate structures as
shown in FIG. 3. If specifically bound ligands are desired, a
linker is optionally used to facilitate the binding, for example to
bind all proteins such that conductive layer 320 is exposed to
solution. To ensure a densely pack binding layer, thiol groups,
Fab, or proteins such as protein A may be used to facilitate the
binding of antibodies or other antiligands along the conductive
layer 320. These and similar substances may be applied to the
conductive layer 320 in a number of ways, including
photolithography, semiconductor processing, or any other
conventional application techniques.
[0173] In addition, some ligands and antiligands may be able to
bind in multiple ways. These ligands typically have a statistically
predominant mode of binding or may be engineered to bind in a
site-specific way. Some antiligands optionally bind the surface in
a site-specific manner. For example, an oligonucleotide might be
bound at one terminus. Genrally, the antiligand will be attached in
a manner which will not impair the function of the antiligand,
e.g., preferably at concentrations that minimize surface
denaturation.
[0174] The concentration of the antiligand on the binding surface
will vary, depending upon the specific analyte. For example,
typical concentrations for proteins are 10.sup.7/cm.sup.2,
10.sup.8/cm.sup.2, 10.sup.9/cm.sup.2, 10.sup.10/cm.sup.2,
10.sup.11/cm.sup.2, 10.sup.12/cm.sup.2, 10.sup.13/cm.sup.2,
10.sup.14/cm.sup.2, 10.sup.15/cm.sup.2, or concentrations ranging
therebetween. Typical concentrations for nucleic acids are
10.sup.7/cm.sup.2, 10.sup.8/cm.sup.2, 10.sup.9/cm.sup.2,
10.sup.10/cm.sup.2, 10.sup.11/cm.sup.2 , 10.sup.12/cm.sup.2,
10.sup.13/cm.sup.2, 10.sup.14/cm.sup.2, 10.sup.15/cm.sup.2,
10.sup.16/cm.sup.2, 10.sup.17/cm.sup.2, 10.sup.18/cm.sup.2,
10.sup.19/cm.sup.2, 10.sup.20/cm.sup.2, or concentrations ranging
therebetween. Typical concentrations for analytes in whole blood
range from 55M, 25M, 10M, 1M, 0.5M, 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, 10.sup.-12M,
10.sup.-13M, 10.sup.-14M, 10.sup.-15M, 10.sup.-16M, 10.sup.-17M,
10.sup.-18M, or concentrations ranging therebetween.
[0175] Enough ligand should adhere within the MBL to alter the
transmission of a signal through the bio-electrical interface. The
quantity of ligands adhering to the binding surface may consist of
1, 10, 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, 10.sup.11, 10.sup.12, 10.sup.13 or
more ligands, as well as any number therebetween depending upon the
surface area of the conductive layer. The ligands need not be
applied in predefined regions along the conductive layer since the
signal responses are determined by inherent dielectric properties
of the MBL as opposed to placement on the bio-assay device or chip.
The MBL will generally have a surface density for smaller molecules
ranging from 10.sup.10 cm to 10.sup.24 cm.sup.2, typically
10.sup.15 cm.sup.2 to 10.sup.20 cm.sup.2. The ligand layer may be
as thin as 1 layer, but 2, 3, 4, 5 or 10 or more layers are
optionally used.
[0176] Once a ligand is bound to the conductive layer, the
chemistry and/or structural biology of the system comes into play.
The ligand's dielectric properties yield a signal response which is
characteristic of the bound structure(s), thereby permitting
binding event detection, as well as detection of other properties
of interest in the structure. The unique response provided by the
binding event will depend on the immobilized antiligand, its target
ligand, and the rearrangement of the nearby solution molecules
(such as water and free ions). The range of molecules that can bind
to the surface include but are not limited to proteins, nucleic
acids, small molecules, saccharides, lipids, and any other molecule
of interest.
[0177] Typically, the molecules of the MBL are disposed within a
solution which may consist of an aqueous solution of water, d-PBS,
Tris, blood, physiological buffer, cerebrospinal fluid, urine,
sweat, saliva, other bodily secretions, organic solvents, and he
like. Other solutions may include gases, emulsions, gels, and
organic and inorganic compounds The secondary binding reaction
occurs at the MBL of the bio-assay device. A ligand in a solution
is transported across the bio-assay device such that it contacts
the antiligand of the binding layer. The concentration of the
ligand in the solution varies and may consist of 10.sup.-1M,
10.sup.-2M, 10.sup.-3M, 10.sup.-4M, 10.sup.-5M, 10.sup.-6M,
10.sup.-7 M, 10.sup.-8M, 10.sup.-9M, 10.sup.-10M, 10.sup.-11M,
10.sup.-12M, 10.sup.-13M, 10.sup.-14M, 10.sup.-15M, 10.sup.-16M,
10.sup.-17M, 10.sup.-18M, 10.sup.-19M, 10.sup.-20M. When an
interaction, such as binding, occurs between the ligand and the
antiligand, the ligand, then optionally becomes part of the binding
layer, as dictated by the chemical equilibrium characteristics of
the binding event.
[0178] The MBL includes the bound ligands and may also include
solution molecules. The bound ligands can be any molecule,
including proteins, carbohydrates, lipids, nucleic acids, and all
other molecules discussed herein. The MBL may further include a
linker to aid in the binding of the antiligand to the binding
surface layer.
[0179] Additionally, the interaction of the antiligand with the
ligand changes the characteristic dielectric response of the
binding layer with only the antiligand attached. For example, if
antiligand A is the antiligand that forms the binding layer, the
dielectric response of a test signal propagating along the
transmission line will reflect the characteristic properties of the
structure of antiligand A. When ligand B binds to antiligand A, the
structure and/or dielectric properties of the binding layer will
change due to the binding of A to B. The structure of A may change
as B binds to it, thus providing a different signal response. The
change in signal due to the binding interaction will be
characteristic of the binding of A to B. Therefore, the presence of
a binding interaction can be determined from the change in the
signal.
[0180] Moreover, information about the type of bond or the
structural and/or conformational changes upon binding is obtained
by noting which parts of the signal response have changed due to
the interaction. Ligand B is optionally detected and identified by
the signal change upon its binding to antiligand A. The binding of
ligand B to antiligand A induces a conformational change, or other
change in the molecular structure or surrounding solution, in
antiligand A and its environs. These changes alter the dielectric
properties of the MBL, thereby altering the signal response of the
test signal propagating along the signal path. The change in the
test signal can be used to detect the ligand B binding event and
the particulars of the change can be used to identify the ligand B.
In as much as the relationship between structure and function of
the molecule is known, for example in the case of enzymes,
antibodies, receptors and the like, the function of the bound
ligand can be deduced from its spectral identification.
[0181] In one embodiment, one type of antiligand is applied to the
binding surface to form a MBL, and a ligand is applied across the
MBL to detect a binding event between the two molecules. In another
embodiment, the antiligand may be a mixture and the ligand that is
applied across the binding layer is a known analyte or antibody. By
detecting specific changes in the signal response, the particular
ligand with which the antiligand interacted can be determined due
to conformational and other changes induced in the ligand or
antiligand, and the spectral response resulting therefrom. Such an
embodiment does not require the spatial isolation of each of the
specific antiligands, but rather derives the desired level of
specificity from the spectral response, so that a given binding
interaction is determined by looking at the electromagnetic
response rather that noting on which part of the assay the binding
event took place.
[0182] In another embodiment, the antiligand may be a known
molecule on the binding layer and the ligand applied across the
bio-assay device as a mixture of unknowns, such as a whole blood
sample. In this case, the presence of a particular ligand such as
an antibody in the blood is detected by the presence or absence of
a particular peak or signal in the spectrum that results from
passing a signal through the bio-assay device. Alternatively it can
be detected due to the changes in the spectrum of the antiligand or
ligand upon binding of the ligand. Such an embodiment increases the
specificity of the detection over that of the binding chemistry
alone, since the signal contains information about the nature of
the binding event. Thus, specific binding may be distinguished over
non-specific binding, and the overall specificity of detection may
be greatly improved over the specificity of the chemistry
alone.
[0183] The system of detection formed through use of the bioassay
device provides a high throughput detection system because
detection optionally occurs in real time and many samples can be
rapidly analyzed. The response period is optionally monitored on a
nanosecond time scale. As soon as the molecules are bound to each
other, detection occurs. More time is optionally required to
measure low concentrations or binding events between molecules with
a low binding affinity. The actual time is optionally limited by
diffusion rates. Other than these potential limitations, thousands
of compounds are optionally run through the system very quickly,
for example, in an hour. For example, using chip fab technologies,
a 10,000 channel device (using some of the emerging microfluidics
technologies) is possible, and with small volumes and thus short
diffusion times, and kinetic measurements measuring only the
beginning of the reaction, 10 million samples per hour are
optionally measured. With known concentrations, the binding
affinity is optionally calculated from the kinetics alone and thus
the device can be probed at a very fast time scale and the affinity
calculated and/or estimated from the slope of the kinetic curve.
References for kinetics and affinities can be found in any standard
biochemistry or chemistry text such as Mathews and van Holde,
Biochemistry, Benjamin Cummings, New York, 1990.
[0184] C. Bio-electrical Interface
[0185] The bio-electrical interface is the structure along which
the MBL and the signal path are formed. As described above, the
signal path may consist of a conductive or dielectric waveguide
structure, a two conductor structure such as a conventional
signal/ground plane structure, or three or more conductor
structures known in the art. Generally, the thickness of the
conductive region of the signal path is designed to provide minimal
signal loss. For example, a typical thickness of gold transmission
line is in the order of 0.1 to 1000.mu.m, preferably about 1-10
.mu.m.
[0186] The signal path is formed along a direction which is
non-orthogonal to the MBL. In one embodiment, the test signal
propagates in parallel to a tangent on the surface on which the MBL
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 MBL 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.
[0187] The operation of the bio-electrical interface will be better
understood by developing an equivalent circuit model for the
interface. The equivalent circuit models presented are shown in a
two-conductor circuit topology, although those of skill in the art
of circuit design will readily appreciate that each may be
implemented in single conductor waveguide topologies, resonant
circuit topologies, as well as circuit topologies with three or
more conductors.
[0188] FIG. 4A illustrates one embodiment of an equivalent circuit
model 420 for the bio-electrical interface structure shown in FIG.
2A. Those of skill in the art of circuit design will appreciate
that the illustrated circuit model is not exhaustive and that other
equivalent circuit models may be derived from the bio-electrical
interface of FIG. 2A.
[0189] The illustrated equivalent circuit model includes series
blocks 422a, 424a, and 426a which models the series electrical
effects of the interface transmission line 232, the MBL 234, and
the solution 260, respectively, all as shown in FIG. 2A. The
interface transmission line, MBL, and solution circuit blocks 422a,
424a, and 426a are coupled in parallel since the interface
transmission line, the MBL, and the solution, each provides a
possible longitudinal signal path along the interface. In an
alternative embodiment where the MBL and solution are located
proximate to the ground plane, the interface transmission line and
the ground planes of the equivalent circuit model 420 are switched.
In the embodiments where the MBL and solution are located proximate
to both the interface transmission line and the ground plane, FIG.
2A represents the top half of the equivalent circuit, the bottom
half (ground plane) of which is identical if the same solution and
MBL is used.
[0190] The equivalent circuit model 420 further includes shunt
circuit blocks 422b, 424b, and 426b which models, respectively, the
shunt electrical effects of the dielectric layer 240, the MBL 234,
and the solution 260, shown in FIG. 2A. The series orientation of
shunt blocks 422b, 424b, and 426b results from the physical
arrangement of each of these elements, occurring serially from
interface transmission line through the MBL, solution, and the
dielectric layer, to the ground plane, the arrangement of which is
shown in FIG. 2A.
[0191] FIG. 4B illustrates one embodiment of a circuit 430
corresponding to the equivalent circuit model shown in FIG. 4A.
Those of skill in the art of circuit design will readily appreciate
that other circuits configurations are possible. The series circuit
blocks 422a, 424a, and 426a each consists of a series-coupled
resistor and inductor. The shunt circuit blocks 422b, 424b, and
426b each consists of a parallel-coupled resistor and capacitor.
Series resistors R.sub.t, R.sub.m, R.sub.S model respectively the
resistivity of the interface transmission line, the MBL, and the
solution. Shunt resistors R.sub.m', R.sub.S', R.sub.d model
respectively the resistivity of the MBL, the solution, and the
dielectric layer. Series inductors L.sub.t, L.sub.m, L.sub.S model
respectively the inductance of the interface transmission line, the
MBL, and the solution. Shunt capacitors C.sub.m, C.sub.S, C.sub.d
model respectively the capacitances of the MBL 234, the solution
260, and the dielectric layer 240. Collectively, the aforementioned
resistors, inductors, and capacitors define the circuit 430 which
transforms the input signal V.sub.i into the output signal
V.sub.o.
[0192] The dielectric properties of the MBL largely determine the
values of the circuit elements corresponding to each of those
layers. For instance, in the illustrated embodiment of FIG. 4B, the
susceptibility of the MBL largely defines the value of the shunt
capacitance C.sub.m. Further, the dispersive properties of the MBL
largely determine the value of the shunt resistance R.sub.m'. The
values of C.sub.m and R.sub.m' define to a significant degree the
signal response of the bio-electrical interface. Thus, the signal
response of the bio-electrical interface is strongly characteristic
of the dielectric properties of the MBL and can be used to detect
and identify molecular binding events, as will be further described
below.
[0193] In embodiments where the solution 260 is an aqueous
solution, the dielectric properties associated therewith are
disadvantageous to signal propagation along the interface
transmission line. Specifically, water and other highly aqueous
solutions such as whole blood, exhibit a relatively high resistance
R.sub.S and a relatively low resistance R.sub.S', as well as
absorptive properties with respect to electromagnetic radiation in
certain areas of the spectrum. The magnitude of these parameters
results in very high signal loss along the interface transmission
line. The location of the MBL between the interface transmission
line and the solution in the present invention serves to insulate
from, or otherwise modulate the coupling with, the signal and the
solution, thereby modulating the signal loss and changing other
parameters of signal propagation.
[0194] FIG. 4C illustrates one embodiment of an equivalent circuit
model 450 for the bio-electrical interface structure shown in FIG.
2B. Those of skill in the art of circuit design will appreciate
that the illustrated circuit model is not exhaustive and that other
equivalent circuit models may be derived from the bio-electrical
interface of FIG. 2B.
[0195] The illustrated equivalent circuit model 450 includes series
and shunt circuit blocks 452a and 452b which electrically model the
interface transmission line. The equivalent circuit model 450 also
includes a MBL circuit block 454 coupled in series with a solution
circuit block 456 which electrically models the MBL and solution.
As explained above, the orientation of the series and shunt blocks
452a and 452b define a conventional transmission line structure.
Additionally, the series orientation of the MBL and solution
circuit blocks 454 and 456 results from signal field lines
extending from interface transmission line, through the MBL, and
into the solution, the arrangement of which is shown in FIG. 2B.
Alternative circuit models may be derived as above for bio-assay
devices implementing a MBL and solution proximate to the ground
plane alternatively, or in addition to their location near the
interface transmission line.
[0196] FIG. 4D illustrates one embodiment of a circuit 470
corresponding to the equivalent circuit model shown in FIG. 4C.
Those of skill in the art of circuit design will readily appreciate
that other circuits configurations are possible. The series and
shunt circuit block 452a and 452b collectively represent the
conventional model for the interface transmission line. The MBL
circuit block 454 is coupled between the interface transmission
line and the solution circuit block and in one embodiment, consists
of a parallel coupled capacitor C.sub.m, resistor R.sub.m and
inductor L.sub.m. Collectively, the aforementioned resistors,
inductors, and capacitors define the circuit 470 which transforms
the input signal V.sub.i into the output signal V.sub.o.
[0197] As explained above, the dielectric properties of the MBL and
solution will affect the values of each of the electrical elements.
In particular, the susceptibility and other dielectric properties
of the MBL will largely determine the value of C.sub.m; the
permittivity, other dielectric properties, and surface morphology
of the MBL will strongly define the value of L.sub.m; and the
dispersive properties as well as conductive and other dielectric
properties of the MBL will significantly determine the value of
R.sub.m. Thus, the signal response of the bio-electrical interface
is strongly characteristic of the dielectric properties of the MBL
and can be used to detect and identify molecular binding events, as
will be further described below.
[0198] D. Specific Embodiments
[0199] FIGS. 5A-5G illustrate specific embodiments of the
bio-electrical interface implemented in a two conductor circuit
topology. Those of skill in the art of circuit design will readily
appreciate that each may be implemented in a single conductor
waveguide topology, as well as three or more conductor circuit
topologies.
[0200] Each of the embodiments consists of a signal plane 520,
dielectric layer 530, and a ground plane 550. Coupled to signal
plane 520, ground plane 550 or both are a MBL 515 and a solution
510. In each of the embodiments, the MBL 520 may either be in
direct contact with the interface transmission line 530, or coupled
thereto. When the signal plane 520 contacts the MBL 515 directly,
it is formed from a material which is capable of both supporting
signal propagation and adhering ligands, such as proteins, nucleic
acids, carbohydrates, enzymes and the like. Such materials include,
but are not limited to gold, 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.
Other materials which can be used will be readily apparent to those
of skill in the art.
[0201] The dielectric layer 530 may consist of air, polyimide,
teflon, woven insulating materials such as Duriod.TM., alumina,
diamond, sapphire, or semiconductor insulating material such as
silicon dioxide or gallium arsenide, or other insulating materials.
The thickness and dielectric constant of the dielectric layer 530
are selected to provide the desired transmission line impedance as
known in the art. The solution 510 may consist of any transporting
medium, such as Dulbecco's phosphate-buffered saline (d-PBS), which
provides the subject molecular structure. The protein, nucleic
acid, or other ligand of interest can be added to the
bio-electrical interface using a variety of techniques such as
wicking, pipeting or through capillary action.
[0202] FIGS. 5A and 5B illustrate cross-sectional views of the
interface realized in a microstrip circuit topology and in which
the solution 510, and MBL 515 are positioned above and below the
interface transmission line 530, respectively. FIGS. 5C and 5D
illustrate cross-sectional views of the interface in which the
solution 510 and MBL 515 are positioned above and below the ground
plane 550, respectively.
[0203] FIG. 5E illustrates a cross-sectional view of the interface
realized in coplanar waveguide topology. In this embodiment, the
solution 510 and MBL 515 are positioned above the interface
transmission line 530. Alternatively, the solution 510 and MBL 515
may be positioned below the interface transmission line 530, or
above or below one or both of the coplanar ground planes 550. FIG.
5F illustrates a cross-sectional view of the interface realized in
a stripline circuit topology. In this configuration, the solution
510 and MBL 515 are positioned above the interface transmission
line 530. In other embodiments, these layers may alternatively or
in addition be place below the interface transmission line 530, or
above or below one or both ground planes 550.
[0204] FIG. 5G illustrates one embodiment of the bio-electrical
interface implemented in a coaxial circuit topology. A first
insulator 530a having a cavity 570 partially circumscribes an
interface center conductor 530. The MBL 515 is positioned in
proximity to the uncovered portion of the interface center
conductor 530. A second insulator 540b is provided between the
outer conductor 550 and the first insulator 540a and circumscribes
the outer conductor 550, forming the cavity 570 in which the
solution 510 resides. The radii and dielectric constants of the
first and second insulators 530a and 530b may be of the same or
differing values and each is selected to provide the desired line
impedance and the requisite measurement sensitivity over the test
signal frequency range. In an alternative embodiment, the MBL 515
is located proximate to the outer conductor 550. In this
embodiment, the second insulator 530b includes a cavity for
allowing the MBL to form proximate to the outer conductor and the
first insulator completely circumscribes the center conductor 320.
Further alternatively, the MBL 515 and solution may be located
outside of the outer conductor 550.
[0205] The bio-electrical interface may be fabricated in a variety
of shapes depending upon the application, for example, squares,
ellipsoids, rectangles, triangles, circles or portions thereof, or
irregular geometric shapes, such as one that would fit into the
bore of a hypodermic needle. The size of the bio-electrical
interface will vary depending upon the application and have sizes
on the order of 10m.sup.2, 1m.sup.2, 10.sup.-1m.sup.2,
10.sup.-2m.sup.2, 10.sup.-3m.sup.2, 10.sup.-4m.sup.2,
10.sup.-5m.sup.2, 10.sup.-6m.sup.2, 10.sup.-7m.sup.2,
10.sup.-8m.sup.2, 10.sup.-10m.sup.2, 10.sup.-11m.sup.2,
10.sup.-12m.sup.2, or orange anywhere therebetween, The
bio-electrical interface may be fabricated to fit into something as
small as a needle bore. The interface may alternatively be modified
to accommodate other diagnostic applications, such as proteomics
chips. The size or shape of the bio-electrical interface need only
be such that signal propagation and molecular binding therealong is
possible.
[0206] V. Measurement Methodology
[0207] A. General Overview
[0208] The measurement methodology of the present invention makes
use of the observation that a vast number of molecules are
distinguishable from one another based upon their unique dielectric
properties which include dispersion effects, resonance effects, and
effects on the solution surrounding said molecules. In the present
invention, when a test signal couples to the MBL, the MBL interacts
with the energy of the test signal, resulting in a unique signal
response. The unique signal response can then be used to detect and
identify the molecules which make up the MBL.
[0209] Those of skill in the art will appreciate that most
molecules exhibit variation in dielectric properties over different
frequencies. For instance, a molecule may exhibit a dramatic change
in its dielectric properties as a function of frequency in one or
more regions of the electromagnetic spectrum. The frequency band
over which the molecule exhibits a dramatic dielectric change is
often referred to as the molecule's dispersion regime. Over these
regimes, the molecule's dielectric constant, permittivity, dipole
and/or multipole moments, and susceptibility will change
dramatically as a function of frequency. These quantities are often
complex, having both real and imaginary parts to account for both
the magnitude and phase changes that occur in the signal response.
The dispersion regimes range over various frequencies, including
the RF, microwave, millimeter wave, far-infrared, and infrared
frequencies.
[0210] The molecule's dielectric properties can be observed by
coupling a test signal to the molecule and observing the resulting
signal. When the test signal excites the molecule at a frequency
within the molecule's dispersion regime, especially at a resonant
frequency, the molecule will interact strongly with the signal, and
the resulting signal will exhibit dramatic variations in its
measured amplitude and phase, thereby generating a unique signal
response. This response can be used to detect and identify the
bound molecular structure. In addition, because most molecules will
exhibit different dispersion properties over the same or different
frequency bands, each generates a unique signal response which can
be used to identify the molecular structure.
[0211] Detection and identification of molecular binding events can
be accomplished by detecting and measuring the dielectric
properties at the molecular level. The dielectric properties at the
molecular level can be defined by the molecule's multipole moments,
the potential energy of which can be represented as an infinite
series as is known in the art: 1 ( x ) = q r + p x r 3 + 1 2 i , j
Q ij x i x j r 5 +
[0212] The infinite series consists of multiple terms, each of
which describes in varying degrees the molecule's dielectric
properties in the presence of an electric, magnetic or an
electro-magnetic field. The first term is referred to as the
monopole moment and represents the scalar quantity of the
electrostatic potential energy arising from the total charge on the
molecule. The second term or "dipole moment" is a vector quantity
and consists of three degrees of freedom. The third term or
"quadrapole moment" is a rank-2 tensor and describes the molecule's
response over 9 degrees of freedom. In general, the N.sup.th term
is a tensor of rank N-1, with 3.sup.N-1 degrees of freedom, though
symmetries may reduce the total number of degrees of freedom. As
one can appreciate, the higher-order moments provide greater detail
about the molecule's dielectric properties and thus reveals more of
the molecule's unique dielectric signature. Since the gradient of
the potential results in the electric field:
E=-.gradient..PHI.(x),
[0213] The field strength of the higher-order moments falls off
rapidly as a function of distance and thus their contribution is
difficult to measure. For instance, the field due to dipole moment
falls off as r.sup.-3 and the field due to the quadrupole moment
falls off as r.sup.-4 . Thus, this approach requires close
proximity between the binding molecules and test signal path and
low signal loss therebetween. Since it is often the case that
molecular binding event detection occurs in strongly
signal-absorbing solutions, such as whole blood samples or ionic
solutions, signal loss between the binding events and signal path
becomes quite high and detection of the higher order moments is
very difficult.
[0214] In addition, each multipole term couples to the electric
field in a different way. This is demonstrated by first looking at
the energy of a given electrostatic system:
W=.intg..rho.(x).PHI.(x)d.sup.3x
[0215] Expanding the electrostatic potential in a Taylor Series
gives 2 ( x ) = ( 0 ) + x ( 0 ) + 1 2 i j x i x j 3 ( 0 ) x i x
j
[0216] Since E=-.gradient..PHI.(x), 3 ( x ) = ( 0 ) - x E ( 0 ) - 1
2 i j x i x j E j x i
[0217] Further, for the external field, .gradient..multidot.E=0, so
that we get 4 ( x ) = ( 0 ) - x E ( 0 ) - 1 6 i j ( 3 x i x j - r 2
ij ) E j x i
[0218] Inserting this back into the equation for the energy given
above yields 5 W = q ( 0 ) - p E ( 0 ) - 1 6 i j Q ij E j x i
[0219] This shows the manner in which each multipole term interacts
with the interrogating field: The total charge q with the
potential, the dipole p with the electric field, the quadrupole
Q.sub.ij with the gradient of the electric field, etc. This
illustrates the second difficulty with the detection of the higher
order multipole moments: It is difficult in a bulk sample to
achieve sufficient field gradients to couple to the higher order
moments.
[0220] The present invention overcomes the aforementioned obstacles
by implementing the described bio-electrical interface. The
interface includes a MBL which is coupled along the signal path.
The MBL consists of a very thin and highly inhomogeneous layer
(from a dielectric standpoint), thus providing the required
proximity to the electromagnetically probing structure as well as
the sufficient field gradients to couple to the higher order
multipole moments. These qualities enable detection of higher order
moments which provide a greatly enhanced view of the molecule's
dielectric properties. The positioning of the MBL proximate to the
signal and/or ground planes serves to isolate the signal
propagating thereon from becoming absorbed into solution, thereby
reducing the signal loss and enabling the usage of higher test
frequencies to more accurately detect and identify the binding
events. In this manner, the present invention enables to a greater
degree the recovery or the signal response including the
contributions from the molecule's dipole and other higher-order
multipole moments.
[0221] Using the described bio-assay device of the present
invention, numerous properties associated with the MBL may be
detected. FIG. 6A illustrates one embodiment of this method.
Initially at step 602 a MBL is formed and coupled along a portion
of a signal path. As described, the MBL may consist of a ligand,
antiligand/ligand complex, etc and be in direct or indirect
physical contact with, or electromangentically coupled to the
signal path. The signal path may consist of the signal plane or
ground plane in a two-conductor transmission topology.
[0222] Next at step 604, a test signal is propagated along the
signal path. The test signal may be any time-varying signal of any
frequency, for instance, a signal frequency of 10 MHz, or a
frequency range from 45 MHz to 20 GHz. Next at step 606, the test
signal couples to the MBL and in response develops a signal
response to the coupling. The signal response is then recovered and
provides information as to one or more properties of the molecular
binding layer.
[0223] The bio-assay device may used to provide information about
numerous properties of the MBL, such as the detection and
identification of molecular binding events, ligand concentrations,
changes in dielectric properties of the MBL, classification of
detected binding events, and the like. In addition, the bio-assay
device includes a self-calibration capability which is useful in
point-of-use quality control and assurance. Each of these methods
and capabilities are further described below. Based upon the
described methods and structures, modifications and additional uses
will be apparent to those skilled in the art.
[0224] The ability to detect and measure molecular dipole,
quadrupole, and higher order multipole moments in solution
represents a significant advance in the art for a number of
reasons. First, many molecules of biomedical interest such as
proteins have very distinct structures, and therefore distinct
multipole moments. Thus identifying the multipole moments for a
given molecule reveals properties of said molecule which are
unique, and thus allows identification of said molecule. Second,
structure and function are intimately related in many molecules of
biomedical relevance, such as proteins. Thus, the ability to detect
properties of a given molecule which relate directly to the
function of said molecule means that functionality may be monitored
for whole ranges of activities. Third, the local physiologic
environment often plays an important role in the structure and
function of a given molecule, so that an ability to detect the
physical properties described above means that molecules may be
used a monitors and probes for the purpose of measuring changes in
a given system. Thus, with the ability to translate complex and
informative properties about molecular and cellular systems into a
detectable electronic data format, whole new possibilities emerge
in the areas discussed herein.
[0225] B. Detecting Bound Molecular Structures
[0226] The bio-assay device described herein enables the detection
of molecular binding events occurring along the signal path.
Detectable binding events include primary, secondary, and
higher-order binding events. For instance, in a two-conductor
bio-electrical interface having no pre-existing MBL, the molecules
of the conductive layer will form the antiligands for binding to
the ligands, the ligands forming the MBL. In another embodiment,
the antiligand and ligand are both included in the MBL. In this
embodiment, the MBL is attached to the signal path surface via
linkers, matrix molecules, insulating layers or a combination of
each as show in FIG. 1D.
[0227] FIG. 6A illustrates one embodiment of this process.
Initially at step 602, a signal path is formed from a material
which can support the propagation of a signal over the desired
frequency of operation. The signal path may consist of a single
port path, a two port path, or a multiple port path within one of
the bio-assay devices described herein. In addition, the signal
path may be realized as a transmission line, resonant cavity, or as
a waveguide structure.
[0228] Next at step 604, a solution is provided which contains the
subject molecule or molecular structure. At step 606, a MBL
consisting of the ligand is formed from the solution and is coupled
between at least a portion of the signal path and the solution.
Next at step 608, a test signal is propagated along the signal
path. Alternatively, the test signal may be launched during the
application of the solution in order to observed in real time the
signal response occurring as a result of the binding events. At
step 610, the test signal propagates over, couples to the MBL and
develops a signal response which indicates the presence of the
ligand. Next at steps 612 and 614, the test signal is recovered,
the response of which indicates detection of the ligand.
[0229] The dielectric properties of the MBL may contribute to
induce any number of signal responses, each of which may be
indicative of molecular binding. For instance, the dispersive
properties of the MBL may vary dramatically over frequency. In this
instance, the test signal response will exhibit large changes in
the amplitude and/or phase response over frequency when molecular
binding events occur along the binding surface, thereby providing a
means for detecting molecular binding events along the binding
surface.
[0230] In another embodiment, the dielectric relaxation properties
of the MBL will vary as a function of pulse period of the input
signal. In this instance, the test signal response will indicate a
change in the amount of power absorbed, or change in some other
parameter of the test signal like phase or amplitude, at or near a
particular pulse period.
[0231] By observing a change in the absorbed power or other
parameters, binding events along the binding surface may be
detected. Other quantities such characteristic impedances,
propagation speed, amplitude, phase, dispersion, loss,
permittivity, susceptibility, frequency, and dielectric constant
are also possible indicators of molecular binding events.
[0232] The above-described method may be used to detect the primary
binding of an antiligand or ligand directly or indirectly along the
signal path. Similarly, the process of FIG. 6A may also be used to
detect secondary binding of a ligand to an antiligand. The method
of FIG. 6A is not limited to detection of primary or secondary
binding events occurring along the signal path. Indeed, tertiary,
and higher-order binding events occurring either along the signal
path or suspended in solution can also be detected using this
method.
[0233] FIG. 6B illustrates a second process for detecting secondary
and higher-order binding events occurring either along the signal
path. Initially at step 620, the primary binding event is detected
and the signal response measured, one embodiment of which is shown
in steps 602-612. Subsequently at step 622, the primary binding
event signal response is stored and used as a baseline response.
Next at step 624, a second molecular solution is added to the
bio-assay device and allowed to circulate over the binding surface.
Next at step 626, steps 608 through 612 of FIG. 6A are repeated to
obtain a second signal response. Next at step 628, the second
signal response and the baseline response are compared. Little or
no change indicates that the two signal responses are very close,
indicating that the structural and dielectric properties of the MBL
have not been altered by the addition of the molecules within the
new solution. In this case, secondary binding has not occurred to a
significant degree (step 630). If the comparison results in a
change outside of a predetermined range, the structure and/or
dielectric properties of the MBL have been altered, thereby
indicating secondary binding events (step 632). Quantities which
can be used to indicate secondary binding events will parallel the
aforementioned quantities, e.g., amplitude, phase, frequency,
dispersion, loss, permittivity, susceptibility, impedance,
propagation speed, dielectric constant as well as other factors.
Tertiary or high-order binding events may be detected using this
approach.
[0234] An alternative method of detecting secondary or higher order
binding events does not required prior knowledge of the specific
primary binding event. In this embodiment, the bio-assay device is
designed in the assay development stage to operate with known
parameters, so that whenever a pre-defined change in one of these
parameters is detected, for example at the point-of-use, the
binding event or events are then known to have occurred. In this
embodiment, the pre-measurement of a primary binding event is not
necessary, as the initial characterization has already been done
either at the time of fabrication or at the time of design.
[0235] Secondary binding events can also be achieved by detecting
changes in the structure of the primary bound molecule. When a
molecule becomes bound, it undergoes conformational and other
changes in its molecular structure relative to its unbound state.
These changes affect the primary binding molecule's dielectric
properties as well as inducing changes in the surrounding solution,
the variation of which can be detected using steps 620-628 of FIG.
6B, described above. Quantities which can be monitored to indicate
a change in the dielectric properties of the primary bound molecule
include the aforementioned quantities, e.g., amplitude, phase,
frequency, dispersion, loss, permittivity, susceptibility,
impedance, propagation speed, dielectric constant as well as other
factors.
[0236] C. Detecting Changes in the Dielectric Properties of the
Molecular Binding Layer
[0237] The bio-assay device described herein may also be used to
measure the dielectric changes of the MBL as a result changes in
temperature, pH, ionic strength and the like.
[0238] FIG. 6C illustrates an exemplary embodiment of the process.
The process closely parallels the disclosed method for identifying
binding events, the exception being that the method allows for the
detection and quantitation of changes in dielectric properties of
the MBL.
[0239] The process begins at step 641, when a solution having an
initial dielectric property is added to the bio-assay device, the
signal response is measured and recorded. In one embodiment, this
step is performed according to steps 602-612. After a predetermined
time or operation, a second measurement is made and a second signal
response is recorded (step 642), again in one embodiment according
to steps 602-612. At step 643, a comparison is then made between
the first and second signals to determine whether the two signals
correlate within a predefined range. If so, the properties of the
solution are deemed to not have undergone any dielectric changes
(step 644).
[0240] If the signal responses do not correlate within a predefined
range, one or more dielectric properties of the solution is deemed
as having undergone (step 645). Optionally the change in dielectric
properties may be quantitated in the following manner. At step 646,
the second signal is stored and correlated to a known signal
response. The closest correlated response will identify the
dielectric property of the solution and the first signal response
can be correlated to the initial value of the dielectric property,
the difference of which can be used to determine the amount by
which the identified dielectric property has been altered (step
647).
[0241] D. Identifying Bound Molecular Structures
[0242] Using the described bio-assay devices, it is possible to
characterize a known ligand and subsequently identify it in a
solution having an unknown ligand make-up. FIG. 6D illustrates one
embodiment of this process. Initially at step 652, a large number
of molecular structures are measured and their responses stored
using one or more of the measurement systems, described below. In
one embodiment, this step is performed according to steps 602-612.
Each stored response may correspond to a single ligand occurring
within the solution or multiple ligands occurring within the same
solution. Subsequently at step 654, a measurement is made of an
unknown solution. In one embodiment, this step is performed
according to steps 602-612. Next at step 656, the signal response
of the solution is compared to the stored signal responses to
determine the degree of correlation therewith. At step 658, the
unknown molecular structure is identified by selecting the stored
response which exhibits the closest correlation to the unknown
response. The comparison may be performed using one or more data
points to determine the correlation between one or more stored
responses, and may involve the use of pattern recognition software
or similar means to determine the correlation. The process may be
used to identify primary, secondary or higher-order bound molecular
structures.
[0243] E. Identifying Classes of Bound Molecular Structures
[0244] It is also possible to characterize known molecular
sub-structures such as domains or other structural homologies that
are common to similar classes of proteins or sequence homologies in
nucleic acids. In one embodiment, the process proceeds as shown in
FIG. 6D, except that in step 652, N number of molecular
sub-structures are measured and their responses stored. Each stored
signal response may correspond to one or more sub-structures. The
process continues as described in steps 654, 656 and 658 until a
sufficient number or structures have been detected and
characterized to identify the unknown compound. Once a sufficient
number of correlations occur, it is then possible to classify the
unknown molecular structure.
[0245] FIG. 6E illustrates another process by which unknown ligands
may be classified. The process identifies the unknown ligand by
detecting binding to structural motifs on the unknown compound.
Initially, at step 660 a bio-assay device is provided which has
multiple addressable arrays, each of which has a antiligand for a
specific ligand sub-structure. Next at step 662, the presence of
particular sub-structures is detected by the binding of each to its
respective antiligand, and subsequent characterization. In one
embodiment, this step is performed according to steps 602-612.
Subsequently at step 664, each of the binding events is then
characterized by identification of qualities such as affinity,
kinetics, and spectral response. At step 666, a correlation is then
made between the known and unknown responses. If each of the
unknown responses correlates to known responses, the ligand is
identified as the ligand corresponding to the known response. If
the sub-structures exhibit both correlated and uncorrelated
responses, the correlated responses may be used to construct a more
general classification of the unknown ligand. This process may be
used to identify any molecular structure, for example proteins,
which occur within the same class or have re-occurring structural
homologies.
[0246] It is also possible that an intensive spectral analysis of a
given unknown compound could lead to insights on structure and
function, as comparisons can be made to known structures, and
extrapolation will lead to some level of classification.
[0247] A. Specific v.s. Non-specific Binding:
[0248] Specific ligand binding is distinguished form non-specific
binding by the spectral fingerprint of the binding event. A given
binding event of interest, for example antibody binding to antigen,
may be first characterized in a purified solution containing just
the ligand of interest and the antiligand specific to said ligand
on the MBL. A broad spectral study is then carried out to see when
in the spectrum the strongest responses are found. The assay is
then repeated in the solutions typically found in the dedicated
applications, for example whole blood, to determine what effects
non-specific binding has on the response. Then various points are
found which are determinate of specific binding, and a separate set
of points are found which are determinate of non-specific binding,
an a subset of these frequency points are chosen for the actual
assay application. By comparing the response due to specific
binding with those due to the non-specific binding, the extent of
specific binding can be determined.
[0249] B. Characterization of a Given Ligand:
[0250] Often it is desirable to determine certain qualities of a
given molecule. Examples in include determining the class to which
a protein belongs, or which type of polymorphism a given gene or
other nucleic acid sequence is. This may be done in a number of
ways. Proteins are often classified by number and types of
structural homologies, or particular substructures which are found
in the same or similar classes of proteins. For example, G-Proteins
commonly found in cell membranes and which mediate signal
transduction pathways between the extra-cellular environment and
the intra-cellular environment, always have a structure which
traverses the cell membrane seven times. Such a structure is
virtually definitive of a G-Protein. Other classes of proteins have
similar structural homologies, and as such, any method which can
distinguish one class of proteins from another on the bases of
these homologies is of enormous use in many of the biomedical
research fields. Given that the dielectric properties of a given
molecule is determined entirely by the geometry of the charge
distribution of said molecule, and further given that most proteins
have a unique structure or geometry, then each protein may be
uniquely determined by measuring the dielectric properties of the
protein. Thus a simple dielectric signature, such as the ones
generated by the present invention, may serve to uniquely identify
a given protein, and further, may allow classification of the
protein into some previously known class of proteins. A further
refinement may be added to the classification methodology by using
a group of antiligands on the bio-assay device which are specific
for particular sub-structures of a given protein. For example, a
group of antibodies which are specific for particular
sub-structures such as domains may be utilized for the
determination of the existence or absence of said sub-structures.
Thus, any given protein may be characterized by determining both
the presence and absence of certain sub-structures as well as the
dielectric properties of the protein itself. Further refinements to
this classification strategy may include looking at temperature,
pH, ionic strength, as well as other environmental effects on the
above-mentioned properties.
[0251] Nucleic acids may also be characterized by following a
similar paradigm. For example, a given gene may be known to have a
certain base pair sequence. Often times in nature there will be
small variations in this sequence. For example, in the gene which
codes for a chloride ion transport channel in many cell membranes
there are common single base-pair mutations, or changes. Such
changes lead to a disease called cystic fibrosis in humans. Thus
characterizing a given nucleic acid sequence with respect to small
variations is of enormous importance. Such variations are often
called polymorphism's, and such polymorphism's are currently
detected by forming complementary strands for each of the known
polymorphism's. Since any given gene may take the form of any one
of hundreds or even thousands of polymorphism's, it is often an
arduous task to generate complementary strands for each
polymorphism. Using the invention described herein,
non-complementary binding or hybridization may be detected and
distinguished by measuring many of the same physical properties as
were described in the previous paragraph: The dielectric properties
of the hybridization event can be characterized and correlated to
known data, thereby determining the type of hybridization which has
occurred-either complete or incomplete. Thus with an antiligand
comprised of a given nucleic acid sequence, hundreds of different
polymorphisms (as ligands) may be detected by the characterization
of the binding event. One of skill in the art will appreciate that
further refinements are possible, such as modifying the stringency
conditions to alter the hybridization process, or varying the
temperature and determining the melting point, which serves as
another indicator of the nature of the hybridization process.
[0252] In a similar manner, drug-receptor interactions may be
characterized to determine is a given binding event results in the
receptor being turned on or off, or some other form of allosteric
effect. For example, a given receptor may be used as an antiligand,
and a known agonist may be used as the first ligand. The
interaction is then characterized according to the dielectric
response, and this response is saved. Subsequently, compounds which
are being screened for drug candidates are then observed with
respect to their binding properties with said receptor. A molecule
which binds and yields a similar dielectric response is then known
to have a similar effect on the receptor as the known agonist, and
therefore will have a much higher probability of being an agonist.
This paradigm may be used to characterize virtually any type of
target-receptor binding event of interest, and represents a
significant improvement over current detection strategies which
determine only if a binding event has occurred or not. Those of
skill in the art will readily appreciate that there are many other
classes of binding events in which the present invention can be
applied.
[0253] Examples of sub-structures which may be used in the above
method include: Protein secondary and tertiary structures, such as
alpha-helices, beta-sheets, triple helices, domains, barrel
structures, beta-turns, and various symmetry groups found in
quaternary structures such as C.sub.2 symmetry, C.sub.3 symmetry,
C.sub.4 symmetry, D.sub.2 symmetry, cubic symmetry, and icosahedral
symmetry. [G. Rose (1979), Heirarchic Organization of Domains in
Globular Proteins, J. Mol. Biol. 134: 447-470] Sub-structures of
nucleic acids which may be analyzed include: sequence homologies
and sequence polymorphisms, A, B and Z forms of DNA, single and
double strand forms, supercoiling forms, anticodon loops, D loops,
and T.psi.C loops in tRNA, as well as different classes of tRNA
molecules. [W. Saenger (1984) Principles of Nucleic Acid Structure.
Springer-Verlag, New York; and P. Schimmel, D. Soll, and J. Abelson
(eds.) (1979) Transfer RNA. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.]
[0254] F. Quantitating Concentrations
[0255] The bio-assay devices described herein may also be used to
quantitate the concentrations of ligands. FIG. 6F illustrates one
embodiment of this process. In the event the device is not
precailbrated (step 679), initially at step 670, antiligands are
chosen having the appropriate binding properties, such as binding
affinity or kinetics, for the measured analyte. These properties
are selected such that the antiligand's equilibrium constant is
near the center of its linear operating region. For applications
where the range of concentration is too wide for the use of a
single antiligand, several antiligands may be used with differing
affinities and/or linear operating ranges, thereby yielding a value
for the concentration over a much wider range.
[0256] Next at steps 672 and 674, the antiligands are attached to
the bio-assay device or chip and the device is connected to the
measurement system. At step 674, a decision is made as to whether
the response requires characterization for maximum specificity. If
so, a spectral analysis is performed in which the frequencies where
analyte binding has maximal binding is determined (step 675a), the
regions where the non-specific binding has maximal effect is
determined (step 675b), and the unique response due to analyte
binding is determined (step 675c). If characterization is not
required, or if so, after its completion, the device is calibrated.
This step is performed in one embodiment by supplying a known
concentration of ligands to the bio-assay device and measuring the
resulting response (step 676a). Alternatively, if more data points
are needed for the calibration (step 676b), then a sample may be
chosen with a different concentration (step 676c), and the response
against this concentration may be measured (step 676a). In one
embodiment, the measurement is made in accordance with steps
602-612. Subsequently at step 677, an extrapolation algorithm is
generated by recording the calibration points from the foregoing
response. Next at step 678, a sample of unknown ligand
concentration is measured. This step is accomplished in one
embodiment by supplying the unknown sample to the bio-assay device,
correlating the response to the titration algorithm, and
determining therefrom the ligand concentration.
[0257] In the event that a given bio-assay device is either
pre-calibrated, or calibrated by design, the only step required is
to apply the ligand or analyte to the surface, and measure the
response. Such a bio-assay device may be realized in many different
ways. For example, some circuit parameter like impedance or
characteristic frequency of a resonant circuit may be designed to
change in a pre-determined way when the binding event occurs, and
the amount by which the parameter changes may further be designed
to have a dose-response. Thus, a measurement of said circuit
parameter will, when analyzed via a suitable algorithm, immediately
yield a quantitative value for the concentration of a given analyte
or ligand.
[0258] G. Bio-assay Device Self-calibration
[0259] The described bio-assay devices possess a self-diagnostic
capability and thus a point-of-use quality control and assurance.
For a given dedication application, a particular antiligand
(primary binding species) will act as an antiligand for some ligand
(the secondarily binding species) of interest in the solution. The
primary binding species may be attached at the point of
fabrication, and the secondary binding species may be attached at
the point-of-use. Thus, variations in fabrication--specially the
attachment of the primary species-will cause variations in the
ability of the device to bind its specific ligand. However, the
amount of ligand bound will be in direct proportion to the amount
of antiligand bound, thus a ratiometic measurement of the two is
possible.
[0260] FIG. 6G illustrates one embodiment of the process. Initially
at step 680, a molecular binding surface is formed along the signal
path by binding the appropriate antibody at various concentrations
and characterising the resulting response for each of these
concentration, yielding some value "x" for each concentration.
Next, at step 682, a similar titration curve is generated for the
ligand by measuring the antibody/ligand binding response for
several different concentrations of ligand, and a ligand titration
curve is predetermined. Next, at step 684 a scale factor A is
generated by taking the ratio of responses of antibody binding to
ligand binding. At the point-of-use, the uncalibrated assay is then
first probed (step 686) to determine the amount of bound antibody
"x" and the scale factor "y" resulting therefrom. The ligand is
then applied to the assay and the response is measured (step 689),
and the response and predetermined titration curve are scaled by
the scale factor "y" (step 690) to determine unknown
concentration.
[0261] The process of FIG. 6F may also be modified to allow
quantitating the amount of ligand in the solution. In the
modification, the binding surface of the bio-assay device includes
antiligands having a predefined affinity and ligand specificity.
The solution is subsequently applied to the device, and a response
is measured. The signal response will be proportional to the amount
of the ligand that has bound. Thus, a titration of any given ligand
may be carried out by choosing an antiligand with an appropriate
linear operating range-the range in which the equilibrium constant
is within a couple of log units of the desired range of
concentrations to be detected. The same ratiometic analysis as
described above can be applied to yield a robust and precise
quantitative assay with internal controls and calibration necessary
to insure reliability.
[0262] Each of the described methods may be practiced in a
multitude of different ways (i.e., software, hardware, or a
combination of both) and in a variety of systems. In one
embodiment, the described method can be implemented as a software
program.
[0263] FIG. 7A illustrates an example of a computer system 710 for
executing a software program designed to perform each of the
described methods. Computer system 710 includes a monitor 714,
screen 712, cabinet 718, and keyboard 734. A mouse (not shown),
light pen, or other I/O interfaces, such as virtual reality
interfaces may also be included for providing I/O commands. Cabinet
718 houses a CD-ROM drive 716, a hard drive (not shown) or other
storage data mediums which may be utilized to store and retrieve
digital data and software programs incorporating the present
method, and the like. Although CD-ROM 716 is shown as the removable
media, other removable tangible media including floppy disks, tape,
and flash memory may be utilized. Cabinet 718 also houses familiar
computer components (not shown) such as a processor, memory, and
the like.
[0264] FIG. 7B illustrates a simplified system block diagram of a
typical computer system 710 used to execute a software program
incorporating the described method. As shown in FIG. 7A, computer
system 710 includes monitor 714 which optionally is interactive
with the I/O controller 724. Computer system 710 further includes
subsystems such as system memory 726, central processor 728,
speaker 730, removable disk 732, keyboard 734, fixed disk 736, and
network interface 738. Other computer systems suitable for use with
the described method may include additional or fewer subsystems.
For example, another computer system could include more than one
processor 728 (i.e., a multi-processor system) for processing the
digital data. Arrows such as 740 represent the system bus
architecture of computer system 710. However, these arrows 740 are
illustrative of any interconnection scheme serving to link the
subsystems. For example, a local bus could be utilized to connect
the central processor 728 to the system memory 726. Computer system
710 shown in FIG. 7B is but an example of a computer system
suitable for use with the present invention. Other configurations
of subsystems suitable for use with the present invention will be
readily apparent to one of ordinary skill in the art.
[0265] V. Measurement Systems
[0266] Various measurement systems may be used to perform the
above-described methods. FIGS. 8-10 illustrate three examples of
possible measurement systems: a frequency domain test system, a
time domain test system and a dielectric relaxation measurement
system.
[0267] A. Frequency Measurement System
[0268] FIG. 8A illustrates one embodiment of a frequency
measurement system in accordance with the present invention. The
system 800 includes a signal source 810 coupled to the bio-assay
device input 852 and a signal detector 890 coupled to the bio-assay
device output 858. Optionally, an additional signal source may be
coupled to the bio-assay device output 858 and an additional signal
detector coupled to the test circuit input 852 for providing
complete two-port measurement capability. The system may be
modified to a one-port test system in which a signal detector is
coupled to the signal path for receiving a reflected signal. In a
specific embodiment, the aforementioned frequency measurement
system consists of a network analyzer such as model number 8510C
from the Hewlett-Packard Company. Other high frequency measurement
systems, such as scalar network analyzers, which provide signal
information based upon transmitted and reflected signals may
alternatively be used.
[0269] Measurements are made according to the aforementioned
methodologies. Initially, an incident signal 860 is launched toward
the test circuit and the transmitted and/or reflected signals 870
and 890, respectively, are subsequently recovered. The resulting
signal responses will take the form of unique frequency responses
or "spectral fingerprints," two examples of which are shown in
FIGS. 8B and 8C. FIG. 8B illustrates one type of frequency response
in which a resonance occurs at frequency f.sub.res. Here, response
870 undergoes a steep fall and rise, indicating little or no signal
energy reaches the output port at this frequency. The resonance is
caused by the dielectric property and impedance of the MBL changing
over frequency f.sub.start to f.sub.stop. Different ligands will
resonate at different frequency points. In addition, some ligands
may exhibit multiple resonant frequency points over the measured
band f.sub.start to f.sub.stop. Once a ligand has been
characterized as having one or more uniquely occurring resonance
points, this data can be used to identify the presence of the
ligand in an unknown solution. This characterization can be
ascertained from empirical data or from theoretical calculations of
multipole moments and resonant frequencies. Furthermore, when
detecting the presence of secondary binding events, this data can
indicate when an analyte is bound to a ligand by a change in the
one or more unique resonance points.
[0270] FIG. 8C illustrates another type of frequency response which
can be used to detect or identify a molecular structure. In this
case, the frequency response exhibits a generally monotonically
increasing or decreasing trend with some degree of amplitude
variation. The response's slope and/or the amplitude variation may
be used to detect and/or uniquely characterize the bound molecule.
Thus in the described manner, the resonant frequency points, slope,
trend, and variation of the test signal's phase may be used to
uniquely identify the molecular binding event. The frequency
response may be measured at the input port 852, at the output port
858 or at both ports to uniquely identify the bound molecular
structure.
[0271] FIG. 9 illustrates a second exemplary embodiment of a
frequency measurement system in accordance with the present
invention. The bio-assay device under test 920 consists of coaxial
topology (shown in FIG. 5G) having a center conductor 921, a first
insulator 922 having a cavity 922a, a second insulator 923, and an
outer conductor 924. Solution 926 occupies cavity 922a. Of course,
devices of other circuit topologies may be tested as well.
[0272] Once the solution 926 is added to the cavity 922a, the
molecules within the solution 926 form a MBL 921 a proximate to the
center conductor 921. During the measurement, a signal source 910
launches an incident test signal 912 to center conductor 921. The
MBL 922a modulates the incident test signal 912, and the reflected
test signal 932 provides a unique signal response which can be used
to identify the ligand. The one-port coaxial configuration may be
realized, for instance, as a sub-cutaneous needle structure.
[0273] B. Time Domain Measurement System
[0274] FIG. 10 illustrates one embodiment of a time domain
measurement system 1000 in accordance with the present invention.
The system includes a pulse source 1002 and a detector 1004 coupled
to the test circuit input 1022. In an alternative embodiment, an
additional pulse source and detector may be coupled to the output
port 1028 to provide complete two-port measurement capability.
Further alternatively, the system may comprise a one-port test
system in which a signal detector is coupled to the signal path for
receiving a reflected signal. In a specific embodiment, the time
domain measurement system consists of a time domain reflectometer
such as model number 11801 manufactured by the Tektronix
Corporation. Other high frequency measurement systems, such as
network analyzers having a time domain measurement mode which
provide signal information based upon transmitted and reflected
signal pulses may alternatively be used.
[0275] In the time domain measurement system, the input test signal
1060 consists of a time domain pulse, the reflected portions of
which can be displayed over time. In the present embodiment, an
incident pulse 1060 is launched toward the portion of the
transmission line which is tightly coupled to the assay surface.
Due to the dielectric property of the MBL, a portion of the
incident pulse 1060 is reflected toward the detector 1004. The
reflected pulse 1070 will exhibit a unique shape and/or time delay
which is characteristic of the MBL's dielectric properties, which
are in turn largely defined by the dielectric properties of the
ligand, antiligand, and the surrounding solution. Thus, the pulse
shape and delay of the reflected pulse 1070 can be used to
characterize and identify the ligand. The time domain test system
may be used separately or in conjunction with the high frequency
test system to identify one or more unknown ligands.
[0276] C. Dielectric Relaxation Measurement System
[0277] As known in the art, the dielectric relaxation frequency of
a ligand is the rate at which the dielectric properties of the
molecular level changes when an electric field is applied to the
molecule. As with the dielectric properties of the ligand, the
dielectric relaxation frequency is primarily defined by the
structure and binding geometries unique to each molecule. Thus once
measured, the dielectric relaxation frequency of a ligand can be
used to identify it.
[0278] The dielectric relaxation frequency can be quantified by
measuring the rate at which the ligand absorbs power over
frequency. FIG. 11 illustrates one embodiment of a system 1100 for
making this measurement. The measurement system 1100 is similar to
the time domain measurement system 1000 illustrated in FIG. 10 and
includes a pulse source 1102 and a detector 1104 coupled to the
test fixture input 1122. An additional pulse source and detector
may be coupled to the output port 1128 to provide complete two-port
measurement capability. In a specific embodiment, the time domain
measurement system consists of a time domain reflectometer such
model number 11801 manufactured by the Tektronix Corporation. Other
high frequency measurement systems, such as network analyzers
having a time domain measurement mode which provide signal
information based upon transmitted and reflected signal pulses may
alternatively be used.
[0279] The input test signal 1160 consists of separate pulse
groups, each group having two or more incident pulses and a
different pulse interval. The pulse groups 1162 and 1164 are
launched toward the portion of the transmission line which is
tightly coupled to the binding surface. If a pulse group 1162 has
an interval substantially equivalent to the dielectric relaxation
period (the reciprocal of the relaxation frequency), the MBL will
absorb successively less energy in succeeding pulses. The decrease
in signal absorption can be measured in the reflected response 1170
at the input port 1122 or at the output port 1128. As an
alternative measurement quantity, the remaining signal power may be
measured either at the input port 1122 or the output port 1128.
[0280] The rate of change of signal absorption and the pulse
interval at which the change occurs can then be plotted and used to
characterize and identify the unknown bound molecule(s). This
system characterization may be used independently or in conjunction
with the above-described time and/or frequency domain test
systems.
[0281] In all of the above systems, one of skill in the art will
readily appreciate that such systems can be scaled down to the chip
level using such technologies as Microwave Monolithic Integrated
Circuits (MMIC) and the like. Such miniaturized systems can be
readily extended to highly parallel systems capable of detecting
and measuring hundreds, thousands, or tens of thousands of
compounds simultaneously. These systems can be configured to yield
"logic gates" which are switched by the binding event itself, such
as by changing a characteristic impedance and thus the transmission
and/or reflection coefficients, or by changing the band pass
properties of such a circuit, and using this as the on/off
gate.
VI. EXAMPLES
A. Example 1
Detection of a Ligand Binding to the Surface.
[0282] Primary binding of urease to an ITO surface was demonstrated
in the bio-assay device as shown in FIG. 2A. The binding surface of
the bio-assay device comprised a cover glass treated with ITO
deposited via chemical vapor deposition (CVD). The ITO transmission
line was carefully examined to ensure that it contained no
microfractures or breaks in it. The transmission line was measured
with a Tektronix 11801 signal analyzer with a TDR module, and found
to have a broadband reference impedance of 32 .OMEGA.. The line
length was about 2.6 nsec in length, the binding surface was found
to have an impedance of 34 .OMEGA., and a length of about 200 psec.
Separation between the top and bottom plates were 10 mils, and the
chamber was 1/2" long. No side walls were used; instead, the
capillary action of the top and bottom plates retained the solution
in place.
[0283] Next, the bio-assay device filed with a solution of d-PBS.
With the bio-assay device filled, baseline transmission loss
(S.sub.21) and return loss (S.sub.11) S-parameter measurements were
made over a test frequency range from 45 MHz to 1 GHz. The
measurements were made and stored using a network analyzer model
number HP 8510B from the Hewlett-Packard Company. Next, urease was
added in a volume excess of 10:1. Transmission loss and return loss
S-parameter measurements was repeated and compared to the baseline
measurement.
[0284] Table 1 below shows these values for 100 MHz and 1 GHz and
the return loss and transmission loss measurement responses are
shown in FIGS. 12A and 12B. The data indicates that the bio-assay
test fixture exhibited a return loss (S.sub.11) change of -0.5 dB
and -0.42 dB, respectively at 100 MHz and 1 GHz between the d-PBS
filled chip and the d-PBS+protein filled device. The fixture
exhibited a transmission loss change (S.sub.21) of +0.325 dB and
+0.450 dB at 100 MHz and 1 GHz, respectively.
[0285] To determine if the signal responses were due to a bulk
effect (proteins in solution), or to proteins binding to the
binding surface, each response was recorded and the protein
solution was flushed with d-PBS in a volume excess of 25:1 (2 mL of
d-PBS to 0.075 mL chamber size). The bio-assay device was then
re-measured from 45 MHz to 1 GHz as described above.
[0286] As can be seen from comparing the last two columns of Table
1, the effect of flushing the protein from the bio-assay device had
minimal effect on the return loss and transmission loss
measurements. This indicates that the measured effect was indeed
due to the urease binding the binding surface within the bio-assay
device. In general, it was noted that the replacement of the
solution containing the ligand with an identical solution without
the ligand caused very little or no change in the response.
1TABLE 1 The Effect of Primary Binding of Urease Frequency Protein
in Solution After d-PBS Flush S.sub.11 100 MHz -500 milli-dB -475
milli-dB 1 GHz -420 milli-dB -200 milli-dB S.sub.21 100 MHz +325
milli-dB +300 milli-dB 1 GHz +450 milli-dB +400 milli-dB
B. Example 2
Identification of Collagenase and Lysozyme through Primary
Binding
[0287] Using a bio-assay device similar to the one cited in example
1 above, and prepared and characterized in a similar manner, we
carried out a series of experiments to examine the differing
responses of different proteins over the frequency range of
1-10GHz. The same device was used for each experiment (to eliminate
small differences in fabrication from one device to another), but
was thoroughly washed with SDS between the application of each of
the proteins. FIGS. 12C and 12D illustrate the transmission loss
measurements of the primary binding effects of collagenase and
lysozyme samples, respectively, over the test frequency range from
1 GHz to 10 GHz. In both instances, the signal response exhibited a
pattern of peaks and valleys which can be used to detect and
identify the ligand uniquely. In particular, the frequency response
of the collagnase sample exhibited a strong positive peak near 5
GHz. The response of the lysozyme sample indicated a relative flat
response near 5 GHz and a strong positive peak near 8 GHz. For each
of the other numerous proteins examined, the response was unique to
each protein, and readily allowed identification of an unknown
protein within the group. Of course, additional spectral points may
also be compared and analyzed to distinguish these and other
molecular substances. The responses may be stored and later
recalled to identify unknown samples. In addition the
less-pronounced peaks may be examined collectively to determine
patterns for particular ligands.
C. Example 3
Detection of Secondary Binding: Concanavalin A to Dextran
[0288] This application provides an example of secondary binding
detection, using a bio-assay device similar to the one cited in
example 1 above, and prepared and characterized in a similar
manner. Concanavalin A (con-A) is a glucose binding protein that
can be found in jack beans, and was used as the primary binding
antiligand The con-A used here was obtained from Sigma Chemical
Company. Dextran, a glucose polysaccharide, was then used as a
ligand to bind con-A, with glucose as a competitive means of
reversing the dextran binding to demonstrate specificity. (Dextran
and glucose were also obtained from Sigma Chemical Company.)
[0289] The transmission line was the same as that discussed in
Example 1, with a nominal 32 .OMEGA. reference impedance, and an
ITO cover glass with a DC resistance of 80 .OMEGA. and a nominal
TDR impedance of 34 .OMEGA.. A concentration of approximately 15
.mu.M solution of con-A was placed directly into the bio-assay
device, and allowed to reached equilibrium. Evaporative losses did
not dry out the chamber as established by visual inspection. After
the system was flushed and stabilized, dextran was added to bind
the con-A. After a change in the signal was detected, the chamber
was flushed with 10 mg/ml d-PBS and the signal response was
measured a second time. This effect is shown in FIG. 12E at 1GHz.
The unbound response being used as the baseline response. As shown,
the bounded response appears to be 0.25 dB less lossy than the
unbound response. Binding specificity was confirmed by competing
off the bound dextran with glucose, followed by a d-PBS flush to
free the glucose. The latter step returned the signal to the
baseline obtained before the dextran had been added to the device,
thus demonstrating specificity of the binding event.
D. Example 4
Detection of Small Molecule Binding.
[0290] Using a bio-assay device similar to the one cited in example
1 above, and prepared and characterized in a similar manner, the
bio-assay test fixture and network analyzer set-up was used to
demonstrate that small molecules binding to large molecules may
also be detected with the present invention. In order to probe the
bio-assay device at higher frequencies, the device was reproducibly
and carefully placed in a Faraday box to shield it from external
influences. This allowed the device to be probed at frequencies up
to 20 GHz. Initially, con-A was added into the bio-assay device and
allowed to bind to the bio-electric interface. A transmission loss
measurement was made, stored, and used as the baseline response
1252 as shown in FIG. 12F.
[0291] Next, a glucose concentration of 10 mg/ml was added to the
bio-assay device and used to bind the con-A antiligand. A
transmission loss measurement was made and plotted relative to the
baseline response 1252 to determine the change in signal response
due to small molecule binding.
[0292] As can be seen from FIG. 12F, the binding response 1254,
which corresponds to the binding of glucose to con-A, is
distinguishable from the baseline measurement 1252. In particular,
the binding response 1254 exhibits 2 large peaks between 16-20 GHz
which is not observed in the baseline response 1252. The difference
in the measured signal responses 1252 and 1254 provides the basis
for detecting when glucose has bound to the con-A antiligand. This
was followed by a flush with the d-PBS buffer only, and the
response was reversed as the bound glucose dissociated from the
con-A. A separate experiment looking at the effect of glucose on
the bare chip (i.e. no con-A as an antiligand) showed that glucose
alone has little if any effect on the response to electromagnetic
interrogation in the above mentioned frequency spectrum, thus
showing that the result shown is due entirely to the effect of
glucose binding to con-A.
[0293] The experiment was repeated for biotin binding to avidin.
Avidin was added to the bio-assay device as the antiligand, and a
transmission loss measurement was made, stored and used as a
baseline response 1262. Next, a 1 .mu.M concentration of biotin was
added, and a transmission loss measurement was made relative to the
baseline measurement. The results are shown in FIG. 12G.
[0294] The binding response 1264 corresponding to the biotin bound
to Avidin indicates a deep null between 14-16 GHz and a large peak
near 20 GHz. The differences between the baseline response
(indicating unbound Avidin) and the binding response 1264
(indicating bound Avidin) is dramatic and can be used to detect the
bound Avidin molecule.
E. Example 5
Quantitation Titrations
[0295] These experiments demonstrate that the magnitude of the
signal change upon a ligand binding to an antiligand is a function
of the number of sites that are occupied. The test system using a
bio-assay device similar to the one cited in example 1 above, and
prepared and characterized in a similar manner, was used with
dextran binding to con-A, with glucose used as a competitive
inhibitor. A series of dilutions was created that centered around
the binding constant of con-A. Dextran as an antiligand was bound
to con-A such that 100% binding occurred. A series of competing
glucose concentrations was used to compete off the dextran, so that
the concentration of dextran on the molecular binding surface was
commensurably decreased.
[0296] The standard transmission line configuration as discussed
above was used. Con-A was bound to the molecular binding layer and
the system was stabilized. The bio-assay device was then flushed
with d-PBS and data obtained at 1 GHz. The results of this
competition titration are shown in FIG. 12H. The results show how
the signal changes as the concentration of glucose is increased
from 0 to 15 mg/dl. The signal of the Con-A changes as the dextran
is released and the glucose is bound (which actually measures the
avidity of the dextran). Specificity was also demonstrated by
reversal by glucose of the dextran binding effect.
[0297] Table 2 shows the magnitude of the change in transmission
loss as a function of the glucose concentration for some selected
concentrations.
2 TABLE 2 Dextran fully bound +320 milli-dB 1 mg/ml glucose +280
milli-dB 1.33 mg/ml glucose +275 milli-dB 2 mg/ml glucose +240
miili-dB 5 mg/ml glucose +115 milli-dB 10 mg/ml glucose -5
miili-dB
[0298] A simple glucose titration was also carried out at a
resonant point in the spectrum of con-A. FIG. 12I shows the change
in the return loss as a function of glucose concentration at this
resonance point, demonstrating two effects: First, glucose has a
dose-response effect as a ligand which is based on the effect it
has on the antiligand (which in this case is con-A). Second, there
are regions in the spectra which show a much more sensitive
response to the ligand/antiligand binding event than other
regions.
[0299] A succession of serial dilutions of the dextran solution
which took the concentration down below one picomolar (10.sub.-5
Molar) showed that even at these low concentrations, a significant
signal response indicating binding occurred. The time required for
the accumulation of the signal ranged from several minutes to ten
minutes, but the response was characteristic of the detection of
dextran at higher concentrations.
F. Example 6
Detection of Nucleic Acids
[0300] In order to demonstrate the ability to detect nucleic acids,
a bio-assay device with polylysine as the antiligand attached to a
gold surface was fabricated. Using a bio-assay device similar to
the one cited in example 1 above except for the gold surface, and
prepared and characterized in a similar manner, a high
concentration solution (about 20 uM) of calf-thymus DNA was
prepared in a d-PBS buffer. The polylysine was placed on the
bio-assay device, and the transmission loss response was measured.
The response was checked for stability over time and saved. The
chamber was then flushed with the buffer, the response again
checked for changes with the flush and stability thereafter, and
the response stored as the baseline response.
[0301] A solution containing the DNA was then placed in the
bio-assay device, and the change in the response was measured by
subtracting the resulting response from the baseline response, and
observed for stability. The bio-assay device was flushed with
buffer to remove the DNA in the bulk, leaving only the
DNA/Polylysine complexes on the bio-assay device surface. The
resulting change is shown in FIG. 12J.
G. Example 7
The Effects of pH and Salinity
[0302] The effects of pH and salinity in the signal were measured
in two different experiments. To investigate the effects of the pH,
a series of buffers or pH ranging from 3.94 to 9.80 were measured.
The 60 Hz conductivity for each buffer was measured to correct for
the change in free ions. Subsequently, transmission loss responses
at 100 MHz, 1 GHz, and 10 GHz was measured. The results are shown
in FIG. 12K.
[0303] A similar experiment was carried out to determine the
effects of changing the ionic concentration of a solution. Several
solutions were made, starting with a simple d-PBS, and adding
various amounts of sodium chloride. The 60 Hz conductivity was then
measured and noted, and the samples were serially placed in the
bio-assay device and the transmission response was measured at 100
MHz, 1 GHz, and 10 GHz. These results are plotted in FIG. 12L.
[0304] As both of these plots show, certain environmental changes
result in changes in the measured parameters.
H. Example 8
Detection in Whole Blood
[0305] The detection of troponin-I (TN-I) was made in whole,
unprocessed human blood was made to verify detection capability in
messy environments. The unprocessed human blood was treated with
sodium citrate to anticoagulate. An anti-TN-I antibody
corresponding to the epitope of TN-I was used for calibration
purposes. The interface transmission line of the bio-assay device
was coated with anti-TN-I Ab (antiligand). A sample of blood was
spiked to a 10 ng/ml concentration of TN-I and a second identical
sample of blood was left unspiked as a control.
[0306] The experiment consisted of attaching the anti-TN-I Ab
antiligand to the device; then first running the unspiked sample
across the device; flushing the sample chamber several times to see
what the noise of exchange was; followed by the spiked sample,
which was also replaced several times to establish a noise floor.
In each case, the change in the transmission loss was measured. As
a check, the anti-TN-I Ab antiligand was removed from the device.
The experiment was subsequently repeated as a control to determine
if any other properties of the two blood samples (assumed identical
except for the TN-I spike) were responsible for the change. The
following table shows the result of this experiment for a probe
signal at 1 GHz.
3 Unspiked sample Spiked Sample Control <20 milli-dB <20
milli-dB Anti-TN-I <20 milli-dB +275 milli-dB
[0307] In a second series of experiments, ten different samples of
blood were obtained from a clinical laboratory, untreated except
for being anticoagulated with heparin. One of the samples was
divided into two parts, and one of the parts was spiked with the
TN-I antigen as described in the previous paragraph. The bio-assay
device was then prepared with the anti-TN-I antibody on the
surface. Each sample was then serially passed through the bio-assay
device, saving the spiked sample for last. The responses for each
of these samples, probed at 1 GHz as in the previous experiment,
and shown in FIG. 12M. The spiked sample was clearly
distinguishable form the rest of the (unspiked) samples.
I. Example 9
Detection of the Quadrupole Moment of a Molecule
[0308] The effect of binding avidin to a gold surface was
investigated to determine the detectablity of a molecule's
quadupole moment. Avidin is a tetramer which has a very small
dipole moment in the unbound state owing to the symmetry of the
molecule in the unbound state. Figure N shows the result of avidin
binding with characteristic peaks as shown in the plot. Note that
these peaks are markedly smaller than the peaks which arise due to
the binding of biotin, as shown in FIG. 12G.
[0309] VII. Applications
[0310] The methods and systems of the present invention may be used
in a variety of applications, examples of which are described
herein.
[0311] The present invention could be used to quantitate the level
of binding between a ligand and an antiligand and thus be used to
determine the effect of other molecules on the activity of an
enzyme. For instance, other molecules in the solution could
decrease or increase the level of the binding and thus the identity
of enzyme inhibitors or inducers could be determined.
[0312] The presence of infectious pathogens (viruses, bacteria,
fungi, or the like) or cancerous tumors can be tested by monitoring
binding of an antiligand to the pathogen, tumor cell, or a
component of the pathogen or tumor, such as a protein, cell
membrane, cell extract, tumor markers like CEA or PSA, other
antigenic epitopes or the like. For example, the invention is
capable of detecting the pathogen or tumor by detecting the binding
of pathogenic or tumor markers in the patient's blood with an
antibody on the bio-assay device. In addition for example, the
binding of an antibody from a patient's blood to a viral protein,
such as an HIV protein is a common test for monitoring patient
exposure to the virus. Another common example is the quantitation
of Prostate Specific Antigen (PSA) in patient blood as a marker for
the progression of prostate cancer.
[0313] Additionally, drug receptor interactions, including both
membrane and non-membrane receptors and receptor conformational
changes as a result of drug binding can be determined with the
present invention. In another aspect, the invention can be used to
provide information on lipid interactions, such as lipo-proteins
binding to lipids, and liposomal interactions with lipids.
[0314] In additional embodiments, the technology of the invention
can be used to provide gene chips for screening nucleic acid
samples and proteomics chips for cataloging and describing
proteins. Such chips can make use of the unique ability of the
invention to measure simultaneously the affinity, kinetics, and
unique dielectric signatures of each binding event; and to make
these measurements at a multiplicity of addressable test sites on
the chip. The exact nature of the addressing will depend on the
applications, but the general strategy is as follows: Define a
vector space by the variables K.sub.eq, k.sub.A, and
.omega.=.omega.1, .omega.2, .omega.3, . . . ) where these variables
represent the equilibrium constant, the kinetic constant, and a
basis set of N frequencies at which the dielectric properties are
probed. An N+2 dimensional space is thus defined into which every
binding event can be mapped. A group of reference molecules is
subsequently chosen which represents a spectrum of binding events
of interest, such as a group of oligonucleotides with different
nucleic acid sequences or a selection of antibodies which are
specific for protein domains or other sub-structures of proteins,
and attach them to addressable points on the chip. A particular
species of molecules or group of species is introduced to the chip,
and each address is then probed for the value of each of the points
in the vector space defined above (or a suitable subset thereof).
Each species can then be represented by an address in the vector
space. The complexity of the system will depend on the size of the
vector space and the total number of different immobilized ligands
on the surface.
[0315] As an example of the above, consider a simple system
comprised of two different nucleic acid probes which are analyzed
at four different frequencies; and further, each of these
frequencies can be parsed into ten different amplitudes. Such a
system would have 100 million possible addresses (10.sup.4 for the
first polymorphism and 10.sup.4 for the second polymorphism). An
unknown placed in the system will be represented by a unique
address of the form [(1,5,3,7)(4,8,6,7)], where the first four
numbers represent the spectral response of the first probe at the
four selected frequencies, and the latter four numbers represent
the spectral response of the second probe at the four selected
frequencies. Thus with just two probes and four frequencies, 100
million unique addresses can be generated.
[0316] "Smart Needle" IV assays, which provide a miniature bioassay
device in the bore of a needle, can also be made to use the
technology of the present invention. This embodiment can be used to
provide cost-effective and safe medical diagnostics devices for use
in emergency rooms and other points-of-care settings and the like.
Examples of uses include: diagnosing acute conditions such as heart
attacks, infectious diseases like bacterial meningitis or Group B
Step infections in the neonatal/perinatal setting, coagulopathies,
fetal and neonatal oxygenation in the intensive care setting;
diagnosing chronic conditions in point-of-care settings such as
health care provider offices and remote locations.
[0317] A bio-assay device bearing a plurality biological binding
partners permits the simultaneous assay of a multiplicity of
analytes in a sample. In addition, the measurement of binding of a
single analyte to a number of different species of biological
binding partners provides a control for non-specific binding. A
comparison of the degree of binding of different analytes in a test
sample permits evaluation of the relative increase or decrease of
the different analytes.
[0318] The bio-assay device of this invention can be used to detect
virtually any analyte in vivo or ex vivo. While in a preferred
embodiment the analyte may be a biological molecule, it need not be
so limited so long as a specific binding partner is available or
some other property of the analyte can be measured in some
embodiment of the invention described herein. Suitable analytes
include virtually any analyte found in biological materials or in
materials processed therefrom. Virtually any analyte that can be
suspended or dissolved preferably in an aqueous solution can be
detected using the methods of this invention. Examples of analytes
of interest include 1) antibodies, such as antibodies to HIV 2),
Helicobacter pylori, hepatitis (e.g., hepatitis A, B and C),
measles, mumps, and rubella; 2) drugs of abuse and their metabolic
byproducts such as cotinine, cocaine, benzoylecgonine,
benzodizazpine, tetrahydrocannabinol, nicotine, ethanol; 3)
therapeutic drugs including theophylline, phenytoin, acetaminophen,
lithium, diazepam, nortryptyline, secobarbital, phenobarbitol, and
the like; 4) hormones and growth factors such as testosterone,
estradiol, 17-hydroxyprogesterone, progesterone, thyroxine, thyroid
stimulating hormone, follicle stimulating hormone, luteinizing
hormone, transforming growth factor alpha, epidermal growth factor,
insulin-like growth factor I and II, growth hormone release
inhibiting factor, and sex hormone binding globulin; and 5) other
analytes including glucose, cholesterol, caffeine, corticosteroid
binding globulin, DHEA binding glycoprotein, and the like.
[0319] As indicated above suitable analytes include, but are not
limited to proteins, glycoproteins, antigen, antibodies, nucleic
acids, sugars, carbohydrates, lectins, and the like. However,
larger, multimolecular, entities, such as cells, cell membranes and
other cellular constituents can also be detected and/or quantified
by the methods of this invention. Thus, for example, microorganisms
(e.g. bacteria, fungi, algae, etc.) having characteristic surface
markers (e.g. receptors, lectins, etc.) can be detected and/or
quantified (e.g. in a biological sample from an animal, or plant).
Similarly, cell types (e.g. cells characteristic of a particular
tissue) having characteristic markers (e.g. tumor cells
overexpressing IL-13 receptor (see, e.g., U.S. Pat. No.
5,614,191)). Thus, cells indicative of particular pathologies,
particular states of differentiation (or lack thereof) or
particular tissue types can be detected and/or quantified.
[0320] Conjugation of the Biological Binding Partner (Ligand or
Antiligand) Effector Molecule "Chip" Surface.
[0321] In one embodiment, the biological binding partner (ligand or
antiligand) is chemically conjugated to the underlying surface
(e.g. the bio-electric interface.) Means of chemically conjugating
molecules are well known to those of skill (see, e.g., Chapter 4 in
Monoclonal Antibodies: Principles and Applications, Birch and
Lennox, eds. John Wiley & Sons, Inc. N.Y. (1995) which
describes conjugation of antibodies to anticancer drugs, labels
including radio labels, enzymes, and the like).
[0322] The procedure for attaching a binding partner (e.g. a
protein, antibody, glycoprotein, nucleic acid, lectin, sugar,
carbohydrate, etc.) to a surface will vary according to the
chemical structure of the binding partner. Polypeptides typically
contain variety of functional groups; e.g., carboxylic acid (COOH)
or free amine (-NH.sub.2) groups, which are available for reaction
with a suitable functional on the surface or linker to which they
are to be bound. Similarly, other biological molecules, e.g.
nucleic acids, sugars, carbohydrates, all contain a variety of
functional groups (e.g. OH, NH2, COOH, --S, etc.) that are suitable
points for linkage.
[0323] Alternatively, the targeting molecule and/or effector
molecule may be derivatized to expose or attach additional reactive
functional groups. The derivatization may involve attachment of any
of a number of linker molecules such as those available from Pierce
Chemical Company, Rockford Illinois.
[0324] A "linker", as used herein, is a molecule that may be used
to join the biological binding partner (e.g. ligand or antiligand)
to the underlying (e.g. apparatus or device) surface. The linker is
capable of forming covalent bonds to both the biological binding
partner and to the underlying surface. Suitable linkers are well
known to those of skill in the art and include, but are not limited
to, straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or peptide linkers.
[0325] A bifunctional linker having one functional group reactive
with a group on the surface, and another group reactive with the
binding partner may be used to form the desired conjugate.
Alternatively, derivatization may involve chemical treatment of the
binding partner and/or the substrate. For example, a silica or
glass substrate can be silanized to create functional group.
Similarly, a protein or glycoprotein, can be derivatized, e.g., by
glycol cleavage of a sugar moiety attached to the protein antibody
with periodate to generate free aldehyde groups. The free aldehyde
groups on the antibody or protein or glycoprotein may be reacted
with free amine or hydrazine groups on athe surface to bind the
binding partner thereto (see U.S. Pat. No. 4,671,958). Procedures
for generation of free sulfhydryl groups on polypeptide, such as
antibodies or antibody fragments, are also known (see U.S. Pat. No.
4,659,839).
[0326] Many procedures and linker molecules for attachment of
various biological molecules to various metal, glass, plastic etc.,
substrates are well known to those of skill in the art. See, for
example, European Patent Application No. 188,256; U.S. Pat. Nos.
4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789;
and 4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47:
4071-4075. Methods of conjugating antibodies, proteins, and
glycoproteins abound in the immunotoxin literature and can be
found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming
the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical
Medicine, Academic Press, pp. 168-190 (1982), Waldmann, Science,
252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and 4,894,443.
[0327] Use of Nucleic Acid Binding Partners.
[0328] Where the binding partner is a nucleic acid (e.g. DNA, RNA,
peptide nucleic acid, etc.) specific binding is preferably achieved
under "stringent" conditions, the more stringent the conditions,
the more specific the hybridization.
[0329] The selection of stringent conditions for any probe/target
combination is routinely accomplished by those of ordinary skill in
the art. Moreover stringency can be determined empirically by
gradually increasing the stringency of the conditions (e.g.
increasing salt, raising temperature, etc.) until the desired level
of specificity is obtained. "Starting points" for stringent
conditions are well known. For example, desired nucleic acids will
hybridize to complementary nucleic acid probes under the
hybridization and wash conditions of 50% formamide at 42.degree. C.
Other stringent hybridization conditions may also be selected.
Generally, stringent conditions are selected to be about 5.degree.
C. lower than the thermal melting point (T.sub.m) for the specific
sequence at a defined ionic strength and pH. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe.
Typically, stringent conditions will be those in which the salt
concentration is at least about 0.02 molar at pH 7 and the
temperature is at least about 60.degree. C. As other factors may
significantly affect the stringency of hybridization, including,
among others, base composition and size of the complementary
strands, the presence of organic solvents and the extent of base
mismatching, the combination of parameters is more important than
the absolute measure of any one. An extensive guide to
hybridization of nucleic acids is found in Ausubel et al., Current
Protocols in Molecular Biology current Protocols, a joint venture
between Greene Publishing Associates, Inc and John Wiley and Sons,
Inc. (supplemented through 1998).
[0330] Oligonucleotides for use as binding partners are chemically
synthesized, for example, according to the solid phase
phosphoramidite triester method first described by Beaucage, S. L.
and Carruthers, M. H., 1981, Tetrahedron Lett., 22(20):1859-1862
using an automated synthesizer, as described in
Needham-VanDevanter, D. R., et al., 1984, Nucleic Acids Res.,
12:6159-6168. Purification of oligonucleotides is by either native
acrylamide gel electrophoresis or by anion-exchange HPLC as
described in Pearson, J. D. and Regnier, F. E. (1983) J. Chrom.
255:137-149. The sequence of the synthetic oligonucleotide can be
verified using the chemical degradation method of Maxam, A. M. and
Gilbert, W. (1980) in Methods Enzymol. 65:499-560.
[0331] The bio-assay device will have a variety of uses, including
for example, screening large numbers of molecules for biological
activity or screening biological samples for the presence or
absence concentration of a particular component or components. To
screen for biological activity, for example, the binding layer is
exposed to one or more receptors, such as antibodies or whole
cells. By detecting an interaction between the binding layer
antiligand and the ligand, the presence and concentration can be
determined. A particular advantage of this technique is that no
labels are needed to detect this interaction. The inherent
properties of the individual molecules are used to detect their
presence and amount, absence, or interaction with other
molecules.
[0332] Other possible applications for the bio-assay device or chip
include diagnostics, in which various antibodies for particular
receptors would be used to form the binding layer, and blood would
be screened for immune deficiencies for example. The bio-assay
device is optionally fabricated such that it fits into a hypodermic
needle bore. Only a tiny blood sample would be necessary to detect
a binding to a pre-applied antiligand on the binding layer. A
diagnostic assay can be made to measure a whole range of clinically
relevant analytes, from pathogens such as viruses or bacteria, to
metabolic activities like glucose concentration or lipid levels, to
the usual sets test for liver enzymes, electrolytes, clotting
factors, specific antibodies like ANA (used in rheumatological
disorders) and allergic response antibodies, arterial blood
oxygenation, drugs of abuse, and the like.
[0333] The bio-assay devices used in this capacity could be
inexpensive disposable chips, since they are easily fabricated and
not limited to semiconductor processing. For example, the chips are
optionally fabricated on cheap materials like plastic or glass
substrates. The chips are then optionally placed in a device as
described below and a signal propagated through the bio-assay
device to detect the binding interactions due to ligands in the
blood. In fact, many different shapes and sizes of the bio-assay
devices could be fabricated containing various binding layers for
the countless biological and chemical applications for which
detection without a label would be useful.
[0334] Unknown and uncharacterized proteins may be classified
and/or identified by detecting binding to structural motifs on the
unknown protein. For example, proteins in the same or similar class
have structural homologies; that is to say, substructures such as
domains that recur within a given class of proteins. By fabricating
a chip with multiple addressable arrays, each of which has a
antiligand for a specific substructure, an unknown molecular
species could be classified and/or identified as follows: The
presence of particular substructures is detected by the binding of
each to its respective antiligand. Each of these sub-structure
binding events is then characterized by such qualities as affinity,
kinetics, and spectral response. Correlation is then made between
the responses of the unknown molecular species and data obtained
from known proteins. In the case that no exact fit is found, much
of the structural details of the unknown compound can be pieced
together in much the same manner as NMR Spectroscopy does for
organic molecules.
[0335] In another embodiment, this technique may be used to develop
gene chips for the detection of nucleic acids. Gene chips are
arrays of nucleic acids that are used for the detection of
complementary nucleic acids in a sample. The existence of the
complementary DNA, as measured by binding to distinct DNA molecules
on the gene chip, is the desired output. In the event that
complementary binding does not occur, partial hybridization can be
detected and characterized by measuring such physical quantities as
affinity, melting point or other stringency conditions, and the
direct spectral response of the signal and correlation with
previously measured data. In this manner, a single antiligand in
the form of a nucleic acid sequence can detect a whole range of
polymorphisms without the need for a separate sequence for each of
the polymorphisms. For example, a chip with just a few hundred
different nucleic acid sequences could detect tens of thousands of
different polymorphisms.
[0336] Gene chips can be designed for the identification of drug
targets, bacterial identification, genotyping, and other
diagnostics needs. The technique requires the attachment of the
requisite nucleic acids, typically as probes, onto a substrate and
a method to measure binding of complementary nucleic acids to that
substrate. Ordinarily, the nucleic acids of the sample need to be
labeled, most commonly with a fluorescent probe. This technology
eliminates the need for labeling the sample DNA and the associated
problems. Gene chips can be developed for specific needs in drug
target identification, molecular diagnostics, and detection and
identification of biological warfare agents. Other types of devices
that could be fabricated and utilized are immunoassay devices, drug
discovery devices, and toxicity testing devices, analytical
devices, and the like.
[0337] The invention described herein can also be used for many
aspects of new drug development, from the initial screening process
all the way though patient typing and therapeutic monitoring. In
the initial stages of drug discovery, the invention can be used to
facilitate target identification, validation, and high throughput
screening (HTS). Target receptors can be the antiligand on the
bio-assay device, and by characterizing the actions of known
agonists, antagonists, or allosteric effectors, initial targets for
the high throughput screening procedure can be rapidly identified
and validated. In the HTS process, hundreds of thousands of
compounds are tested to determine which of them can bind to the
target. The invention described herein can be miniaturized, so that
highly parallel screening platforms can be realized; platforms
which are capable of screening hundreds or thousands of compounds
simultaneously, and at the same time determining the effect of
binding (e.g. agonist or antagonist), affinity, kinetics, etc.
Additionally, such miniature systems require very small amounts of
compound, thus greatly saving costs in purchasing said compounds
from combinatorial libraries. The system of detection formed
through use of the bio-assay device provides a high throughput
detection system because detection optionally occurs in real time
and many samples can be rapidly analyzed. The response period is
optionally monitored on a nanosecond time scale. As soon as the
molecules are bound to each other, detection occurs. More time is
optionally required to measure low concentrations or binding events
between molecules with a low binding affinity. The actual time is
optionally limited by diffusion rates. Other than these potential
limitations, thousands of compounds are optionally run through the
system very quickly, for example, in an hour. For example, using
chip fabrication technologies, a 10,000 channel device (using some
of the emerging microfluidics technologies) is possible, and with
small volumes and thus short diffusion times, and kinetic
measurements measuring only the beginning of the reaction, 10
million samples per hour are optionally measured. With known
concentrations, the binding affinity is optionally calculated from
the kinetics alone and thus the device can be probed at a very fast
time scale and the affinity calculated and/or estimated from the
slope of the kinetic curve. References for kinetics and affinities
can be found in any standard biochemistry or chemistry text such as
Mathews and van Holde, Biochemistry, Benjamin Cummings, New York,
1990.
[0338] The invention may be easily extended into cell-based assays,
since the detection may not require sample purification and
amplification. In these classes of applications, cellular systems
may be monitored for various changes either by detecting external
expressions or by lysing the cell to release the cytosolic
constituents and detect the presence of one or more analytes of
interest.
[0339] The invention may also be adapted to "Laboratory-on-a-Chip"
applications. Because of the ease of miniaturization, very small
chips with thousands or tens of thousands of addressable bio-assay
devices contained therein may be realized. The detector may be
realized as a sort of "logic gate" in which the presence of a
particular ligand or analyte has the effect of either turning on
the gate or turning off the gate, as is appropriate for a given
application. Such a gate may be realized in any number of ways
which translate the binding event into an electromagnetic signal
which can be assigned to one of two possible states corresponding
to off and on, 1 or 0, and the like. The two states could be
different frequencies of a resonant cavity or waveguide
corresponding to bound and unbound, or amplitude changes in a
transmission line or waveguide which correspond to bound and
unbound, or changes in the band-pass of a particular circuit, or
the like.
[0340] While the above is a complete description of possible
embodiments of the invention, various alternatives, modifications,
and equivalents may be used. For instance a person skilled in the
art will appreciate that the signal path of foregoing bio-assay
device is not limited to a transmission line. Other transmission
mediums, such as conductive or dielectric waveguides may
alternatively be used. Further, all publications and patent
documents recited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication and patent document was so individually
denoted. The above description should be view as only exemplary
embodiments of the invention, the boundaries of which are
appropriately defined by the metes and bounds of the following
claims.
* * * * *