U.S. patent application number 14/822737 was filed with the patent office on 2016-06-09 for real time microarrays.
The applicant listed for this patent is California Institute of Technology. Invention is credited to Arjang Hassibi, Babak Hassibi, Jose Luis Riechmann, Haris Vikalo.
Application Number | 20160160271 14/822737 |
Document ID | / |
Family ID | 38802311 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160160271 |
Kind Code |
A1 |
Hassibi; Arjang ; et
al. |
June 9, 2016 |
Real Time Microarrays
Abstract
This invention provides methods and systems for measuring the
binding of analytes in solution to probes bound to surfaces in
real-time.
Inventors: |
Hassibi; Arjang; (Sunnyvale,
CA) ; Hassibi; Babak; (San Marino, CA) ;
Vikalo; Haris; (Pasadena, CA) ; Riechmann; Jose
Luis; (Pasadena, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
California Institute of Technology |
Pasadena |
CA |
US |
|
|
Family ID: |
38802311 |
Appl. No.: |
14/822737 |
Filed: |
August 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11758621 |
Jun 5, 2007 |
9133504 |
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14822737 |
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60840060 |
Aug 24, 2006 |
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60811064 |
Jun 5, 2006 |
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Current U.S.
Class: |
506/9 ; 435/6.11;
436/501 |
Current CPC
Class: |
C12Q 1/6818 20130101;
C12Q 1/6837 20130101; C12Q 1/6818 20130101; C12Q 1/6837 20130101;
C12Q 2561/113 20130101; C12Q 2565/501 20130101; C12Q 2561/113
20130101; C12Q 2565/101 20130101; C12Q 2565/101 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1.-66. (canceled)
67. A system for determining at least one parameter associated with
a binding interaction between at least one analyte and at least one
probe, comprising: a substrate comprising a surface having said at
least one probe coupled to said surface; a signal source that
provides an input signal to said surface; a chamber in fluid
communication with said surface, wherein during use, said chamber
retains a fluid comprising said at least one analyte under
conditions that are sufficient to permit said binding interaction
between said at least one analyte and said at least one probe; a
detector that detects output signals from said surface in response
to said input signal from said signal source, which output signals
are detected at multiple time points in real time during said
binding interaction and are indicative of said binding interaction;
and a controller in communication with said detector, wherein said
controller (i) determines said at least one parameter associated
with said binding interaction based at least in part on said output
signals from said detector, and (ii) provides an output indicative
of said at least one parameter.
68. The system of claim 67, further comprising an electronic
storage device that stores said output.
69. The system of claim 67, wherein said at least one probe
provides a fluorescence signal, a luminescence signal, or an
absorption signal.
70. The system of claim 67, wherein said at least analyte provides
a fluorescence signal, a luminescence signal, or an absorption
signal.
71. The system of claim 67, wherein said input signal is an
electromagnetic signal.
72. The system of claim 67, wherein said at least one parameter
comprises: a forward binding reaction rate; a backward binding
reaction rate; C which is an original quantity of said at least one
analyte in said fluid; C-(P.sub.o-P(t)) which is an available
analyte density at time t, wherein P.sub.o is a number of unbound
molecules of said at least one probe, and wherein P(t) is a number
of unbound molecules of said at least one probe at time t; t which
is a time constant; P.sub..infin. which is a steady-state value of
P(t); n(t), which is a number of analytes in said fluid that are
specific to said probe molecule; P.sub.near, which is a probability
that said at least one analyte is in close proximity to said probe
molecule; P.sub.h, which is a probability that, in a unit interval
of time, said at least one analyte binds to said probe once said
analyte is near; P.sub.r, which is a probability that, in a unit
interval of time, said at least one analyte bound to said probe
molecule is released; N, which is a total number of analyte
molecules; q(t), which is a number of unbound molecules of said at
least one analyte as a function of time; a reaction rate between
said at least one analyte and said at least one probe; and a
cross-hybridization parameter.
73. The system of claim 67, wherein said substrate comprises a
plurality of different probes coupled to said surface.
74. The system of claim 67, wherein said fluid is configured to
contain a plurality of different analytes, including said at least
one analyte.
75. The system of claim 67, wherein said at least one probe is
labeled with a donor.
76. The system of claim 67, wherein said at least one analyte is
labeled with an acceptor.
77. The system of claim 76, wherein said acceptor is a
quencher.
78. The system of claim 67, wherein said at least one analyte is
not labeled with a metal cluster.
79. The system of claim 67, wherein said binding interaction is
non-competitive.
80. The system of claim 67, wherein during use, said output signals
are detected while said fluid is in contact with said surface.
81. The system of claim 80, wherein during use, said output signals
are detected by said detector without washing said fluid from
surface.
82. The system of claim 67, wherein said surface comprises a
plurality of probe spots, and wherein a given probe spot of said
plurality of probe spots includes said at least one probe.
83. The system of claim 67, wherein during use, said output signals
are generated in the absence of fluorescent resonance energy
transfer.
84. The system of claim 67, wherein said detector is integrated
with said substrate.
85. The system of claim 67, wherein said signal source and said
detector are disposed adjacent to a same side of said
substrate.
86. The system of claim 67, wherein during use, said fluid
comprises a plurality of different analytes.
87. The system of claim 67, further comprising an assembly that is
capable of changing a temperature of said fluid and/or said
substrate.
Description
CROSS-REFERENCE
[0001] This application claims priority to U.S. Provisional
Application Nos. 60/811,064, filed Jun. 5, 2006, and 60/840,060
filed on Aug. 24, 2006, which are incorporated herein by reference
in their entirety.
BACKGROUND OF THE INVENTION
[0002] Affinity-based biosensors exploit selective binding and
interaction of certain bio-molecules (recognition probes) to detect
specific target analytes in biological samples. The essential role
of the biosensor platforms and the parallel and miniaturized
version of them as microarrays are to exploit specific bindings of
the probe-target complexes to produce detectable signals, which
correlate with the presence of the targets and conceivably their
abundance. The essential components of such a system include the
molecular recognition layer (capturing probes) integrated within or
intimately associated with a signal-generating physiochemical
transducer and a readout device.
[0003] To generate target-specific signal, the target analytes in
the sample volume generally first need to collide with the
recognition layer, interact with the probes, bind to the correct
probes, and ultimately take part in a transduction process. The
analyte motion in typical biosensor settings (e.g., aqueous
biological buffers) can be dominated by diffusion spreading, which
from a microscopic point of view is a probabilistic mass-transfer
process (modeled as a random walk for each analyte molecule).
Accordingly, analyte collisions with probes become a stochastic
process. Moreover, because of the quantum-mechanical nature of
chemical bond forming, the interaction between the probes and the
analytes molecules is also probabilistic, thus further contributing
to uncertainty and noise corruption of the measured data in
biosensors and microarrays. We view such phenomena as inherent
noise in the detection system, which results in unavoidable
uncertainties even when the measurements are noiseless. Such
inherent noise is essentially inevitable since it originates from
the stochastic nature of molecular-level interactions. Its examples
include Poisson noise sources in microarrays and image sensor
detection shot-noise.
[0004] Beside the inherent noise, other non-idealities also corrupt
the signal obtained by the microarray experiments. Examples of such
phenomena include probe density variations, sample preparation
systematic errors, and probe saturation. We define systematic
errors as the unwanted deviations from the intended detection
procedure. If these errors are accurately evaluated, in theory,
they can be compensated by post experiment data processing.
[0005] Gene expression microarrays are a widely used microarray
platform. These systems can measure the expression level of
thousands of genes simultaneously, providing a massively-parallel
affinity-based detection platform in life science research.
Unfortunately, the uncertainty originating from both inherent noise
sources and systematic errors in each experiment can obscure some
of the important characteristics of the biological processes of
interest. The expression level uncertainty (overall measurement
error) in microarrays, can originate from the probabilistic
characteristics of detection process as mentioned before, all the
way from sample extraction and mRNA purification to hybridization
and fluorescent intensity measurements. Currently, there are
various techniques which attempt to increase the accuracy and
signal-to-noise ratio (SNR) of the estimated values. Nonetheless,
these techniques often rely on comparative methods, redundant
spots, or mathematical algorithms which introduce confidence zones
by excluding the unreliable data and outliers. Independent of the
method utilized, the degree in which the SNR can be improved in
such approaches can still be limited by the inherent microarray
noise and systematic errors.
[0006] The interfering signals originating from non-specific
bindings in microarrays are generally referred to as "background
signals." Traditionally in microarray analysis, background signals
and their fluctuations are all considered as corruptive noise
without any signal content. Users often implement a sub-optimal yet
widely adopted approach. This technique defines a confidence
threshold level for the signal intensity in view of the background,
which effectively divides the signals into irrelevant (below
threshold) and relevant (above threshold) regimes. This particular
approach is theoretically valid and optimal only when there is a
global background signal which is constant everywhere. In practical
microarray experiments, this assumption may not, be not valid since
the background and fluctuation level varies between spots. The
approach can thus be sub-optimal. Even when local background
subtraction methods are employed, the intensity data are
sub-optimally processed, as the background signal that is present
in the immediate vicinity of a given microarray probe spot may not
actually be the same as the background signal from within the spot.
The major outcome of background subtraction, regardless of the
method that is used, is that the minimum detectable level (MDL) is
higher than necessary. It also contributes more errors in ratio
analysis approaches, since low level signals are basically
truncated away. Both of these effects in turn can reduce the
microarray detection dynamic range.
[0007] Beside all the uncertainties within the measurement results,
there is also one major question in microarrays and all essentially
affinity-based biosensor systems, and that is of the necessary
incubation time (hybridization time for DNA microarrays). Since the
incubation kinetics in the microarrays experiments is a function of
analyte diffusion, reaction chamber size, temperature and binding
kinetics of every analyte species, as well as the unknown analyte
concentrations, the settling time of the system is quite complex
and unpredictable. Although all these questions can, to some
extent, be empirically addressed, they are still major impediments
in microarray technology and platform-to-platform inconsistencies
can be caused by them.
[0008] In conventional fluorescent-based microarrays and other
extrinsic reporter-based (label-based) biosensors assays, the
detection of captured analytes is usually carried out after the
incubation step. In some cases, proper fluorescent and reporter
intensity measurements are compromised in the presence of a large
concentration of floating (unbound) labeled species in the
incubation solution, whose signal can overwhelm the target-specific
signal from the captured targets. When the incubation is ceased and
the solution is removed from the surface of the array, the washing
artifacts often occur that make the analysis of the data even more
challenging. Thus there exists a need for affinity based sensors
that are able to simultaneously obtain high quality measurements of
the binding characteristics of multiple analytes, and that are able
to determine the amounts of those analytes in solution.
SUMMARY OF THE INVENTION
[0009] One aspect of the invention is a method comprising measuring
binding of analytes to probes on a microarray in real-time. In one
embodiment, the method comprises the steps of: contacting a fluid
volume comprising a plurality of different analytes with a solid
substrate comprising a plurality of different probes, wherein the
probes are capable of specifically binding with the analytes; and
measuring signals at multiple time points while the fluid volume is
in contact with the substrate, wherein the signals measured at
multiple time points can be correlated with the amount of binding
of the analytes with the probes. One embodiment of the method
further comprises using the signals measured at multiple time
points to determine the concentration of an analyte in the fluid
volume. In some embodiments, a change in the signals with time
correlates with the amount of the analytes bound to the probes.
[0010] In some embodiments, the signals comprise electrochemical,
electrical, mechanical, magnetic, acoustic, or electromagnetic
signals. In some embodiments, the signals are electromagnetic
signals comprising fluorescence, absorption, or luminescence. In
some embodiments, the analytes comprise quenching moieties, and the
electromagnetic signals measured at multiple time points correlate,
at least in part to the quenching of fluorescence. In some
embodiments, the fluorescence that is quenched is due at least in
part to fluorescent moieties bound to the probes. In some
embodiments, the fluorescence that is quenched is due at least in
part to fluorescent moieties bound to the solid substrate, wherein
such fluorescent moieties are not covalently bound to the probes.
In some embodiments, the fluorescence that is quenched is due at
least in part to fluorescent moieties that are non-covalently bound
to the probe. In some embodiments, the optical signals measured at
multiple time points are due, at least in part, to fluorescent
resonance energy transfer (FRET).
[0011] In some embodiments, the different probes are located on the
solid substrate at different addressable locations. In some
embodiments there are at least about 3, 4, 5 10, 50, 100, 500,
1000, 10,000, 100,000, or 1,000,000 probes. In some embodiments,
the solid substrate comprises one or more beads.
[0012] In some embodiments, the analytes and/or the probes comprise
the chemical species of nucleic acids or nucleic acid analogs,
proteins, carbohydrates, or lipids. In some embodiments, the
analytes and the probes are the same type of chemical species. In
some embodiments, the analyte and the probe are each a different
type of chemical species. In some embodiments, the analyte and the
probe comprise nucleic acids or nucleic acid analogs. In some
embodiments, the probes comprise proteins, which in some cases can
comprise antibodies or enzymes.
[0013] In some embodiments, the at least two time points are
measured at a time when the amount of binding of an analyte to a
probe corresponding to that signal is less than 50% of
saturation.
[0014] In some embodiments, two or more time points are measured
when the fluid volume is at one temperature, and two or more time
points are measured when the fluid volume is at a second
temperature. In some embodiments, two or more time points are
measured when the analyte in the fluid volume is at one
concentration, and two or more time points are measured when the
fluid volume is at a second concentration. In some embodiments, the
concentration of the analyte is enriched between the sets of time
points. In some embodiments, the concentration of the analyte is
diluted between the sets of time points.
[0015] In some embodiments, the solid substrate also comprises
control spots.
[0016] In some embodiments, the method comprises the use of
multiple fluorescent species, with different excitation and/or
emission spectra. In some embodiments, the method comprises the use
of multiple quencher species, with different quenching
properties.
[0017] In some embodiments the method further comprises determining
binding of a probe with two or more analytes comprising subjecting
binding data corresponding to signal and time to an algorithm which
determines the contribution of each of the two or more analytes to
the binding data. In some embodiments, the probes comprise a
fluorescent moiety and the initial surface concentration of probes
is determined by measuring fluorescence.
[0018] One aspect of the invention is a method of claim 2 wherein:
(i) the analytes comprise nucleic acid molecules labeled with a
quencher; (ii) the probes comprise nucleic acid molecules; (iii)
the substrate is substantially planar and comprises an array of
discrete locations at different addresses on the substrate, wherein
the locations have attached thereto different probes and a
fluorescent label that produces the signal, wherein the fluorescent
label is in sufficient proximity to the probe whereby hybridization
between an analyte and probe at the location causes FRET and/or
quenching of the signal. In some embodiments, the fluorescent label
is attached to the surface through the probe. In some embodiments,
the fluorescent label is non-covalently attached to the probe. In
some embodiments, the fluorescent label is not attached to the
surface through the probe. In some embodiments, the concentration
of the analyte in the fluid based on the kinetics of change of the
signal over time.
[0019] In some embodiments of the method of the invention, the
fluid volume further comprises competitor molecules labeled with a
quencher, wherein the competitor molecules compete with the
analytes for binding with the probes.
[0020] One aspect of the invention is a method of detecting binding
between analyte molecules and an array of probe molecules
comprising: (a) incubating the analyte molecules with the array of
probe molecules, wherein binding between a analyte molecule and a
probe molecule results in a change in a signal from the array; and
(b) measuring the signal from the array over time during
incubation. In some embodiments, binding between a target molecule
and a probe molecule results in a decrease in a signal from the
array. In some embodiments, binding between a target molecule and a
probe molecule results in an increase in a signal from the
array.
[0021] One aspect of the invention is a method of detecting binding
between an analyte molecule and a probe comprising: (a) incubating
an analyte molecule comprising a quencher of a donor-quencher pair
with a probe immobilized on a surface of a solid substrate under
conditions whereby the analyte molecule can bind to the probe,
wherein the donor of the donor-quencher pair is immobilized on the
surface at a distance from the immobilized probe and whereby
binding between the analyte molecule and the probe quenches a
signal from the donor, and wherein the donor is not directly
coupled to the immobilized probe; and (b) measuring the signal. In
some embodiments, the method comprises incubating a plurality of
different analyte molecules with a plurality of different probes
immobilized to different addressable locations on the surface of
the substrate.
[0022] One aspect of the invention is a system comprising: (a) a
device with (i) a solid support having a surface and (ii) a
plurality of different probes, wherein the different probes are
immobilized to the surface; (b) a fluid volume comprising an
analyte wherein the fluid volume is in contact with the solid
support, and (c) a detector assembly comprising means to detect
signals measured at multiple time points from each of a plurality
of spots on the microarray while the fluid volume is in contact
with the solid support. In some embodiments, the signals measured
at multiple time points detected by the detectors can be correlated
with the binding of analyte to the probes.
[0023] In some embodiments the system further comprises: (d) an
assembly that controls temperature of the solid support and/or the
fluid volume. In some embodiments, the different probes are
immobilized at different addressable locations.
[0024] In some embodiments the system further comprises: (e) a data
acquisition system for acquiring and storing the data and (f) a
computing system for analyzing the signals. In some embodiments,
the plurality of detectors comprises an array of transducers. In
some embodiments, the array of transducers is capable of measuring
an electrochemical, electrical, mechanical, magnetic, acoustic, or
optical signal. In some embodiments, the solid substrate is in
contact with the array of transducers. In some embodiments, the
transducer array spaced away from the solid substrate. In some
embodiments, one or more transducers correspond to an addressable
location. In some embodiments, the transducer array is an optical
transducer array which is optically coupled to the solid substrate.
In some embodiments, the optical transducer array is optically
coupled to the solid substrate via one or more lenses.
[0025] In some embodiments, the system is capable of measuring a
plurality of binding rates simultaneously. In some embodiments, the
plurality of binding rates is used to determine the concentration
of a plurality of analytes.
[0026] In some embodiments, the signal detected by the detector
comprises an electrochemical, electrical, mechanical, magnetic,
acoustic, or optical signal. In some embodiments, the signal is
generated from cyclic voltammetry, impedance spectroscopy, or
surface plasmon resonance systems. In some embodiments, the signal
is an optical signal. In some embodiments, the signal is from
fluorescence, absorption, or luminescence. In some embodiments, the
signals measured at multiple time points, correlating to the
binding of analyte are due, at least in part, to fluorescent
resonance energy transfer (FRET). In some embodiments, the signals
measured at multiple time points, correlating to the binding of
analyte are due, at least in part, to quenching of a fluorescent
signal. In some embodiments, the signals measured at multiple time
points are due, at least in part, to the interaction between an
analyte comprising a quenching moiety, and probe comprising a
fluorescent moiety. In some embodiments, the moiety comprising the
optical signal is covalently bound to the probe molecule. In some
embodiments, the moiety comprising the optical signal is
non-covalently bound, to the probe molecule. In some embodiments,
the moiety comprising the optical signal is bound to the probe
molecule through another molecule. In some embodiments, the moiety
comprising the optical signal is bound to the substrate.
[0027] One aspect of the invention is a system comprising: an assay
assembly comprising means to engage a microarray and means to
perform an assay on a surface of the microarray; and a detector
assembly comprising means to detect signals measured at multiple
time points from each of a plurality of spots on the microarray
during the performance of the assay. In some embodiments, the means
to perform the assay comprise means to provide a compartment
wherein the surface of the microarray comprises a floor of the
compartment and means to deliver reagents and analytes into the
compartment.
[0028] One aspect of the invention is a device comprising: a solid
substrate having a surface and a plurality of different probes,
wherein the different probes are immobilized to the surface at
different addressable locations, the addressable locations comprise
optical signal moieties bound to the surface, the optical signal
moieties are not bound directly to the probes, and the optical
signal from the optical signal moieties is capable of changing upon
binding of an analyte to the probes. In some embodiments, the
optical signal moiety comprises a dye, a luminescent moiety, or a
fluorescent moiety.
[0029] One aspect of the invention comprises software comprising:
(a) a computer executable code that accesses information about
signals measured at multiple time points at each of a plurality of
known locations on a microarray, wherein the signal intensity is a
function of the number of binding events between analyte molecules
and probe molecules attached to the microarray at known locations.
In some embodiments, the software further comprises: (b) code that
executes an algorithm that uses the information to determine the
expected number of binding events before binding has reached
saturation, the existence and number of analyte molecules in the
solution and the existence of cross-hybridization. The algorithm
furthermore can suppress the effects of cross-hybridization on the
acquired data.
INCORPORATION BY REFERENCE
[0030] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0032] FIG. 1 shows conventional detection procedure in
affinity-based biosensors where capturing probes are used to
capture target analyte in the incubation phase, and detection is
carried out in a dry-phase after completion of the incubation
[0033] FIG. 2 shows conventional detection after completing
incubation at time t.sub.1 and the uncertainty associated with
it.
[0034] FIG. 3 shows that in real-time microarray systems of the
present invention, multiple measurement of the number of captured
analytes can be carried out, without the necessity of stopping the
incubation step.
[0035] FIG. 4 shows a block diagram of the errors associated with
conventional DNA microarrays.
[0036] FIG. 5 shows a nucleic acid based real-time microarray
system of the present invention where the probes are labeled with
fluorescent moieties.
[0037] FIG. 6 shows a nucleic acid based real-time microarray
system of the present invention where the probes are labeled with
fluorescent moieties, the analytes are labeled with quenchers, and
the fluorescent intensity on various spots can be used to measure
the amount of analyte specifically bound to probe.
[0038] FIG. 7 shows a block diagram of a real-time microarray
system of the present invention.
[0039] FIG. 8 shows an example of a real-time microarray system An
example of a real-time microarray system where binding of BHQ2
quencher-labeled cDNA molecules were detected using a fluorescent
laser-scanning microscope.
[0040] FIG. 9 shows a real-time microarray system where the
detection system comprises a sensor array in intimate proximity of
the capturing spots.
[0041] FIG. 10 shows the layout of a 6.times.6 DNA microarray of
the present invention
[0042] FIG. 11 shows a few samples of the real-time measurements of
the microarray experiment where control target analytes are added
to the system
[0043] FIGS. 12-15 each show data for 4 different spots with
similar oligonucletide capturing probes. The target DNA analyte is
introduced in the system at time zero and quenching (reduction of
signal) occurs only when binding happens.
[0044] FIG. 16 shows the signals measured during two real-time
experiments wherein a target analyte (target 2) is applied to the
microarray, at 2 ng and at 0.2 ng.
[0045] FIG. 17 shows the signal versus time measured in a real-time
oligonucleotide array, and the fit of the data to an algorithm,
where 80 ng/50 .mu.l of the target is applied to the array.
[0046] FIG. 18 shows he signal measured in a real-time
oligonucleotide array, and the fit of the data to an algorithm,
where 16 ng/50 .mu.l of the target is applied to the array.
[0047] FIG. 19 shows the results of a simulation indicating the
potential of suppression of cross-hybridization over 3 orders of
magnitude.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0048] The methods, devices, and systems disclosed herein concern
the real-time measurements of target binding events in microarrays
and parallel affinity-based biosensors. The methods and systems
described herein can be referred to as real-time microarray
(RT-.mu.Array) systems. Real-time measurement of the kinetics of
multiple binding events allows for an accurate and sensitive
determination of binding characteristics or of analyte
concentration for multiple species simultaneously. One aspect of
present invention is the evaluation of the abundance of target
analytes in a sample by the real-time detection of target-probe
binding events. In certain embodiments, RT-.mu.Array detection
systems measure the concentration of the target analytes by
analyzing the binding rates and/or the equilibrium concentration of
the captured analytes in a single and/or plurality of spots. Some
applications of RT-.mu.Arrays fall within the field of Genomics and
Proteomics, in particular DNA and Protein microarrays. Some of the
advantages of RT-.mu.Array systems over conventional microarray
platforms are in their higher detection dynamic range, lower
minimum detection level (MDL), robustness, and lower sensitivity to
array fabrication systematic errors, analyte binding fluctuation,
and biochemical noise, as well as in the avoidance of the washing
step required for conventional microarrays.
[0049] One aspect of the invention is a method of measuring binding
of analytes to a plurality of probes on surface in "real time". As
used herein in reference to monitoring, measurements, or
observations of binding of probes and analytes of this invention,
the term "real-time" refers to measuring the status of a binding
reaction while that reaction is occurring, either in the transient
phase or in biochemical equilibrium. Real time measurements are
performed contemporaneously with the monitored, measured, or
observed binding events, as opposed to measurements taken after a
reaction is fixed. Thus, a "real time" assay or measurement
contains not only the measured and quantitated result, such as
fluorescence, but expresses this at various time points, that is,
in hours, minutes, seconds, milliseconds, nanoseconds, etc. "Real
time" includes detection of the kinetic production of signal,
comprising taking a plurality of readings in order to characterize
the signal over a period of time. For example, a real time
measurement can comprise the determination of the rate of increase
or decrease in the amount of analyte bound to probe.
[0050] The measurements are performed in real time with a plurality
of probes and analytes. The invention is useful for measuring
probes and analytes that bind specifically to one another. A probe
and an analyte pair that specifically bind to one another can be a
specific binding pair.
[0051] The methods and systems of the invention can be used for
measuring the binding of multiple specific binding pairs in the
same solution at the same time. In one embodiment of the invention
a plurality of probes which are members of a specific binding pair
are attached to a surface, and this surface is used to measure the
binding kinetics of a plurality of analytes which comprise the
other member of the specific binding pair.
[0052] One aspect of the invention is a method of measuring binding
between analyte and probe which lowers, and in some cases
eliminates the noise which is present in conventional microarrays
and which decreases the quality of the analyte-probe binding
information. In conventional microarrays and most of the
affinity-based biosensors, the detection and incubation phases of
the assay procedure are carried out at different times. As shown in
FIG. 1, conventional detection is carried out in a dry-phase at the
point where a scanning and/or imaging technique used to assess the
captured targets.
[0053] The following analysis illustrates inherent problems with
conventional microarray analysis techniques, and the advantages of
the real time microarray systems of the present invention in
improving the quality of the binding measurement. Let x(t) denote
the total number of captured analyte in a given spot of the
microarray and/or affinity-based biosensor at a given time instant
t. Furthermore, let x(t) denote the expected value of x(t) when the
incubation process has reached biochemical equilibrium. A typical
microarray procedure is focused on estimating x(t), and using its
value as an indication of the analyte concentration in the sample;
in fact, most data analysis techniques deduce their results based
on x(t). Nevertheless, if we measure the number of captured
analytes at time t.sub.1 in the equilibrium, for any given
microarray spot it holds that x(t.sub.1).noteq.x(t). This is due to
the inherent biochemical noise and other uncertainties of the
system. This phenomenon is illustrated in FIG. 2, where the number
of captured analytes in each spot of the microarray fluctuates in
time, even in biochemical equilibrium. Accordingly, a single
measurement taken at time t.sub.1, which is what conventional
microarray experiments provide, essentially samples a single point
of the random process of analyte binding.
[0054] Now, consider the case where we are able measure x(t)
multiple times, in real-time without the necessity of stopping the
incubation and analyte binding reaction. This platform, which we
call the real-time microarrays (RT-.mu.Arrays), has many advantages
over the conventional method. In some embodiments of RT-.mu.Arrays,
the kinetic of the bindings can be observed. Therefore, one can
test whether the system has reached biochemical equilibrium or not.
In other embodiments, multiple samples of x(t) are measured (see
FIG. 3), and different averaging techniques and/or estimation
algorithms can be used to estimate x(t) and other characteristics
of process x(t).
[0055] FIG. 4 shows a block diagram of the errors associated with
conventional DNA microarrays. These may be categorized into three
stages: pre-hybridization (steps 1 and 2), hybridization (step 3),
and post-hybridization (steps 4 and 5). The pre-hybridization
errors arise from sample purification variations and the errors or
variations in reverse transcribing mRNA to cDNA, in generating in
vitro transcribed RNA complementary to the cDNA (cRNA, or IVT RNA),
and or in labeling the analytes (step 1), and the errors arising
from non-uniform probe spotting and or synthesis on the array (step
2). The hybridization errors arise from the inherent biochemical
noise, cross-hybridization to similar targets, and the probe
saturation (step 3). Post-hybridization errors include washing
artifacts, image acquisition errors (step 4), and suboptimal
detection (step 5). The most critical of these are probe density
variations (step 2), probe saturation and cross-hybridization (step
3) and washing artifacts (step 4).
[0056] The methods and systems of the present invention can
compensate for all the above errors except for those of sample
preparation (step 1). Probe density variations can be measured
prior to incubation and therefore accounted for in post-processing
(step 5), incubation noise can be reduced by taking many samples
(rather than a single one), as mentioned earlier, probe saturation
can be avoided by estimating target concentrations from the
reaction rates, and finally washing is avoided altogether.
[0057] One aspect of the invention is a method of measuring the
binding of a plurality of analytes to a plurality of probes with a
higher dynamic range than obtained with conventional microarrays.
In some embodiments of the invention, the dynamic range is 3, 4, 5
or more orders of magnitude.
Methods
[0058] One aspect of the invention is a method comprising measuring
binding of analytes to probes on a microarray in real-time. In one
embodiment, the method comprises the steps of: contacting a fluid
volume comprising a plurality of different analytes with a solid
substrate comprising a plurality of different probes, wherein the
probes are capable of specifically binding with the analytes; and
measuring signals at multiple time points while the fluid volume is
in contact with the substrate, wherein the signals measured at
multiple time points can be correlated with the amount of binding
of the analytes with the probes. One embodiment of the method
further comprises using the signals measured at multiple time
points to determine the concentration of an analyte in the fluid
volume.
[0059] In one embodiment the method involves the use of probe
arrays in which each addressable location emits a signal that is
quenchable upon binding of an analyte. For example, the quenchable
moiety (e.g., a fluorescent moiety) is attached to the probe on the
array or in close physical proximity thereto. The surface of such
array will emit signal from each addressable location which can be
detected using, for example, a microscope and a light detector
(e.g., a CCD camera or CMOS image sensor). The analytes in the
sample are tagged with a quencher moiety that can quench the signal
from the quenchable moiety. When the quencher does not, itself,
emit a light signal, there is no signal from the fluid to interfere
with the signal from the array. This diminishes the noise at the
array surface. During the course of a binding reaction between
analytes and substrate-bound probes, the signal at each addressable
location is quenched. The signal at each addressable location is
measured in real time, for example, by a CCD camera focused on the
array surface. As the signal at any location changes as a result of
binding and quenching, the change is measured. These measurements
over time allow determination of the kinetics of the reaction
which, in turn, allows determination of the concentration of
analytes in the sample.
[0060] Alternatively if the analytes are labeled with a
light-emitting reporter, such as a fluorescent label, signal at the
surface of array resulting from binding of the labeled analyte
molecules can be detected by properly focusing the detector at the
array surface, thereby minimizing the noise from signal in
solution.
[0061] In another embodiment, the probes are attached to the
surface of an array comprising sensors, such as a CMOS sensor
array, which produce electrical signals that change as a result of
binding events on the probes. This also affords real time
measurement of a plurality of signals on an array (Hassibi and Lee,
IEEE Sensors journal, 6-6, pp. 1380-1388, 2006, and Hassibi, A.
"Integrated Microarrays," Ph.D. Thesis Stanford University,
2005.
[0062] Accordingly, the methods of this invention allow real time
measurements of a plurality of binding events on an array of probes
on a solid support.
Analyte and Probe
[0063] The terms "probe" and "analyte" as used herein refer to
molecular species that bind to one another in solution. A single
probe or a single analyte is generally one chemical species. That
is, a single analyte or probe may comprise many individual
molecules. In some cases, a probe or analyte may be a set of
molecules that are substantially identical. In some cases a probe
or analyte can be a group of molecules all of which have a
substantially identical binding region. A "probe" and/or "analyte"
can be any pair of molecules that bind to one another, including
for example a receptor/ligand pair, or a hybridizing pair of
nucleic acids. In probes of the present invention are bound to a
solid surface. The analyte is in solution, and can also be referred
to as the target or the target analyte. Thus, while the probe and
analyte can interchangeably be the different members of any binding
pair, in some cases it is more advantageous for one or the other to
be the probe or the analyte, for instance where the molecule is
more easily coupled to the surface, it can be advantageous for that
molecule to be the probe, or where a molecule is more soluble in
the solution of interest, it can be advantageous for that molecule
to be the analyte.
[0064] The probes or analytes can be any type of chemical species.
The probes or analytes are generally biomolecules such as nucleic
acids, proteins, carbohydrates, lipids, or small molecules. The
probe and analyte which bind to one another can each be the same or
different types of species. The analyte or probe may be bound to
another type of molecule and may comprise different molecules. For
example, an analyte could be a protein carbohydrate complex, or a
nucleic acid connected to protein. A probe-analyte pair can also be
a receptor-ligand pair. Where the chemical species is large or made
of multiple molecular components, the probe or analyte may be the
portion of the molecule that is capable of binding, or may be the
molecule as a whole. Examples of analytes that can be investigated
by this invention include, but are not restricted to, agonists and
antagonists for cell membrane receptors, toxins and venoms, viral
epitopes, hormones (e.g., opiates, steroids, etc.), hormone
receptors, peptides, enzymes, enzyme substrates, substrate analogs,
transition state analogs, cofactors, drugs, proteins, antibodies,
and hybridizing nucleic acids.
[0065] The term "probe" is used herein to refer to the member of
the binding species that is attached to the substrate. For
instance, the probe consists of biological materials deposited so
as to create spotted arrays; and materials synthesized, deposited,
or positioned to form arrays according to other current or future
technologies. Thus, microarrays formed in accordance with any of
these technologies may be referred to generally and collectively
hereafter for convenience as "probe arrays." Moreover, the term
"probe" is not limited to probes immobilized in array format.
Rather, the functions and methods described herein may also be
employed with respect to other parallel assay devices. For example,
these functions and methods may be applied with respect to
probe-set identifiers that identify probes immobilized on or in
beads, optical fibers, or other substrates or media. The
construction of various probe arrays of the invention are described
in more detail below.
[0066] In some embodiments, the probe and/or the analyte comprises
a polynucleotide. The terms "polynucleotide," "oligonucleotide,"
"nucleic acid" and "nucleic acid molecule" as used herein include a
polymeric form of nucleotides of any length, either ribonucleotides
(RNA) or deoxyribonucleotides (DNA). This term refers only to the
primary structure of the molecule. Thus, the term includes triple-,
double- and single-stranded DNA, as well as triple-, double- and
single-stranded RNA. It also includes modifications, such as by
methylation and/or by capping, and unmodified forms of the
polynucleotide. More particularly, the terms "polynucleotide,"
"oligonucleotide," "nucleic acid" and "nucleic acid molecule"
include polydeoxyribonucleotides (containing 2-deoxy-D-ribose),
polyribonucleotides (containing D-ribose), any other type of
polynucleotide which is an N- or C-glycoside of a purine or
pyrimidine base, and other polymers containing nonnucleotidic
backbones. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and U.S. Pat. No. 5,644,048),
phosphorodithioate (Briu et al., J. Am. Chem. Soc. 11 1:2321
(1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), and peptide nucleic acid backbones and linkages
(see Carlsson et al., Nature 380:207 (1996)). Other analog nucleic
acids include those with positive backbones (Denpcy et al., Proc.
Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat.
Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);
Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et
al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,
Bioorganic & Medicinal Chem. Left. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Left. 37:743 (1996))
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
169-176). These modifications of the ribose-phosphate backbone may
be done to facilitate the addition of labels, or to increase the
stability and half-life of such molecules in physiological
environments.
[0067] In some embodiments of the invention, oligonucleotides are
used. An "oligonucleotide" as used herein is a single-stranded
nucleic acid ranging in length from 2 to about 1000 nucleotides,
more typically from 2 to about 500 nucleotides in length. In some
embodiments, it is about 10 to about 100 nucleotides, and in some
embodiments, about 20 to about 50 nucleotides.
[0068] In some embodiments of the invention, for example,
expression analysis, the invention is directed toward measuring the
nucleic acid concentration in a sample. In some cases the nucleic
acid concentration, or differences in nucleic acid concentration
between different samples, reflects transcription levels or
differences in transcription levels of a gene or genes. In these
cases it can be desirable to provide a nucleic acid sample
comprising mRNA transcript(s) of the gene or genes, or nucleic
acids derived from the mRNA transcript(s). As used herein, a
nucleic acid derived from an mRNA transcript refers to a nucleic
acid for whose synthesis the mRNA transcript or a subsequence
thereof has ultimately served as a template. Thus, a cDNA reverse
transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA
amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all derived from the mRNA transcript and detection of
such derived products is indicative of the presence and/or
abundance of the original transcript in a sample. Thus, suitable
samples include, but are not limited to, mRNA transcripts of the
gene or genes, cDNA reverse transcribed from the mRNA, cRNA
transcribed from the cDNA, DNA amplified from the genes, RNA
transcribed from amplified DNA, and the like.
[0069] In some embodiments, where it is desired to quantify the
transcription level (and thereby expression) of one or more genes
in a sample, the nucleic acid sample is one in which the
concentration of the mRNA transcript(s) of the gene or genes, or
the concentration of the nucleic acids derived from the mRNA
transcript(s), is proportional to the transcription level (and
therefore expression level) of that gene. Similarly, it is
preferred that the hybridization signal intensity be proportional
to the amount of hybridized nucleic acid. While it is preferred
that the proportionality be relatively strict (e.g., a doubling in
transcription rate results in a doubling in mRNA transcript in the
sample nucleic acid pool and a doubling in hybridization signal),
one of skill will appreciate that the proportionality can be more
relaxed and even non-linear. Thus, for example, an assay where a 5
fold difference in concentration of the target mRNA results in a 3
to 6 fold difference in hybridization intensity is sufficient for
most purposes. Where more precise quantification is required
appropriate controls can be run to correct for variations
introduced in sample preparation and hybridization as described
herein. In addition, serial dilutions of "standard" target mRNAs
can be used to prepare calibration curves according to methods well
known to those of skill in the art. Of course, where simple
detection of the presence or absence of a transcript or large
differences of changes in nucleic acid concentration are desired,
no elaborate control or calibration is required.
[0070] In the simplest embodiment, such a nucleic acid sample is
the total mRNA or a total cDNA isolated and/or otherwise derived
from a biological sample. The term "biological sample", as used
herein, refers to a sample obtained from an organism or from
components (e.g., cells) of an organism. The sample may be of any
biological tissue or fluid. Frequently the sample will be a
"clinical sample" which is a sample derived from a patient. Such
samples include, but are not limited to, sputum, 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.
[0071] The nucleic acid (either genomic DNA or mRNA) may be
isolated from the sample according to any of a number of methods
well known to those of skill in the art. One of skill will
appreciate that where alterations in the copy number of a gene are
to be detected genomic DNA is preferably isolated. Conversely,
where expression levels of a gene or genes are to be detected,
preferably RNA (mRNA) is isolated.
[0072] Methods of isolating total mRNA are well known to those of
skill in the art. For example, methods of isolation and
purification of nucleic acids are described in detail in Chapter 3
of Laboratory Techniques in Biochemistry and Molecular Biology:
Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic
Acid Preparation, P. Tijssen, ed. Elsevier, N.Y. (1993) and Chapter
3 of Laboratory Techniques in Biochemistry and Molecular Biology:
Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic
Acid Preparation, P. Tijssen, ed. Elsevier, N.Y. (1993)).
[0073] Frequently, it is desirable to amplify the nucleic acid
sample prior to hybridization. One of skill in the art will
appreciate that whatever amplification method is used, if a
quantitative result is desired, care must be taken to use a method
that maintains or controls for the relative frequencies of the
amplified nucleic acids.
[0074] In some embodiments, the probe and or the analyte may
comprise a polypeptide. As used herein, the term "polypeptide"
refers to a polymer of amino acids and does not refer to a specific
length of the product; thus, peptides, oligopeptides, and proteins
are included within the definition of polypeptide. This term also
does not refer to or exclude post expression modifications of the
polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like. The "peptide" refers to polypeptides
of no more than about 50 amino acids, while term "protein" refers
to longer polypeptides, typically with three-dimensional
structures. Non-natural polypeptides containing one or more analogs
of an amino acid (including, for example, unnatural amino acids,
etc.), can also be useful in the invention, as can polypeptides
with substituted linkages, as well as other modifications known in
the art, both naturally occurring and non-naturally occurring.
Polypeptides and proteins can have specific binding properties. For
instance, an enzyme can have a region that binds specifically with
a substrate, and/or has regions that bind to other proteins, such
as the binding of enzyme subunits. Antibodies, which can have very
specific binding properties are also polypeptides.
[0075] In some embodiments the probe and/or analyte can comprise a
carbohydrate such as a polysaccharide. The term polysaccharide, as
used herein, refers to a carbohydrate which is a polyhydroxy
aldehyde or ketone, or derivative thereof, having the empirical
formula (CH.sub.2O).sub.n wherein n is a whole integer, typically
greater than 3. Monosaccharides, or simple sugars, consist of a
single polyhydroxy aldehyde or ketone unit. Monosaccharides
include, but are not limited to, ribose, 2-deoxy-ribose, glucose,
mannose, xylose, galactose, fucose, fructose, etc. Disaccharides
contain two monosaccharide units joined by a glycosidic linkage.
Disaccharides include, for example, sucrose, lactose, maltose,
cellobiose, and the like. oligosaccharides typically contain from 2
to 10 monosaccharide units joined in glycosidic linkage.
Polysaccharides (glycans) typically contain more than 10 such units
and include, but are not limited to, molecules such as heparin,
heparan sulfate, chondroitin sulfate, dermatan sulfate and
polysaccharide derivatives thereof. The term "sugar" generally
refers to mono-, di- or oligosaccharides. A saccharide may be
substituted, for example, glucosamine, galactosamine,
acetylglucose, acetylgalactose, N-acetylglucosamine,
N-acetyl-galactosamine, galactosyl-N-acetylglucosamine,
N-acetylneuraminic acid (sialic acid), etc. A saccharide may also
reside as a component part of a larger molecule, for example, as
the saccharide moiety of a nucleoside, a nucleotide, a
polynucleotide, a DNA, an RNA, etc.
[0076] In some embodiments, the analyte and/or probe is a small
molecule. Generally the small molecule will be an organic molecule,
for example, biotin or digoxigenin, but in some cases, the analyte
can be inorganic, for example an inorganic ion such as lithium,
sodium, ferric, ferrous, etc. The small molecule can also be an
organometallic compound, having both inorganic and organic
components.
Probes on a Solid Substrate
[0077] For the methods of the present invention, the probes are
attached to a solid substrate. The solid substrate may be
biological, nonbiological, organic, inorganic, or a combination of
any of these, existing as particles, strands, precipitates, gels,
sheets, tubing, spheres, containers, capillaries, pads, slices,
films, plates, slides, semiconductor integrated chips etc. The
solid substrate is preferably flat but may take on alternative
surface configurations. For example, the solid substrate may
contain raised or depressed regions on which synthesis or
deposition takes place. In some embodiments, the solid substrate
will be chosen to provide appropriate light-absorbing
characteristics. For example, the substrate may be a polymerized
Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP,
SiO.sub.2 SiN.sub.4, modified silicon, or any one of a variety of
gels or polymers such as (poly)tetrafluoroethylene,
(poly)vinylidendifluoride, polystyrene, polycarbonate, or
combinations thereof. The solid support and the chemistry used to
attach the solid support are described in detail below.
[0078] The substrate can be a homogeneous solid and/or unmoving
mass much larger than the capturing probe where the capturing
probes are confined and/or immobilized within a certain distance of
it. The mass of the substrate is generally at least 100 times
larger than capturing probes mass. In certain embodiments, the
surface of the substrate is planar with roughness of 0.1 nm to 100
nm, but typically between 1 nm to 10 nm. In other embodiments the
substrate can be a porous surface with roughness of larger than 100
nm. In other embodiments, the surface of the substrate can be
non-planar. Examples of non-planar substrates are spherical
magnetic beads, spherical glass beads, and solid metal and/or
semiconductor and/or dielectric particles.
[0079] For the methods of the present invention, the plurality of
probes may be located in one addressable region and/or in multiple
addressable regions on the solid substrate. In some embodiments the
solid substrate has about 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100,
100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-50,000,
50,000-100,000, 100,000-500,000, 500,000-1,000,000 or over
1,000,000 addressable regions with probes.
[0080] In some embodiments it is also useful to have addressable
regions which do not contain probe, for example, to act as control
spots in order to increase the quality of the measurement, for
example, by using binding to the spot to estimate and correct for
non-specific binding.
Analyte/Probe Binding
[0081] The methods of the present invention are directed toward
measuring the binding characteristics of multiple probes and
analytes in real time. The method is particularly useful for
characterizing the binding of probes and analytes which
specifically bind to one another. As used herein, a probe
"specifically binds" to a specific analyte if it binds to that
analyte with greater affinity than it binds to other substances in
the sample.
[0082] The binding between the probe and the analyte in the present
invention occurs in solution. Usually the probe and analyte are
biomolecules and the solution is an aqueous solution. An aqueous
solution is a solution comprising solvent and solute where the
solvent is comprised mostly of water. The methods of the invention,
however, can be used in any type of solution where the binding
between a probe and an analyte can occur and be observed.
[0083] Molecular recognition assays generally involve detecting
binding events between two types of molecules. The strength of
binding can be referred to as "affinity". Affinities between
biological molecules are influenced by non-covalent intermolecular
interactions including, for example, hydrogen bonding, hydrophobic
interactions, electrostatic interactions and Van der Waals forces.
In multiplexed binding experiments, such as those contemplated
here, a plurality of analytes and probes are involved. For example,
the experiment may involve testing the binding between a plurality
of different nucleic acid molecules or between different proteins.
In such experiments analytes preferentially will bind to probes for
which they have the greater affinity. Thus, determining that a
particular probe is involved in a binding event indicates the
presence of an analyte in the sample that has sufficient affinity
for the probe to meet the threshold level of detection of the
detection system being used. One may be able to determine the
identity of the binding partner based on the specificity and
strength of binding between the probe and analyte.
[0084] The binding may be a receptor-ligand, enzyme-substrate,
antibody-antigen, or a hybridization interaction. The probe/analyte
binding pair or analyte/probe binding pair can be nucleic acid to
nucleic acid, e.g. DNA/DNA, DNA/RNA, RNA/DNA, RNA/RNA, RNA. The
probe/analyte binding pair or analyte/probe binding pair can be a
nucleic acid and a polypeptide, e.g. DNA/polypeptide and
RNA/polypeptide, such as a sequence specific DNA binding protein.
The probe/analyte binding pair or analyte/probe binding pair can be
any nucleic acid and a small molecule, e.g. RNA/small molecule,
DNA/small molecule. The probe/analyte binding pair or analyte/probe
binding pair can be any nucleic acid and synthetic DNA/RNA binding
ligands (such as polyamides) capable of sequence-specific DNA or
RNA recognition. The probe/analyte binding pair or analyte/probe
binding pair can be a protein and a small molecule or a small
molecule and a protein, e.g. an enzyme or an antibody and a small
molecule.
[0085] The probe/analyte binding pair or analyte/probe binding pair
can be a carbohydrate and protein or a protein and a carbohydrate,
a carbohydrate and a carbohydrate, a carbohydrate and a lipid, or
lipid and a carbohydrate, a lipid and a protein, or a protein and a
lipid, a lipid and a lipid.
[0086] The analyte/probe binding pair can comprise natural binding
compounds such as natural enzymes and antibodies, and synthetic
binding compounds. The probe/analyte binding pair or analyte/probe
binding pair can be synthetic protein binding ligands and proteins
or proteins and synthetic binding ligands, synthetic carbohydrate
binding ligands and carbohydrates or carbohydrates and synthetic
carbohydrate binding ligands, synthetic lipid binding ligands or
lipids and lipids and synthetic lipid binding ligands.
Nucleic Acid Systems
[0087] One particularly useful aspect of the present invention
involves specific hybridization between an analyte and a probe,
where both comprise nucleic acids.
[0088] As used herein an "oligonucleotide probe" is an
oligonucleotide capable of binding to a target nucleic acid of
complementary sequence through one or more types of chemical bonds,
usually through complementary base pairing, usually through
hydrogen bond formation. The oligonucleotide probe may include
natural (i.e. A, G, C, or T) or modified bases (7-deazaguanosine,
inosine, etc.). In addition, the bases in oligonucleotide probe may
be joined by a linkage other than a phosphodiester bond, so long as
it does not interfere with hybridization. Thus, oligonucleotide
probes may be peptide nucleic acids in which the constituent bases
are joined by peptide bonds rather than phosphodiester linkages.
The oligonucleotide probes can also comprise locked nucleic acids
(LNA), LNA, often referred to as inaccessible RNA, is a modified
RNA nucleotide. The ribose moiety of the LNA nucleotide is modified
with an extra bridge connecting 2' and 4' carbons. The bridge
"locks" the ribose in 3'-endo structural conformation, which is
often found in A-form of DNA or RNA. LNA nucleotides can be mixed
with DNA or RNA bases in the oligonucleotide. Such oligomers are
commercially available. The locked ribose conformation can enhance
base stacking and backbone pre-organization, and can increase the
thermal stability (melting temperature) of oligonucleotides.
[0089] The term "nucleic acid analyte" or "target nucleic acid" or
"target" refers to a nucleic acid (often derived from a biological
sample and hence referred to also as a sample nucleic acid), to
which the oligonucleotide probe specifically hybridizes. It is
recognized that the target nucleic acids can be derived from
essentially any source of nucleic acids (e.g., including, but not
limited to chemical syntheses, amplification reactions, forensic
samples, etc.). It is either the presence or absence of one or more
target nucleic acids that is to be detected, or the amount of one
or more target nucleic acids that is to be quantified. The target
nucleic acid(s) that are detected preferentially have nucleotide
sequences that are complementary to the nucleic acid sequences of
the corresponding probe(s) to which they specifically bind
(hybridize). The term target nucleic acid may refer to the specific
subsequence of a larger nucleic acid to which the probe
specifically hybridizes, or to the overall sequence (e.g., gene or
mRNA) whose abundance (concentration) and/or expression level it is
desired to detect. The difference in usage will be apparent from
context.
[0090] In the present invention, the specific hybridization of an
oligonucleotide probe to the target nucleic acid can be measured in
real-time. An oligonucleotide probe will generally hybridize, bind,
or duplex, with a particular nucleotide sequence under stringent
conditions even when that sequence is present in a complex mixture.
The term "stringent conditions" refers to conditions under which a
probe will hybridize preferentially to its target subsequence, and
to a lesser extent to, or not at all to, other sequences.
[0091] For nucleic acid systems, the oligonucleotide probes of the
present invention are designed to be complementary to a nucleic
acid target sequence, such that hybridization of the target
sequence and the probes of the present invention occurs. This
complementarity need not be perfect; there may be any number of
base pair mismatches which will interfere with hybridization
between the target sequence and the single stranded nucleic acids
of the present invention. However, if the number of mutations is so
great that no hybridization can occur under even the least
stringent of hybridization conditions, the sequence is not a
complementary target sequence. Thus, an oligonucleotide probe that
is not substantially complementary to a nucleic acid analyte will
not hybridize to it under normal reaction conditions.
[0092] The methods of the present invention thus can be used, for
example, to determine the sequence identity of a nucleic acid
analyte in solution by measuring the binding of the analyte with
known probes. The sequence identity can be determined by comparing
two optimally aligned sequences or subsequences over a comparison
window or span, wherein the portion of the polynucleotide sequence
in the comparison window may optionally comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical subunit (e.g. nucleic
acid base or amino acid residue) occurs in both sequences to yield
the number of matched positions, dividing the number of matched
positions by the total number of positions in the window of
comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0093] The methods of the current invention when applied to nucleic
acids, can be used for a variety of applications including, but not
limited to, (1) mRNA or gene expression profiling, involving the
monitoring of expression levels for example, for thousands of genes
simultaneously. These results are relevant to many areas of biology
and medicine, such as studying treatments, diseases, and
developmental stages. For example, microarrays can be used to
identify disease genes by comparing gene expression in diseased and
normal cells; (2) comparative genomic hybridization (Array CGH),
involving the assessment of large genomic rearrangements; (3) SNP
detection arrays for identifying for Single Nucleotide
Polymorphisms (SNP's) in the genome of populations; and chromatin
immunoprecipitation (chIP) studies, which involve determining
protein binding site occupancy throughout the genome, employing
ChIP-on-chip technology.
[0094] The present invention can be very sensitive to differences
in binding between nucleic acid species, in some cases, allowing
for the discrimination down to a single base pair mismatch. And
because the present invention allows the simultaneous measurement
of multiple binding events, it is possible to analyze several
species simultaneously, where each is intentionally mismatched to
different degrees. In order to do this, a "mismatch control" or
"mismatch probe" which are probes whose sequence is deliberately
selected not to be perfectly complementary to a particular target
sequence can be used, for example in expression arrays. For each
mismatch (MM) control in an array there, for example, exists a
corresponding perfect match (PM) probe that is perfectly
complementary to the same particular target sequence. In "generic"
(e.g., random, arbitrary, haphazard, etc.) arrays, since the target
nucleic acid(s) are unknown, perfect match and mismatch probes
cannot be a priori determined, designed, or selected. In this
instance, the probes can be provided as pairs where each pair of
probes differs in one or more pre-selected nucleotides. Thus, while
it is not known a priori which of the probes in the pair is the
perfect match, it is known that when one probe specifically
hybridizes to a particular target sequence, the other probe of the
pair will act as a mismatch control for that target sequence. It
will be appreciated that the perfect match and mismatch probes need
not be provided as pairs, but may be provided as larger collections
(e.g., 3, 4, 5, or more) of probes that differ from each other in
particular preselected nucleotides. While the mismatch(s) may be
located anywhere in the mismatch probe, terminal mismatches are
less desirable as a terminal mismatch is less likely to prevent
hybridization of the target sequence. In a particularly preferred
embodiment, the mismatch is located at or near the center of the
probe such that the mismatch is most likely to destabilize the
duplex with the target sequence under the test hybridization
conditions. In a particularly preferred embodiment, perfect matches
differ from mismatch controls in a single centrally-located
nucleotide.
[0095] It will be understood by one of skill in the art that
control of the characteristics of the solution such as the
stringency are important in using the present invention to measure
the binding characteristics of a analyte-probe pair, or the
concentration of a nucleic acid analyte (target nucleic acid). A
variety of hybridization conditions may be used in the present
invention, including high, moderate and low stringency conditions;
see for example Maniatis et al., Molecular Cloning: A Laboratory
Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology,
ed. Ausubel, et al, hereby incorporated by reference. Stringent
conditions are sequence-dependent and will be different in
different circumstances. Longer sequences hybridize specifically at
higher temperatures. An extensive guide to the hybridization of
nucleic acids is found in Tijssen, Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes,
"Overview of principles of hybridization and the strategy of
nucleic acid assays" (1993). In some embodiments, highly stringent
conditions are used. In other embodiments, less stringent
hybridization condition; for example, moderate or low stringency
conditions may be used, as known in the art; see Maniatis and
Ausubel, supra, and Tijssen, supra. The hybridization conditions
may also vary when a non-ionic backbone, i.e. PNA is used, as is
known in the art.
[0096] Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences tend to
hybridize specifically at higher temperatures. Generally, stringent
conditions can be 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, pH, and nucleic acid concentration)
at which 50% of the probes complementary to the target sequence
hybridize to the target sequence at equilibrium. (As the target
analyte sequences are generally present in excess, at T.sub.m, 50%
of the probes are occupied at equilibrium). Typically, stringent
conditions will be those in which the salt concentration is at
least about 0.01 to 1.0 M Na ion concentration (or other salts) at
pH 7.0 to 8.3 and the temperature is at least about 30.degree. C.
for short probes (e.g., 10 to 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide.
[0097] In some embodiments, the probe and or the analyte may
comprise an antibody. As used herein, the term "antibody" refers to
an immunoglobulin molecule or a fragment of an immunoglobulin
molecule having the ability to specifically bind to a particular
molecule, referred to as an antigen. The antibody may be an
anti-receptor antibody specific for the receptor used in the assay.
Thus, the antibody may be capable of specifically binding the
receptor as the antigen. Antibodies and methods for their
manufacture are well known in the art of immunology. The antibody
may be produced, for example, by hybridoma cell lines, by
immunization to elicit a polyclonal antibody response, or by
recombinant host cells that have been transformed with a
recombinant DNA expression vector that encodes the antibody.
Antibodies include but are not limited to immunoglobulin molecules
of any isotype (IgA, IgG, IgE, IgD, IgM), and active fragments
including Fab, Fab', F(ab').sub.2, Facb, Fv, ScFv, Fd, V.sub.H and
V.sub.L. Antibodies include but are not limited to single chain
antibodies, chimeric antibodies, mutants, fusion proteins,
humanized antibodies and any other modified configuration of an
immunoglobulin molecule that comprises an antigen recognition site
of the required specificity.
[0098] The preparation of antibodies including antibody fragments
and other modified forms is described, for example, in
"Immunochemistry in Practice," Johnstone and Thorpe, Eds.,
Blackwell Science, Cambridge, Mass., 1996; "Antibody Engineering,"
2.sup.nd edition, C. Borrebaeck, Ed., Oxford University Press, New
York, 1995; "Immunoassay", E. P. Diamandis and T. K. Christopoulos,
Eds., Academic Press, Inc., San Diego, 1996; "Handbook of
Experimental Immunology," Herzenberg et al., Eds, Blackwell
Science, Cambridge, Mass., 1996; and "Current Protocols in
Molecular Biology" F. M. Ausubel et al., Eds., Greene Pub.
Associates and Wiley Interscience, 1987, the disclosures of which
are incorporated herein. A wide variety of antibodies also are
available commercially.
[0099] In some embodiments, the probe and or the analyte may
comprise two proteins. Protein-protein interactions can enable two
or more proteins to associate. A large number of non-covalent bonds
can form between the proteins when two protein surfaces are
precisely matched, and these bonds account for the specificity of
recognition. Protein-protein interactions are involved, for
example, in the assembly of enzyme subunits; of multiprotein
enzymatic complexes, or of molecular machines; in enzyme-substrate
reactions; in antigen-antibody reactions; in forming the
supramolecular structures of ribosomes, filaments, and viruses; in
transport; and in the interaction of receptors on a cell with
growth factors and hormones. Products of oncogenes can give rise to
neoplastic transformation through protein-protein interactions. For
example, some oncogenes encode protein kinases whose enzymatic
activity on cellular target proteins leads to the cancerous state.
Another example of a protein-protein interaction occurs when a
virus infects a cell by recognizing a polypeptide receptor on the
surface, and this interaction has been used to design antiviral
agents. In some cases, protein-protein interactions can be
dependent on protein modifications. For example, histone proteins
can be modified at different positions with different chemical tags
(e.g. phosphorylation, or methylation), and the modifications
themselves be required or involved in the recognition by other
proteins (e.g chromatin remodeling and associated proteins).
Binding Kinetics
[0100] One aspect of the current invention is the use of the
measurement of the binding kinetics to characterize binding of
multiple probes and analytes in solution. The term "binding
kinetics" as used herein refers to the rate at which the binding of
the analyte to the probe occurs in a binding reaction. The term
"binding reaction" as used herein describes the reaction between
probes and analytes. In some cases, binding reaction refers to the
concurrent binding reactions of multiple analytes and probes, and
in other cases, the term binding reaction refers to the reaction
between a single probe with a single analyte. The meaning will be
clear from the context of use. The kinetic measurements can be
expressed as the amount of analyte bound to the probe as a function
of time. The binding kinetics can provide information about the
characteristics of the probe-analyte binding such as the strength
of binding, the concentration of analyte, the competitive binding
of an analyte, the density of the probes, or the existence and
amount of cross-hybridization.
[0101] In order to determine binding kinetics, the signal at
multiple time points must be determined. The signal at at least two
time points is required. In most cases, more than two time points
will be desired in order to improve the quality of the kinetic
information. In some embodiments the signal at, 2-10, 10-50,
50-100, 100-200, 200-400, 400-800, 800-1600, 1600-3200, 3200-6400,
6400-13000, or higher than 13,000 time points will be measured. One
of ordinary skill in the art can determine the effective number of
points for a given embodiment. For example, where few points are
obtained, the quality of information about the binding event can be
low, but where the number of data points is very high, the data
quality may be high, but the handling, storage, and manipulation of
the data can be cumbersome.
[0102] The frequency at which the signal is measured will depend on
the kinetics of the binding reaction or reactions that are being
monitored. As the frequency of measurements gets lower, the time
between measurements gets longer. One way to characterize a binding
reaction is to refer to the time at which half of the analyte will
be bound (t.sub.1/2). The binding reactions of the invention can
have a (t.sub.1/2) from on the order of milliseconds to on the
order of hours, thus the frequency of measurements can vary by a
wide range. The time between measurements will generally not be
even over the time of the binding reaction. In some embodiments, a
short time between of measurements will be made at the onset of the
reaction, and the time between measurements will be longer toward
the end of the reaction. One advantage of the present invention is
the ability to measure a wide range of binding rates. A high
initial frequency of measurements allows the characterization of
fast binding reactions which may have higher binding, and lower
frequency of measurements allows the characterization of slower
binding reactions. For example, points can initially be measured at
a time between points on the order of a microsecond, then after
about a millisecond, points can be measured at a time between
points on the order of a millisecond, then after about a second,
time points can be measured at a time between points on the order
of a second. Any function can be used to ramp the change in
measurement frequency with time. In some cases, as described below,
changes in the reaction conditions, such as stringency or
temperature changes will be made during a reaction, after which it
may be desirable to change the frequency of measurements to measure
the rates of reaction which will be changed by the change in
reaction condition.
[0103] In some embodiments, a probe will have substantially no
analyte bound to it at the beginning of the binding reaction, then
the probe will be exposed to a solution containing the analyte, and
the analyte will begin to bind, with more analyte bound to the
probe with time. In some cases, the reaction will reach saturation,
the point at which all of the analyte that is going to bind has
bound. Generally, saturation will occur when a reaction has reached
steady state. At steady state, in a given time period, just as many
analytes are released as new analytes are bound (the on rate and
off rate are equal). In some cases, with very strong binding, where
the off-rate for the analyte is essentially zero, saturation will
occur when substantially all of the analyte that can bind to the
probe will have bound, has bound. Thus, while it is advantageous to
measure a change in signal with time that can be correlated with
binding kinetics, the measurement of a signal that does not change
with time also provides information in the context of a real-time
experiment, and can also be useful in the present invention. For
example, in some cases the absence of a change in the signal will
indicate the level of saturation. In other cases the absence of a
change in signal can indicate that the rate of the reaction is very
slow with respect to the change in time measured. It is thus a
beneficial aspect of this invention to measure binding event in
real time both where signals change with time and where the signals
do not change with time.
[0104] One aspect of the methods of the present invention is the
measurement of concentration of an analyte from the measurement of
binding kinetics. Since analyte binding rate can be
concentration-dependant, we can estimate the analyte abundance in
the sample solution using binding rates.
[0105] In some embodiments, the concentration of analyte can be
determined by equations relating to the kinetics of the
hybridization process. For example, suppose that the number of
probes at a particular spot on the array prior to any hybridization
is given by P.sub.0. The probability of a specific target binding
to the probe site is given by
Prob(binding)=kProb(target and probe in close proximityProb(probe
is free), (1)
[0106] where k.ltoreq.1 depends of the bonds between the probe and
the target and essentially a function of temperature, incubation
conditions, and probe density. Here, the first probability is
proportional to the number of target molecules available whereas
the second probability is
Probe ( probe is free ) = P ( t ) P 0 , ( 2 ) ##EQU00001##
where P(t) is the number of available probes at time t, i.e., those
are not yet bound to any target. If we thus denote the forward and
backwards target/probe binding reaction rates by K.sub.+ and K,
respectively, we may write the following differential equation for
the available probe concentration P(t):
P ( t ) t = - K + P 0 P ( t ) ( C - P 0 - P ( t ) ) + K - ( P 0 - P
( t ) ) ( 3 ) ##EQU00002##
where C is the original target quantity in the solution so that
C-(P.sub.0-P(t)) represents the available target density at time t.
The above is a Riccati differential equation that can be solved in
closed form. However, instead of doing so, we can note that for
small values oft we have P(t).apprxeq.P.sub.0, so that the
differential equation becomes
P ( t ) t = - K + P 0 P ( t ) C . ( 4 ) ##EQU00003##
This a first-order linear differential equation with time constant
.tau.=P.sub.0/K.sub.+C. Accordingly, the target density can be
determined from the reaction rate (or time constant) of P(t). In
other words, using many sample measurements of P(t) at different
times and fitting them to the curve
P ( t ) = P 0 exp ( - K + P 0 C ) t ( 5 ) ##EQU00004##
allows us to estimate the target quantity C. In this case, the
reaction rate (or inverse of the time constant) is proportional to
the target concentration and inversely proportional to the probe
density, something that has been observed in experiments.
[0107] One can also attempt to estimate C from the steady-state
value of P(t), i.e., P.sub..infin.. This can be found by setting
dP(t)/dt=0 in the original Riccati equation which leads to a
quadratic equation for P.sub..infin.. In some simple cases, the
solution to this quadratic equation can be considerably
simplified.
[0108] When the target concentration is low: In this case, we can
assume P.sub.0>>C, so that we obtain
P.sub..infin.=P.sub.0-C, (6)
i.e., the reduction in available probes is equal to the target
concentration.
[0109] When the target concentration is high: In this case, we can
assume that P.sub.0<<C, so that we obtain
P .infin. = K - K + P 0 2 C . ( 6 ) ##EQU00005##
[0110] In this case, the number of remaining probes is inversely
proportional o the target concentration. This corresponds to probe
saturation, which generally is not as good a method of determining
C as determining C based on the reaction rate near the beginning of
the reaction.
[0111] One aspect of the present invention is the determination of
the binding of analyte to probe by measuring the rate near the
beginning of the reaction. In addition to providing a more reliable
estimate of C, measurement near the beginning of the reaction can
shorten the time that is required to measure analyte binding over
the time required for measuring binding from saturation. In some
embodiments of the invention, the binding is measured during the
time for less than about the first 0.1, 0.5, 1, 2, 3, 4, 5, 6, 8,
10, 12, 14, 18, 20, 25, 30, 40, 50, 60, 70, 80, or 90 percent of
the analyte to bind as compared to the amount of analyte bound at
saturation. In some embodiments, the binding kinetics are
determined in a time for less than about the first 20% of the
analyte to bind. In some embodiments, the binding kinetics are
determined in a time for less than about the first 1-2% of the
analyte to bind.
Changing Conditions During the Binding Experiment
[0112] One aspect of the methods of the present invention is a step
of changing the conditions during the binding experiment. In
conventional microarrays where only the end-point is determined,
only a single set of binding conditions can be tested. In the
methods of the present invention, the binding conditions can be
changed in order to explore multiple sets of binding conditions
during the same binding experiment. The condition which is changed
can be, for example, any condition that affects the rate of binding
of analyte to probe. The condition which is changed can be, for
example, temperature, pH, stringency, analyte concentration, ionic
strength, an electric field, or the addition of a competitive
binding compound.
[0113] In some embodiments, the condition that is changed changes
the rate of binding or hybridization. When measuring the binding of
multiple analytes to probes in the same binding medium, as in the
present invention, the kinetics of binding can vary widely for
different analyte-probe combinations. The binding rate conditions
can be varied, for example, by changing the temperature,
concentration, ionic strength, pH, or by applying an electric
potential. The binding rates for the different analytes can in some
cases vary by many orders of magnitude, making it difficult and
time consuming to measure the binding of all the analytes in one
binding experiment. This ability to change the rate conditions can
be used to improve the measurement of binding for multiple analytes
that bind at different rates, for example by performing the initial
part of the experiment under slower rate conditions, such that
rapidly binding analytes can be readily measured, then raising the
rate conditions such that more slowly binding analytes can be
readily measured. This method of changing the binding rate during
the binding experiment can also be used for better characterization
of a single analyte or single set of analytes in solution, for
instance, using binding rate conditions to measure the initial
portion of the binding kinetics, then increasing the binding rate
conditions to measure the later portion of the binding kinetics for
a single analyte, for example, to establish the level of
saturation. It will be understood by those of skill in the art that
this method of changing the rate conditions can result in both
improved quality of measurements, such as the measurement of
analyte concentration, and/or in a savings of time. With the
present method, for example by measuring the kinetics of binding,
then changing the conditions to increase the rate of binding of
weaker binding species, the time of the binding experiment can be
reduced by greater than about 10%, 20%, 50%, 75%, or by a factor of
2, 4, 8, 10, 50, 100, 1000 or greater than 1000 over the times
needed to obtain the same quality information using end-point
binding methods.
[0114] In some embodiments, the condition that is changed is the
stringency. As described above, the stringency can be changed by
many factors including temperature, ionic strength, and the
addition of compounds such as formamide. In some embodiments of the
present invention, the medium is at one stringency at the beginning
of the binding reaction, and at a later point the stringency of the
medium is changed. This method can be used where different analytes
or sets of analytes have different hybridization characteristics,
for example, allowing the measurement of the binding of one set of
analytes with a high stringency, then allowing the measurement of
another set of analytes at a lower stringency in the same medium as
part of the same binding experiment. This method can also be used
for the characterization of binding for a single analyte by, for
instance, measuring binding at high stringency at an initial
portion of the binding reaction, then lowering the stringency and
measuring a later portion of the binding reaction. The ability to
change stringency can also be used to create conditions where a
bound analyte becomes unbound, allowing, for instance, the
measurement of the kinetics of binding at one stringency, followed
by the measurement of release of the analyte into solution upon
raising the stringency. This method also allows the binding of an
analyte to a probe to be measured multiple times, for example, by
measuring the kinetics of binding of the analyte under one set of
stringency conditions, changing the stringency to release the
analyte, for instance, by raising the stringency, then measuring
the kinetics of binding of the analyte a subsequent time by
changing the stringency conditions again, for example by lowering
the stringency. Thus the ability to change the stringency during
the binding reaction allows for the measurement of any number of
binding and unbinding reactions with the same set of probes and
analytes.
[0115] In some embodiments of the method of the present invention,
an electric potential is applied during the binding reaction to the
fluid volume to electrically change the stringency of the medium.
In some embodiments, the system will provide an electrical stimulus
to the capturing region using an electrode structure which is
placed in proximity of the capturing region. If the analyte is an
electro-active species and/or ion, the electrical stimulus can
apply an electrostatic force of the analyte. In some embodiments
the electrical potential is direct current (DC). In some
embodiments, the electric potential is time-varying. In some
embodiments the electric potential has both DC and time varying
aspects. Their amplitude of the applied potential can be between 1
mV to 10V, but typically between 10 mV to 100 mV. The frequency of
time-varying signal is between 1 Hz to 1000 MHz, but typically
between 100 Hz to 100 kHz.
[0116] In some embodiments, the change in conditions is the
addition of a competitive binding agent. For example, initially, a
sample can be introduced which contains analyte that binds to a
particular probe. The binding of that analyte can be monitored as
described herein. Then, at any point during the binding of that
analyte, an analyte that competes for the binding of that analyte
to the probe can be added. The rate of displacement of the analyte
by the competitive binding agent can then be measured, providing
more information to characterize the binding of analyte to
probe.
Detection of Signals
[0117] For the methods of the present invention, a signal is
detected that can be correlated with the binding of analytes to the
plurality of probes. The type of signals appropriate for the
invention is any signal that can be amount of analyte bound to the
plurality of probes. Appropriate signals include, for example,
electrical, electrochemical, magnetic, mechanical, acoustic, or
electromagnetic (light) signals. Examples of electrical signals
useful in the present invention that can be correlated with analyte
binding are capacitance and/or impedance. For example, analytes
labeled with metals or metal clusters can change the capacitance
and/or the impedance of a surface in contact with a fluid, allowing
the amount of analyte bound to the probe on the surface to be
determined. The electrical measurement can be made at any frequency
including DC, 0-10 Hz, 10-100 Hz, 100-1000 Hz, 1 KHz-10 KHz, 10
KHz-100 KHz, 100 KHz-1 MHz, 1 MHz-10 MHZ, 10 MHz-100 MHz, 100 MHz-1
GHz, or above 1 GHz. In some embodiments, impedance spectroscopy
can be used which obtains impedance versus frequency for any range
of frequencies within the range of frequencies described above.
Examples of electrochemical signals useful in the present invention
that can be correlated with analyte binding include amperometric
and voltammetric measurements, and/or measurements that involve the
oxidation or reduction of redox species. For example, the analyte
can be labeled with a compound which undergoes an oxidation or
reduction reaction at a known redox potential, and the oxidative or
reductive current can be correlated with the amount of analyte
bound to surface probes. Examples of mechanical signals include the
use of microelectomechanical (MEMS) devices. For example, the
binding of analyte to probe on the surface of a small surface
feature, such as a cantilever, can change the mass of the surface
feature, the vibration frequency of which can then be correlated
with the amount of analyte bound to the probe. Generally, the
higher the mass, the lower the vibration frequency. Examples of
acoustic signals include surface acoustic wave (SAW), and surface
plasmon resonance signals. A surface acoustic wave (SAW) is an
acoustic wave traveling along the surface of a material having some
elasticity, with amplitude that typically decays exponentially with
the depth of the substrate. The binding of labeled or unlabeled
analyte to probe on a surface can change the SAW characteristics,
e.g. amplitude, frequency in a manner that can be correlated with
the amount of analyte bound to a probe. Surface plasmon resonance
relies on surface plasmons, also known as surface plasmon
polaritons, which are surface electromagnetic waves that propagate
parallel, usually along a metal/dielectric interface. Since the
wave is on the boundary of the metal and the external medium (water
for example), these oscillations are very sensitive to any change
of this boundary, such as the adsorption of molecules to the metal
surface. The binding of labeled or unlabeled analyte to a probe
attached to the surface can change the frequency of the resonant
surface plasmon in a manner that can be correlated with the amount
of analyte bound to the probes.
[0118] Particularly useful signals for the methods of the present
invention are electromagnetic (light) signals. Examples of optical
signals useful in the present invention are signals from
fluorescence, luminescence, and absorption. As used herein, the
terms "electromagnetic" or "electromagnetic wave" and "light" are
used interchangeably. Electromagnetic waves of any frequency and
wavelength that can be correlated to the amount of analyte bound to
probe on the surface can be used in the present invention including
gamma rays, x-rays, ultraviolet radiation, visible radiation,
infrared radiation, and microwaves. While some embodiments are
described with reference to visible (optical) light, the
descriptions are not meant to limit the embodiments to those
particular electromagnetic frequencies.
[0119] For the methods of the present invention it is desired that
the signal changes upon the binding of the analyte to the probe in
a manner that correlates with the amount of analyte bound. In some
cases, the change in signal will be a change in intensity of the
signal. In some embodiments, the signal intensity will increase as
more analyte is bound to probe. In some embodiments, the signal
intensity will decrease as more analyte is bound to probe. In some
embodiments, the change in signal is not a change in intensity, but
can be any other change in the signal that can be correlated with
analyte binding to probe. For example, the change in signal upon
binding of the probe can be a change in the frequency of the
signal. In some embodiments, the signal frequency will increase as
more analyte is bound to probe. In some embodiments, the signal
frequency will decrease as more analyte is bound to the probe.
[0120] The signal that is measured is generally the signal in the
region of the solid surface. In some embodiments, signal from
moieties attached to the surface is used as the signal that can be
correlated with the amount of analyte bound to the probe. In some
embodiments signal from the solution is used as the signal that can
be correlated with the amount of analyte bound to the probe.
[0121] In some embodiments of the methods of the present invention,
labels are attached to the analytes and/or the probes. Any label
can be used on the analyte or probe which can be useful in the
correlation of signal with the amount of analyte bound to the
probe. It would be understood by those of skill in the art that the
type of label with is used on the analyte and/or probe will depend
on the type of signal which is being used, for example, as
described above, a dense label for a mechanical signal, or a redox
active label for a voltammetric measurement.
[0122] In some embodiments, the signal that can be correlated to
the amount of analyte bound to probe is due to the buildup of label
at the surface as more analyte is bound to the probes on the
surface. For example, where the analyte has a fluorescent label, as
more analyte binds, the intensity of the fluorescent signal can
increase in a manner that can be correlated with the amount of
analyte bound to probe on the surface. In some embodiments, the
signal that can be correlated to the amount of analyte bound to
probe is due to the release of label from the surface. For example,
where the probe has a fluorescent label and the label is released
into solution upon the binding of the analyte to the probe, the
fluorescent intensity at the surface will decrease as more analyte
is bound and more fluorescent label is released.
[0123] In some embodiments, the signal that can be correlated to
the amount of analyte bound to probe is due to a change in the
signal from label on the surface upon binding of the analyte to the
probe. For example, where a fluorescent label is on the surface,
and the analyte is labeled with a compound capable of changing the
fluorescent signal of the surface fluorescent label upon binding of
the analyte with the probe, the change in signal can be correlated
with the amount of analyte bound to probe. In some embodiments, the
analyte is labeled with a quencher, and the decrease in intensity
from the surface fluorescent label due to quenching is correlated
to the increased amount of analyte bound to probe. In some
embodiments, the analyte is labeled with a fluorescent compound
which can undergo energy transfer with the fluorescent label on the
surface such that the increase in fluorescence from the analyte
fluorescent label and/or the decrease in fluorescence from the
surface fluorescent label can be correlated with the amount of
analyte bound to probe. In some embodiments the surface fluorescent
label is bound directly, e.g. covalently to the probe. In some
embodiments, the surface fluorescent label is bound to the surface,
is not bound to the probe, but is in sufficient proximity that the
binding of the analyte to the probe produces a change in signal
from the surface fluorescent label that can be correlated with the
amount of analyte bound to probe.
[0124] In some embodiments, the analyte is unlabeled, and the
binding characteristics and or concentration of the analyte is
determined by competitive binding with another labeled species,
which competes with the analyte for biding to a probe. For example,
where we have a solution with an analyte, A, whose concentration we
want to determine, and we have a competitive binding species, B,
whose binding characteristics with probe and whose concentration
are known, then using the present invention, we can use, for
example, an array of probes on a surface to determine the
concentration of A by determining the amount of competitive binding
of B to a probe. For example, the probe is attached to a surface
that is fluorescently labeled, and B is labeled with a quencher
such that the level of quenching of the surface fluorescence can be
correlated with the amount of B bound to the probe. The rate of
binding of B to the probe is measured in real time, and the
concentration of A is determined by knowing the characteristics of
A as a competitive binder. In some embodiments, the amount of the
competitive binding species does not need to be known beforehand.
For instance, the kinetics of binding of be can be measured in the
fluid volume, then the conditions can be changed, (e.g. increasing
the stringency) such that B is released from the probe, then the
analyte A is added, and the binding of B under competition with A
is measured. This example illustrates an advantage of the being
able to change the conditions of the medium during one experiment.
In some cases, A and B can be the same species, where B is labeled,
and the amount of B is known, and the amount of A can be determined
by the kinetics of the binding of B. In some cases, A and B are not
the same species, but compete for binding with a probe: This
competitive binding real-time assay can be done with all types of
molecular species described herein including nucleic acids,
antibodies, enzymes, binding proteins, carbohydrates and
lipids.
Electromagnetic Signals--Optical Methods
[0125] The use of optical detection provides a variety of useful
ways of implementing the methods of the present invention. Optical
methods include, without limitation, absorption, luminescence, and
fluorescence.
[0126] Some embodiments of the invention involve measuring light
absorption, for example by dyes. Dyes can absorb light within a
given wavelength range allowing for the measurement of
concentration of molecules that carry that dye. In the present
invention, dyes can be used as labels, either on the analyte or on
the probe. The amount of dye can be correlated with the amount of
analyte bound to the surface in order to determine binding
kinetics. Dyes can be, for example, small organic or organometallic
compounds that can be, for example, covalently bound to the analyte
to label the analyte. Dyes which absorb in the ultraviolet,
visible, infrared, and which absorb outside these ranges can be
used in the present invention. Methods such as attenuated total
reflectance (ATR), for example for infrared, can be used to
increase the sensitivity of the surface measurement.
[0127] Some embodiments of the invention involve measuring light
generated by luminescence. Luminescence broadly includes
chemiluminescence, bioluminescence, phosphorescence, and
fluorescence. In some embodiments, chemiluminescence, wherein
photons of light are created by a chemical reaction such as
oxidation, can be used. Chemiluminescent species useful in the
invention include, without limitation, luminol, cyalume, TMAE
(tetrakis(dimethylamino)ethylene), oxalyl chloride, pyrogallol
(1,2,3-trihydroxibenzene), lucigenin. In some embodiments,
bioluminescence is used. Where the luminescence is bioluminescence,
creation of the excited state derives from an enzyme catalyzed
reaction. Bioluminescence derives from the capacity of living
organisms to emit visible light through a variety of
chemiluminescent reaction systems. Bioluminescence generally
include three major components: a luciferin, a luciferase and
molecular oxygen. However other components may also be required in
some reactions, including cations (Ca.sup.++ and Mg.sup.++) and
cofactors (ATP, NAD(P)H). Luciferases are enzymes that catalyze the
oxidation of a substrate, luciferin, and produce an unstable
intermediate. Light is emitted when the unstable intermediate
decays to its ground state, generating oxyluciferin. Any of the
different unrelated types of luciferin can be used herein including
those from phyla which use a luciferin, known as coelenterazine,
which contains a ring formed by three amino acids (2 tyrosines, and
a phenylalanine). Photoproteins from animals such as jellyfish can
be used where the "photoprotein" of the luciferin/luciferase system
emits light upon calcium binding. Other bioluminescent systems as
described in U.S. Patent Application 2007/0065818, and including
bioluminescence resonance energy transfer (BRET) as described in
U.S. Patent Application 2007/0077609 can be used in the present
invention.
Fluorescent Systems
[0128] A useful embodiment of the present invention involves the
use of fluorescence. As used herein, fluorescence refers to the
process wherein a molecule relaxes to its ground state from an
electronically excited state by emission of a photon. As used
herein, the term fluorescence also encompasses phosphorescence. For
fluorescence, a molecule is promoted to an electronically excited
state by generally by the absorption of ultraviolet, visible, or
near infrared radiation. The excited molecule then decays back to
the ground state, or to a lower-lying excited electronic state, by
emission of light. An advantage of fluorescence for the methods of
the invention is its high sensitivity. Fluorimetry may achieve
limits of detection several orders of magnitude lower than for
absorption. Limits of detection of 10.sup.-10 M or lower are
possible for intensely fluorescent molecules; in favorable cases
under stringently controlled conditions, the ultimate limit of
detection (a single molecule) may be reached.
[0129] A wide variety of fluorescent molecules can be utilized in
the present invention including small molecules, fluorescent
proteins and quantum dots. Useful fluorescent molecules
(fluorophores) include, but are not limited to: 1,5 IAEDANS;
1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;
5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein;
5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM
(5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy
Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA
(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G;
6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);
7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine;
ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine);
Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;
Acriflavin Feulgen SITSA; Aequorin (Photoprotein);
AFPs--AutoFluorescent Protein--(Quantum Biotechnologies); Alexa
Fluor 350.TM.; Alexa Fluor 430.TM.; Alexa Fluor 488.TM.; Alexa
Fluor 532.TM.; Alexa Fluor 546.TM.; Alexa Fluor 568.TM.; Alexa
Fluor 594.TM.; Alexa Fluor 633.TM.; Alexa Fluor 647.TM.; Alexa
Fluor 660.TM.; Alexa Fluor 680.TM.; Alizarin Complexon; Alizarin
Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA
(Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;
Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC
(Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red
4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL;
Atabrine; ATTO-TAG.TM. CBQCA; ATTO-TAG.TM. FQ; Auramine;
Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole);
BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta
Lactamase; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC;
Blancophor FFG; Blancophor SV; BOBO.TM.-1; BOBO.TM.-3; Bodipy
492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy
530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy
576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy
665/676; Bodipy FI; Bodipy FL ATP; Bodipy FI-Ceramide; Bodipy R6G
SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy
TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO.TM.-1; BO-PRO.TM.-3;
Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue;
Calcium Crimson.TM.; Calcium Green; Calcium Green-1 Ca.sup.2+Dye;
Calcium Green-2 Ca.sup.2+; Calcium Green-5N Ca.sup.2+; Calcium
Green-C18 Ca.sup.2+; Calcium Orange; Calcofluor White;
Carboxy-X-rhodamine (5-ROX); Cascade Blue.TM.; Cascade Yellow;
Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A;
Chromomycin A; CL-NERF; CMFDA; Coumarin Phalloidin; C-phycocyanine;
CPM Methylcoumarin; CTC; CTC Formazan; Cy2.TM.; Cy3.1 8; Cy3.5.TM.;
Cy3.TM.; Cy5.1 8; Cy5.5.TM.; Cy5.TM.; Cy7.TM.; cyclic AMP
Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl
Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;
Dapoxyl; Dapoxyl 2; Dapoxyl 3' DCFDA; DCFH
(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine
123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP);
Dichlorodihydrofluorescein Diacetate (DCFH); DiD--Lipophilic
Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil
(DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7));
DM-NERF (high pH); DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF
97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium
homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III)
chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF
(Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3;
Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald;
Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43.TM.;
FM 4-46; Fura Red.TM. (high pH); Fura Red.TM./Fluo-3; Fura-2;
Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow
10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2);
Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258;
Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin;
Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high
calcium; Indo-1, low calcium; Indodicarbocyanine (DiD);
Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1;
LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;
Leucophor SF; LeucophorWS; Lissamine Rhodamine; Lissamine Rhodamine
B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow;
Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green;
Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor
Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B);
Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green;
Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant
Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;
Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange;
Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane
(mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene);
NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline;
Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G;
Oregon Green; Oregon Green 488-X; Oregon Green.TM.; Oregon
Green.TM. 488; Oregon Green.TM. 500; Oregon Green.TM. 514; Pacific
Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;
PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red);
Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine
3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26
(Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3;
PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium lodid
(PL); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin
7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin;
RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine
5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B
extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine
Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT;
Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); S65A; S65C;
S65L; S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron
Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron
Yellow L; SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic
Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1;
Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum
Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene;
Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 11; SYTO 12;
SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO
21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42;
SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO
63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85;
SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline;
Tetramethylrhodamine (TRITC); Texas Red.TM.; Texas Red-X.TM.
conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole
Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte;
Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1;
TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC
TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite;
Uranine B; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange;
Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1;YOYO-3, Sybr Green,
Thiazole orange (interchelating dyes), or combinations thereof.
[0130] Some embodiments of the present invention include the Alexa
Fluor dye series (from Molecular Probes/Invitrogen) which cover a
broad spectrum and match the principal output wavelengths of common
excitation sources such as Alexa Fluor 350, Alexa Fluor 405, 430,
488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660,
680, 700, and 750. Some embodiments of the present invention
include the Cy Dye fluorophore series (GE Healthcare), also
covering a wide spectrum such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7.
Some embodiments of the present invention include the Oyster dye
fluorophores (Denovo Biolabels) such as Oyster-500, -550, -556,
645, 650, 656. Some embodiments of the present invention include
the DY-Labels series (Dyomics), for example, with maxima of
absorption that range from 418 nm (DY-415) to 844 nm (DY-831) such
as DY-415, -495, -505, -547, -548, -549, -550, -554, -555, -556,
-560, -590, -610, -615, -630, -631, -632, -633, -634, -635, -636,
-647, -648, -649, -650, -651, -652, -675, -676, -677, -680, -681,
-682, -700, -701, -730, -731, -732, -734, -750, -751, -752, -776,
-780, -781, -782, -831, -480XL, -481XL, -485XL, -510XL, -520XL,
-521XL. Some embodiments of the present invention include the ATTO
fluorescent labels (ATTO-TEC GmbH) such as ATTO 390, 425, 465, 488,
495, 520, 532, 550, 565, 590, 594, 610, 611X, 620, 633, 635, 637,
647, 647N, 655, 680, 700, 725, 740. Some embodiments of the present
invention include CAL Fluor and Quasar dyes (Biosearch
Technologies) such as CAL Fluor Gold 540, CAL Fluor Orange 560,
Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red
635, Quasar 670. Some embodiments of the present invention include
quantum dots such as the EviTags (Evident Technologies) or quantum
dots of the Qdot series (Invitrogen) such as the Qdot 525, Qdot565,
Qdot585, Qdot605, Qdot655, Qdot705, Qdot 800. Some embodiments of
the present invention include fluorescein, rhodamine, and/or
phycoerythrin.
FRET and Quenching
[0131] In some embodiments of the invention, fluorescence resonance
energy transfer is used to produce a signal that can be correlated
with the binding of the analyte to the probe. FRET arises from the
properties of certain fluorophores. In FRET, energy is passed
non-radiatively over a distance of about 1-10 nanometers between a
donor molecule, which is a fluorophore, and an acceptor molecule.
The donor absorbs a photon and transfers this energy
non-radiatively to the acceptor (Forster, 1949, Z. Naturforsch. A4:
321-327; Clegg, 1992, Methods Enzymol. 211: 353-388). When two
fluorophores whose excitation and emission spectra overlap are in
close proximity, excitation of one fluorophore will cause it to
emit light at wavelengths that are absorbed by and that stimulate
the second fluorophore, causing it in turn to fluoresce. The
excited-state energy of the first (donor) fluorophore is
transferred by a resonance induced dipole--dipole interaction to
the neighboring second (acceptor) fluorophore. As a result, the
excited state lifetime of the donor molecule is decreased and its
fluorescence is quenched, while the fluorescence intensity of the
acceptor molecule is enhanced and depolarized. When the
excited-state energy of the donor is transferred to a
non-fluorophore acceptor, the fluorescence of the donor is quenched
without subsequent emission of fluorescence by the acceptor. In
this case, the acceptor functions as a quencher.
[0132] Pairs of molecules that can engage in fluorescence resonance
energy transfer (FRET) are termed FRET pairs. In order for energy
transfer to occur, the donor and acceptor molecules must typically
be in close proximity (up to 7 to 10 nanometers. The efficiency of
energy transfer can falls off rapidly with the distance between the
donor and acceptor molecules.
[0133] Molecules that can be used in FRET include the fluorophores
described above, and includes fluorescein, 5-carboxyfluorescein
(FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
rhodamine, 6-carboxyrhodamine (R6G),
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA),
6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo) benzoic
acid (DABCYL), and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic
acid (EDANS). Whether a fluorophore is a donor or an acceptor is
defined by its excitation and emission spectra, and the fluorophore
with which it is paired. For example, FAM is most efficiently
excited by light with a wavelength of 488 nm, and emits light with
a spectrum of 500 to 650 nm, and an emission maximum of 525 nm. FAM
is a suitable donor fluorophore for use with JOE, TAMRA, and ROX
(all of which have their excitation maximum at 514 nm).
[0134] In some embodiments of the methods of the present invention,
the acceptor of the FRET pair is used to quench the fluorescence of
the donor. In some cases, the acceptor has little to no
fluorescence. The FRET acceptors that are useful for quenching are
referred to as quenchers. Quenchers useful in the methods of the
present invention include, without limitation, Black Hole Quencher
Dyes (Biosearch Technologies such as BHQ-0, BHQ-1, BHQ-2, BHQ-3,
BHQ-10; QSY Dye fluorescent quenchers (from Molecular
Probes/Invitrogen) such as QSY7, QSY9, QSY21, QSY35, and other
quenchers such as Dabcyl and Dabsyl; Cy5Q and Cy7Q and Dark Cyanine
dyes (GE Healthcare), which can be used, for example, in
conjunction with donor fluors such as Cy3B, Cy3, or Cy5;
DY-Quenchers (Dyomics), such as DYQ-660 and DYQ-661; and ATTO
fluorescent quenchers (ATTO-TEC GmbH), such as ATTO 540Q, 580Q,
612Q.
[0135] In some embodiments of the methods of the invention, both
the analytes and the probes have labels that are members of a FRET
pair, and the labels are attached such that when an analyte binds
to a probe, FRET will occur between the labels, resulting in a
change in signal that can be correlated with the binding of analyte
to probe in real-time. The change in signal can be the decrease in
the intensity of the donor and/or the increase in the intensity of
the acceptor. The FRET pair can be chosen such that emission
wavelength of the donor fluorophore is far enough from the emission
wavelength of the acceptor fluorophore, that the signals can be
independently measured. This allows the measurement of both the
decrease in signal from the donor and the increase in signal from
the acceptor at the same time, which can result in improvements in
the quality of the measurement of binding. In some cases, the probe
will have a label that is the donor of the donor-acceptor pair. In
some cases, the analyte will have a label that is the donor of the
donor acceptor pair.
[0136] In some embodiments of the methods of the invention, the
analyte will have a fluorescent label that is a member of a FRET
pair, and the other member of the FRET pair will be attached to the
surface, wherein the member of the FRET pair attached to the
surface is not covalently linked to the probe. In some cases, the
analyte will have a label that is the donor of the donor-acceptor
pair. In some cases, the analyte will have a label that is the
acceptor of the donor acceptor pair. In some embodiments, the
member of the FRET pair that is attached to the surface is attached
to an oligonucleotide which is attached to the surface (a
surface-bound label). The oligonucleotide that is labeled with the
FRET pair can be a nucleotide sequence that does not have a
sequence anticipated to specifically bind to an analyte. The use of
a surface-bound label allows for the labeling of multiple areas of
an array without having to label each specific binding probe. This
can simplify the production of the array and reduce costs. We have
found that even though the surface-bound FRET pairs are not
covalently bound to the probe, they can be sensitive to the binding
of the analyte labeled with the other member of the FRET pair in a
manner that allows the change in signal to be correlated with the
amount of analyte bound to probe.
[0137] In some embodiments of the methods of the present invention,
the analyte is labeled with a quencher, and the probe is labeled
with a donor fluorophore. The analyte is labeled with the quencher
such that when analyte binds with the probe, the fluorescence from
the fluorescent label on the probe is quenched. Thus, the signal,
measured in real-time, can be correlated with the amount of binding
of the analyte and the probe, allowing for the measurement of the
kinetics of the binding. In some embodiments of the methods of the
present invention, the analyte is labeled with a quencher, and the
probe is labeled with a donor fluorophore, that is not covalently
attached to it. The quencher is labeled such that when analyte
binds with the probe, the fluorescence from the fluorescent label
on the probe is quenched. Thus, the signal, measured in real-time,
can be correlated with the amount of binding of the analyte and the
probe, allowing for the measurement of the kinetics of the
binding.
[0138] In some embodiments of the methods of the present invention,
the analyte is labeled with a quencher, and the surface is labeled
with a donor fluorophore wherein the donor fluorophore is not
covalently linked to the probe (e.g. with a surface bound
fluorescent label). The quencher is labeled such that when analyte
binds with the probe, the fluorescence from the fluorescent label
on the surface is quenched. Thus, the signal, measured in
real-time, can be correlated with the amount of binding of the
analyte and the probe, allowing for the measurement of the kinetics
of the binding.
[0139] Where the probe is labeled with a fluorophore, one aspect of
the invention is the use of an image of the fluorescently labeled
probe on the surface obtained before binding has occurred in order
to effectively establish a baseline signal for the state where no
binding of analyte to probe has occurred. In conventional arrays,
in which unlabeled probe is treated with labeled analyte, and the
signal is measured after hybridization and washing, it can be
difficult to know exactly how much probe is actually on the array
in the region of interest. Thus, differences in array manufacture
can affect the quality of the data. In the present invention, where
the probe is labeled with fluorophore, the image of the labeled
probe on the surface provides a measurement of the amount of probe
actually on the surface, increasing the quality and reliability of
the binding measurement.
[0140] One exemplary embodiment of the method of the invention is
illustrated in FIGS. 5 and 6. FIG. 5B shows a top view of a 4 by 4
microarray that has 16 independently addressable spots, each spot
having bound DNA probes, wherein the probes are labeled with
fluorescent label. FIG. 5C shows a close up view of one of the
spots illustrating the attached probe of sequence (A), each probe
having a fluorescent label. FIG. 5D shows the close up view of a
second spot with attached probes of sequence (B), each probe having
a fluorescent label. FIG. 5A shows a side view of the array,
showing that the array is in contact with the hybridization
solution. FIG. 5 represents a time at which no analyte is bound to
probe on the array.
[0141] FIG. 6 illustrates the same array as in FIG. 5 after
hybridization for some time with target analytes (targets) having a
quencher attached. FIGS. 6A and 6B shows a side view and top view
of the array, still in contact with the hybridization solution. The
different spots on the array in FIG. 6B have different light
intensities, indicating that there is a different amount of binding
of analyte at each spot, and therefore a different amount of
fluorescence from the spots. FIG. 6C shows a close up view
illustrating that a small amount of target (A) has specifically
bound (hybridized) to probe (A) resulting in quenching of each
molecule of probe to which analyte is bound. FIG. 6D illustrates
that a larger amount of analyte (B) has specifically bound
(hybridized) to probe (B), resulting in a higher level of quenching
than observed for spot (A). The signal from each of the spots on
the array can be measured at various time points during the binding
reaction between analytes and probes, while the solution containing
the analyte is in contact with the solid surface of the microarray,
allowing a real-time measurement of the amount of analyte-probe
binding, and allowing the measurement of binding kinetics at each
spot.
Measuring Cross-Hybridization
[0142] One aspect of the methods of the present invention is the
step of performing an algorithm on real-time binding data to
determine cross-hybridization for multiple probes on a substrate.
One embodiment involves improving the quality of analyte-probe
binding measurements by determining and correcting for
cross-hybridization.
[0143] In its early stages, the probe-target binding kinetics can
be described by a simple first-order differential equation whose
time-domain solution is given by an exponential function; the rate
of decay of the exponential function is determined by the binding
reaction rate. Non-specific binding can have an adverse effect on
the accuracy of microarray platforms, especially if the amount of
the non-specific target is high relative to the specific target.
One reason is that the specific and non-specific targets will
compete for the same probe, and even though the probability of
non-specific binding is much lower it may have an effect if the
amount of the non-specific target is much higher. Fortunately,
specific and non-specific bindings have different reaction rates,
by virtue of the fact that the binding probabilities and target
amounts are different; therefore, if both specific and non-specific
(i.e., interfering) targets bind to a probe on a microarray or a
parallel affinity-based biosensor, the signal measured by a
RT-.mu.Array system can be represented by a sum of
exponentials,
.mu.(t)=.SIGMA..alpha..sub.jexp(.beta..sub.jt), (1)
where .SIGMA..alpha..sub.j gives the initial intensity of the probe
spots, the .beta..sub.j are the rates of decay for the specific
(j=1) and non-specific (j=2,3, . . . ) bindings, and where the
.alpha..sub.j themselves depend on the reaction rates and target
densities. In certain embodiments of the invention, we employ the
so-called Prony method or one of its modifications. This method
essentially represents the signal .mu.(t) in terms of the
coefficients of the original differential equation. These
coefficients are computed by finding eigenvalues of an appropriate
covariance matrix. In other embodiments of the invention, algebraic
characterization using singular value decomposition of the
correlation matrix of the data is employed. In yet another
embodiment of the invention, information-theoretic or Bayesian
techniques can be used to detect a presence of the species that
bind non-specifically, estimate the number of such species, and
quantify their amounts.
The Basic Algorithm
[0144] The hybridization process in general satisfies a nonlinear
differential equation. To see this, let p(t) denote the number of
available probe molecules at a given spot at time t, and let n(t)
denote the number of analytes in the solution that are specific to
this spot. Thus, if the probability that an analyte be in close
proximity to a probe molecule is P.sub.near, the probability that
it binds to the probe once it is near in a unit interval of time is
P.sub.h, and the probability that an analyte bound to a probe
molecule is released in a unit interval of time is P.sub.r, then we
may write
p(t+.delta.)-p(t)=.delta.(p.sub.0-p(t))P.sub.r-.delta.p(t)n(t)P.sub.near-
P.sub.h, (1)
where p.sub.0 denotes the initial number of probe molecules on this
probe spot of the array. If we denote the total number of analyte
molecules by N, then it is clear that n(t)=N-p.sub.0+p(t). Letting
.delta..fwdarw.0, therefore gives
q ( t ) t = ( p 0 - p ( t ) ) P r - p ( t ) ( N - p 0 + p ( t ) ) P
near P h . ( 2 ) ##EQU00006##
[0145] Upon rearranging terms, we have
p ( t ) t = p 0 P r - p ( t ) [ P r + ( N - p 0 ) P near P h ] - p
2 ( t ) P near P h , ( 3 ) ##EQU00007##
which is the nonlinear equation we were seeking. This is a Riccati
equation and can be solved in closed form (however, we shall not do
so here). In any event, it is clear that by looking at the
trajectory of p(t) one can glean information about the parameters
N, p.sub.0, P.sub.h, P.sub.r. For example, the steady-state of
p(t), i.e., p.sub..infin.=p(.infin.), satisfies the quadratic
equation
P.sub.near=P.sub.hp.sub..infin..sup.2+[P.sub.r+(N-p.sub.0)P.sub.nearP.su-
b.h]p.sub..infin.-p.sub.0P.sub.r=0 (4)
and so measuring it, gives us information about the parameters of
interest.
Determining the Analyte Concentration
[0146] The equations are often more instructive when written in
terms of the number of probe molecules that have bound to analytes,
i.e., q(t)=p.sub.0-p(t). In this case we may write
- q ( t ) t = q ( t ) P r - ( p 0 - q ( t ) ) ( N - q ( t ) ) P
near P h , ( 5 ) ##EQU00008##
which upon a rearrangement of terms becomes
q ( t ) t = Np 0 P near P h - [ P r + ( N + p 0 ) P near P h ] q (
t ) + P near P h q 2 ( t ) . ( 6 ) ##EQU00009##
[0147] In the early phase of the hybridization process, i.e., when
q(t) is very small, we may ignore the quadratic term in the
differential equation and write
q ( t ) t .apprxeq. Np 0 P near P h - [ P r + ( N + p 0 ) P near P
h ] q ( t ) . ( 7 ) ##EQU00010##
[0148] This has the solution
q ( t ) .apprxeq. Np 0 P near P h P r + ( N + p 0 ) P near P h ( 1
- exp ( - t [ P r + ( N + p 0 ) P near P h ] ) ) . ( 8 )
##EQU00011##
[0149] The above formula has several different ramifications; first
the slope of increase of q(t), equivalently, the slope of decay of
p(t), is given by
q ( t ) t | t = 0 = Np 0 P near P h , ( 9 ) ##EQU00012##
which is proportional to the number of analytes in the solution,
N.
[0150] Second, the reaction rate also has information on the number
of analytes. In fact, the reaction rate is simply the coefficient
of t in the exponential function for q(t), which is readily seen to
be
reaction_rate=P.sub.r+(N+p.sub.0)P.sub.nearP.sub.h. (10)
[0151] Though this is not quite a linear relationship for N, it can
still be used to estimate the number of analytes.
[0152] Of course, one can estimate N jointly from both the initial
slope and the reaction rate. In particular, it is often true that
P.sub.r is very small and can be ignored compared to other terms.
Therefore we shall take
reaction_rate=(N+p.sub.0)P.sub.nearP.sub.h (11)
Determining and Suppressing Cross-Hybridization
[0153] The other advantage of looking at the reaction rate is that
it allows for one to deal with cross-hybridization and to suppress
its effect. As mentioned earlier, in the early phase of the
hybridization process, the number of available probe molecules, or
equivalently the light intensity of a probe spot, decays
exponentially with time. For example
C.sub.kexp(-r.sub.kt), (12)
where C.sub.k is a constant and r.sub.k is the reaction rate that
can be determined from the parameters of the experiment
(probability of hybridization, number of analytes, number of probe
molecules, etc.). In particular, as seen from (11), r.sub.k is
linear in the number of target analytes, N.sub.k, say.
[0154] If, in addition to hybridization of the target of interest,
a number of other targets cross-hybridize to the same probe spot,
the light intensity of the probe spot will decay as the sum of
several exponentials, i.e.,
I ( t ) = k = 0 K - 1 C k exp ( - r k t ) , ( 13 ) ##EQU00013##
where k=0 corresponds to the desired target and k=1, . . . , K-1
corresponds to the K-1 cross-hybridizing analytes. The whole point
is that the reaction rates for the different analytes differ (due
to different numbers of analytes, binding probabilities, etc.) so
that if we can estimate the reaction rates from (13), we should be
able to determine the number of molecules for each different
analyte.
[0155] The RT-uArray system samples the signal (i.e., the light
intensity) of the probe spots at certain time intervals (multiples
of .DELTA., say) and thus obtains the sequence
y n = I ( n .DELTA. ) + v ( n .DELTA. ) = k = 0 K - 1 C k exp ( - n
.DELTA. r k ) + v ( n .DELTA. ) ( 14 ) ##EQU00014##
for n=0, 1, . . . T-1, where T is the total number of samples and
v(t) represents the measurement noise. Defining
u.sub.k=exp(-.DELTA.r.sub.k), we may write
y n = k = 0 K - 1 C k u k n + v n . ( 15 ) ##EQU00015##
[0156] The goal is to (i) determine the value of K (i.e., how many
analytes are binding to the probe spot), (ii) to estimate the
values of the pairs {C.sub.k, u.sub.k} for all k=1, . . . , K-1 and
(iii) to determine the number of each analyte N.sub.k (recall from
(9) that C.sub.k is proportional to the number of analytes and that
from (11) the reaction rate is linear in N.sub.k).
[0157] The problem of determining the number of exponential signals
in noisy measurements, and estimating the individual rates, is a
classical one in signal processing and is generally referred to as
system identification. (There are a multitude of books and papers
on this subject.) The basic idea is that, when y.sub.n is the sum
of K exponentials, it satisfies a K th order recurrence
equation
y.sub.n+h.sub.1y.sub.n-1+ . . . .
+h.sub.K-1y.sub.n-K+1+h.sub.Ky.sub.n-K=0. (16)
[0158] Furthermore, the u.sub.k are the roots of the polynomial
H(z)=z.sup.K+h.sub.1z.sup.Kn-1+ . . . +h.sub.K-1z+h.sub.k. (17)
[0159] In practice, since one observes a noisy signal, one first
uses the measurements to form the so-called Hankel matrix
( y T / 2 y T / 2 - 1 y 1 y 0 y T / 2 + 1 y T / 2 y 2 y 1 y T y T -
1 y T / 2 + 1 y T / 2 ) ( 18 ) ##EQU00016##
[0160] When y.sub.n is the sum of K exponentials, the above Hankel
matrix has rank K, i.e., only K nonzero eigenvalues. When y.sub.n
is noisy, the standard practice is to compute the singular values
of the Hankel matrix and estimate K as being the number of
significant singular values.
[0161] Once K has been determined, one forms the
(T-K+1).times.(K+1) Hankel matrix
( y K y K - 1 y 1 y 0 y K + 1 y K y 2 y 1 y T y T - 1 y T - K + 1 y
T - K ) ( 19 ) ##EQU00017##
and then identifies the vector (1 h.sub.1 . . . h.sub.k).sup.t with
the smallest right singular vector of (19).
[0162] As mentioned earlier, the roots of H(z) are the desired
u.sub.k, from which we determine the rates r.sub.k and thereby the
amounts of targets present. While the algorithm outlined above can
be used, a variety of different techniques to find the u.sub.k,
including, but not limited to, total least squares, ESPRIT, Prony's
method, may be used.
[0163] In addition to the algorithm described above, other
algorithmic solutions can be used, including the methods overviewed
in (Petersson et al., Applied Mathematics and Computation, vol.
126, no. 1, February 2002, pp. 31-61). The methods described above
provide the ability to quantify the amounts of the species that
bind whether specifically or non-specifically.
Arrays
[0164] One aspect of the invention is an array that has a solid
surface with a plurality of probes attached to it, where the array
can be used for the real-time measurement of binding of analyte to
the plurality of probes.
[0165] The arrays of the present invention comprise probes attached
to a solid substrate. The solid substrate may be biological,
nonbiological, organic, inorganic, or a combination of any of
these, existing as particles, strands, precipitates, gels, sheets,
tubing, spheres, containers, capillaries, pads, slices, films,
plates, slides, semiconductor integrated chips, etc. The solid
substrate is preferably flat but may take on alternative surface
configurations. For example, the solid substrate may contain raised
or depressed regions on which synthesis or deposition takes place.
In some embodiments, the solid substrate will be chosen to provide
appropriate light-absorbing characteristics. For example, the
substrate may be a polymerized Langmuir Blodgett film,
functionalized glass, Si, Ge, GaAs, GaP, SiO.sub.2, SiN.sub.4,
modified silicon, or any one of a variety of gels or polymers such
as (poly)tetrafluoroethylene, (poly)vinylidendifluoride,
polystyrene, polycarbonate, or combinations thereof.
[0166] The substrate can be a homogeneous solid and/or unmoving
mass much larger than the capturing probe where the capturing
probes are confined and/or immobilized within a certain distance of
it. The mass of the substrate is generally at least 100 times
larger than capturing probes mass. In certain embodiments, the
surface of the substrate is planar with roughness of 0.1 nm to 100
nm, but typically between 1 nm to 10 nm. In other embodiments the
substrate can be a porous surface with roughness of larger than 100
nm. In other embodiments, the surface of the substrate can be
non-planar. Examples of non-planar substrates are spherical
magnetic beads, spherical glass beads, and solid metal and/or
semiconductor and/or dielectric particles.
[0167] In some embodiments the substrate is optically clear,
allowing light to be transmitted through the substrate, and
allowing excitation and or detection to occur from light passing
through the substrate. In some embodiments the substrate is opaque.
In some embodiments, the substrate is reflective, allowing for
light to pass through the surface layer containing probes and
reflect back to a detector.
[0168] In some embodiments, glass slides are used to prepare
biochips. The substrates (such as films or membranes) can also be
made of silica, silicon, plastic, metal, metal-alloy, anopore,
polymeric, and nylon. The surfaces of substrates can be treated
with a layer of chemicals prior to attaching probes to enhance the
binding or to inhibit non-specific binding during use. For example,
glass slides can be coated with self-assembled monolayer (SAM)
coatings, such as coatings of as aminoalkyl silanes, or of
polymeric materials, such as acrylamide and proteins. A variety of
commercially available slides can be used. Some examples of such
slides include, but are not limited to, 3D-link.RTM. (Surmodics),
EZ-Rays.RTM. (Mosaic Technologies), Fastslides.RTM. (Schleicher and
Schuell), Superaldehyde.RTM., and Superamine.RTM. (CEL
Technologies).
[0169] Probes can be attached covalently to the solid surface of
the substrate (but non-covalent attachment methods can also be
used). In one embodiment, similar substrate, coating, and
attachment chemistries are used for all three--UniScreen.TM.,
ProScreen.TM., NuScreen.TM.--devices. In another embodiment,
different chemistries are applied.
[0170] A number of different chemical surface modifiers can be
added to substrates to attach the probes to the substrates.
Examples of chemical surface modifiers include N-hydroxy
succinimide (NHS) groups, amines, aldehydes, epoxides, carboxyl
groups, hydroxyl groups, hydrazides, hydrophobic groups, membranes,
maleimides, biotin, streptavidin, thiol groups, nickel chelates,
photoreactive groups, boron groups, thioesters, cysteines,
disulfide groups, alkyl and acyl halide groups, glutathiones,
maltoses, azides, phosphates, and phosphines. Glass slides with
such chemically modified surfaces are commercially available for a
number of modifications. These can easily be prepared for the rest,
using standard methods (Microarray Biochip Technologies, Mark
Schena, Editor, March 2000, Biotechniques Books).
[0171] In one embodiment, substrate surfaces reactive towards
amines are used. An advantage of this reaction is that it is fast,
with no toxic by-products. Examples of such surfaces include
NHS-esters, aldehyde, epoxide, acyl halide, and thio-ester. Most
proteins, peptides, glycopeptides, etc. have free amine groups,
which will react with such surfaces to link them covalently to
these surfaces. Nucleic acid probes with internal or terminal amine
groups can also be synthesized, and are commercially available
(e.g., from IDT or Operon). Thus, nucleic acids can be bound (e.g.,
covalently or non-covalently) to surfaces using similar
chemistries.
[0172] The substrate surfaces need not be reactive towards amines,
but many substrate surfaces can be easily converted into
amine-reactive substrates with coatings. Examples of coatings
include amine coatings (which can be reacted with bis-NHS
cross-linkers and other reagents), thiol coatings (which can be
reacted with maleimide-NHS cross-linkers, etc.), gold coatings
(which can be reacted with NHS-thiol cross linkers, etc.),
streptavidin coatings (which can be reacted with bis-NHS
cross-linkers, maleimide-NHS cross-linkers, biotin-NHS
cross-linkers, etc.), and BSA coatings (which can be reacted with
bis-NHS cross-linkers, maleimide-NHS cross-linkers, etc.).
Alternatively, the probes, rather than the substrate, can be
reacted with specific chemical modifiers to make them reactive to
the respective surfaces.
[0173] A number of other multi-functional cross-linking agents can
be used to convert the chemical reactivity of one kind of surface
to another. These groups can be bifunctional, tri-functional,
tetra-functional, and so on. They can also be homo-functional or
hetero-functional. An example of a bi-functional cross-linker is
X-Y-Z, where X and Z are two reactive groups, and Y is a connecting
linker. Further, if X and Z are the same group, such as NHS-esters,
the resulting cross-linker, NHS-Y-NHS, is a homo-bi-functional
cross-linker and would connect an amine surface with an amine-group
containing molecule. If X is NHS-ester and Z is a maleimide group,
the resulting cross-linker, NHS-Y-maleimide, is a
hetero-bi-functional cross-linker and would link an amine surface
(or a thiol surface) with a thio-group (or amino-group) containing
probe. Cross-linkers with a number of different functional groups
are widely available. Examples of such functional groups include
NHS-esters, thio-esters, alkyl halides, acyl halides (e.g.,
iodoacetamide), thiols, amines, cysteines, histidines, di-sulfides,
maleimide, cis-diols, boronic acid, hydroxamic acid, azides,
hydrazines, phosphines, photoreactive groups (e.g., anthraquinone,
benzophenone), acrylamide (e.g., acrydite), affinity groups (e.g.,
biotin, streptavidin, maltose, maltose binding protein,
glutathione, glutathione-S-transferase), aldehydes, ketones,
carboxylic acids, phosphates, hydrophobic groups (e.g., phenyl,
cholesterol), etc. Such cross-linkers can be reacted with the
surface or with the probes or with both, in order to conjugate a
probe to a surface.
[0174] Other alternatives include thiol reactive surfaces such as
acrydite, maleimide, acyl halide and thio-ester surfaces. Such
surfaces can covalently link proteins, peptides, glycopeptides,
etc., via a (usually present) thiol group. Nucleic acid probes
containing pendant thiol-groups can also be easily synthesized.
[0175] Alternatively, one can modify glass surfaces with molecules
such as polyethylene glycol (PEG), e.g. PEGs of mixed lengths
[0176] Other surface modification alternatives (such as
photo-crosslinkable surfaces and thermally cross-linkable surfaces)
are known to those skilled in the art. Some technologies are
commercially available, such as those from Mosiac Technologies
(Waltham, Mass.), Exiqon.TM. (Vedbaek, Denmark), Schleicher and
Schuell (Keene, N.H.), Surmodics.TM. (St. Paul, Minn.),
Xenopore.TM. (Hawthorne, N.J.), Pamgene (Netherlands), Eppendorf
(Germany), Prolinx (Bothell, Wash.), Spectral Genomics (Houston,
Tex.), and Combimatrix.TM. (Bothell, Wash.).
[0177] Surfaces other than glass are also suitable for such
devices. For example, metallic surfaces, such as gold, silicon,
copper, titanium, and aluminum, metal oxides, such as silicon
oxide, titanium oxide, and iron oxide, and plastics, such as
polystyrene, and polyethylene, zeolites, and other materials can
also be used. The devices can also be prepared on LED (Light
Emitting Diode) and OLED (Organic Light Emitting Diode) surfaces.
An array of LEDs or OLEDs can be used at the base of a probe array.
An advantage of such systems is that they provide easy
optoelectronic means of result readout. In some cases, the results
can be read-out using a naked eye.
[0178] Probes can be deposited onto the substrates, e.g., onto a
modified surface, using either contact-mode printing methods using
solid pins, quill-pins, ink-jet systems, ring-and-pin systems, etc.
(see, e.g., U.S. Pat. Nos. 6,083,763 and 6,110,426) or non-contact
printing methods (using piezoelectric, bubble-jet, syringe,
electro-kinetic, mechanical, or acoustic methods. Devices to
deposit and distribute probes onto substrate surfaces are produced
by, e.g., Packard Instruments. There are many other methods known
in the art. Preferred devices for depositing, e.g., spotting,
probes onto substrates include solid pins or quill pins
(Telechem/Biorobotics).
[0179] The arrays of the present invention can also be
three-dimensional arrays such as porous arrays. Such as devices
consisting of one or more porous gel-bound probes in an array or an
array of arrays format. A device can have one or more such
structures and the structures can be of any geometric shape and
form. The structures can also be vertically straight, angled, or
twisted. Thus, each device denotes a (multiplexed) reaction site.
The device can be used to perform reactions simultaneously or
sequentially. Any of the known substrates and chemistries can be
used to create such a device. For example, glass, silica, silicon
wafers, plastic, metals; and metal alloys can all be used as the
solid support (see. e.g., Stillman B A, Tonkinson J L, Scleicher
and Schuell; Biotechniques, 29(3), 630-635, 2000; Rehmna et. al;
Mosaic Technologies Inc., Nucleic Acids Research, 27(2), 649-655,
1999). In other embodiments, the intermediate species can be
immobilized to the substrate using mechanical and/or electrostatic
and/or and magnetic forces. Examples are magnetic beads with
magnetic fields and glass beads with electrostatic fields. Bead
based methods are described, for example in Gunderson et al.,
Genome Research, 870-877, 2004; Michael et al., Anal. Chem. 70,
1242-1248, 1998; Han et al., Nat. Biotechnol, 19, 631-635, 2001;
and Lockhart et al., Nat. Biotechnol. 19, 1122-1123, 2001.
[0180] In other embodiments, the microarrays are manufactured
through the in-situ synthesis of the probes. This in-situ synthesis
can be achieved using phosphoramidite chemistry and/or
combinatorial chemistry. In some cases, the deprotection steps are
performed by photodeprotection (such as the Maskless Array
Synthesizer (MAS) technology, (NimbleGen; or the photolithographic
process, by Affymetrix). In other cases, deprotection can be
achieved electrochemically (such as in the Combimatrix procedure).
Microarrays for the present invention can also be manufactured by
using the inkjet technology (Agilent).
[0181] For the arrays of the present invention, the plurality of
probes may be located in one addressable region and/or in multiple
addressable regions on the solid substrate. In some embodiments the
solid substrate has about 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100,
100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-50,000,
50,000-100,000, 100,000-500,000, 500,000-1,000,000 or over
1,000,000 addressable regions with probes.
[0182] The spots may range in size from about 1 nm to 10 mm, in
some embodiments from about 1 to 1000 micron and more in some
embodiments from about 5 to 100 micron. The density of the spots
may also vary, where the density is generally at in some
embodiments about 1 spot/cm.sup.2, in some embodiments at least
about 100 spots/cm.sup.2 and in other embodiments at least about
400 spots/cm.sup.2, where the density may be as high as 10.sup.6
spots/cm.sup.2 or higher.
[0183] The shape of the spots can be square, round, oval or any
other arbitrary shape.
[0184] One aspect of the invention is an array that comprises a
solid substrate having a surface and a plurality of different
probes, wherein (a) the different probes are immobilized to the
surface at different addressable locations, (b) the addressable
locations comprise optical signal moieties bound to the surface,
(c) the optical signal moieties are not bound directly to the
probes, and (d) the optical signal from the optical signal moieties
is capable of changing upon binding of an analyte to the probes.
For these arrays, the optical signal moiety, for example, a
fluorescent moeity is bound directly to the surface, but is not
covalently bound to a probe, and in these cases the probe need not
be labeled. The fluorescent moiety can be bound to the surface or
synthesized in-situ by an of the methods described above for
probes. The fluorescent moiety can be attached to an
oligonucleotide that is not a probe, for example, having a sequence
that is not complementary to target analytes in solution.
[0185] In one embodiment, a fluorescent moiety on the surface
(surface-bound label) can be brought to the proximity of the probe
via post-probe-synthesis or post-probe-deposition methods.
[0186] In some embodiments, the label can be bound to the probe by
non-covalent means, such as by hybridization. For example, in
certain embodiments of the present invention, some or all of the
probes on the microarray may contain two different sequence
segments: one segment that consists of a sequence that is specific
to the probe and specific for the detection of a given target
analyte, and another segment that is a sequence that is common to
all or many of the probes on the microarray. These two sequence
segments can be immediately adjacent to each other on the probe, or
separated by a linker. In this embodiment, the microarray is first
hybridized with a (labeled oligonucleotide that is complementary to
the common sequence segment, thus resulting in a microarray in
which the spots or features where the probes are located also now
contain fluorescent labels. These non-covalently bound labels can
be bound to the probe such that FRET and or quenching of the label
occurs upon binding of an analyte to the specific portion of the
probe. This method can be advantageous, for instance by (1)
lowering the cost of manufacturing microarrays that can be used in
the real-time platform and/or (2) enabling the use of in-situ
synthesized arrays in the real time platform. The labeled
oligonucleotide can be a locked nucleic acid (LNA) oligonucleotide.
LNA oligonucleotides can be useful because the LNA modification can
result in enhanced hybridization properties (for example,
diminishing the sequence length that is needed to achieve a certain
Tm) (Jepsen et al., Oligonucleotides. 2004; 14(2):130-46).
Systems
[0187] One aspect of the invention is a system comprising: (a) a
device with (i) a solid support having a surface and (ii) a
plurality of different probes, wherein the different probes are
immobilized to the surface; (b) a fluid volume comprising an
analyte wherein the fluid volume is in contact with the solid
support, and (c) a detector assembly comprising means to detect
signals measured at multiple time points from each of a plurality
of spots on the microarray while the fluid volume is in contact
with the solid support.
[0188] The fluid volume can be introduced and held in the system by
any method that will maintain the fluid in contact with the solid
support. In many cases the fluid is held in a chamber. In some
embodiments the chamber is open on one face, in other embodiments
the chamber will mostly enclose the fluid. In some embodiments, the
chamber will have one or more ports for introducing and/or removing
material (usually fluids) from the chamber. In some embodiments one
side of the chamber comprises the solid substrate on which the
probes are attached. In some embodiments the chamber is integral to
the solid substrate. In some embodiments, the chamber is a
sub-assembly to which the solid substrate with probes can be
removably attached. In some embodiments, some or all of the fluid
chamber is an integral part of the instrument that comprises the
detector. The chamber can be designed such that the signal that can
be correlated with analyte-probe binding can be detected by a
detector outside of the chamber. For instance, all or a portion of
the chamber can be transparent to light to allow light in or out of
the chamber to facilitate excitation and detection of
fluorophores.
[0189] The detector assembly can comprise a single detector or an
array of detectors or transducers. As used herein, the terms
detector and transducer are used interchangeably, and refer to a
component that is capable of detecting a signal that can be
correlated with the amount of analyte-probe binding. Where the
detector system is an array of transducers, in some embodiments,
the detector system is a fixed array of transducers, wherein one or
more transducers in the transducer array corresponds to one
independently addressable area of the array. In some embodiments,
the detector or the array of transducers scans the array such that
a given detector or transducer element detects signals from
different addressable areas of the array during a binding
reaction.
[0190] In some embodiments the detector array is in contact with
the solid substrate. In some embodiments, the detector is at a
distance away from the substrate. Where the detector is a distance
away from the substrate, in some embodiments, the detector or
detector array is capable of scanning the substrate in order to
measure signal from multiple addressable areas. In some
embodiments, the detector is an optical detector which is optically
coupled to the substrate. The detector can be optically coupled to
the substrate, for example with one or more lenses or
waveguides.
[0191] FIG. 9 shows an example of a real-time microarray system
where the detection system comprises a sensor array in intimate
proximity of the capturing spots. In this embodiment, individual
sensors detect the binding events of a single capturing spots.
[0192] In some embodiments, the detector is optically coupled
through spatially confined excitation. This method is useful to
optically couple the substrate to detector for a small region of
substrate with probes. This method generally requires only a single
detector, since only one region can create signal at a time. The
method can be used in scanning systems, and is applicable in assays
which an excitation is required for detection, such as fluorescence
spectroscopy or surface plasmon resonance (SPR) methods.
[0193] In some embodiments, the detector is optically coupled
through imaging using focal plane detector arrays: In this method
the signal generated from the system is focused on a focal point
detector array. This approach useful for optical detection systems
where signal focusing can be carried out using lenses and other
optical apparatus. Examples of detectors in these embodiments are
complementary metal oxide semiconductor (CMOS) and charge coupled
device (CCD) image sensors.
[0194] In some embodiments, the detector is optically coupled
through surface imaging: In this method the detectors are placed in
intimate proximity of the capturing probes such that the signal
generated from the capturing region can only be observed by the
dedicated detector. If a microarray with multiple capturing spots
is used, multiple detectors are used, each dedicated to an
individual spot. This method can be used in electrochemical-,
optical-, and magnetic-based biosensors.
[0195] In some embodiments, the detector is optically coupled
through surface imaging using signal couplers: In this method the
detectors are not place in proximity of the capturing spots,
however a signal coupler is used to direct signal from the
capturing region to a detector. This method is generally used in
optical detection systems where the signal coupling elements is a
plurality of optical waveguide. Examples of signal coupling
elements include fiber optic cables, fiber optic bundles, fiber
optic faceplates, and light pipes.
[0196] The detectors of the present invention must be capable of
capturing signal at multiple time points in real time, during the
binding reaction. In some embodiments the detector is capable of
measuring at least two signals in less than about 1 psec, 5 psec,
0.01 nsec, 0.05 nsec, 0.1 nsec, 0.5 nsec, 1 nsec, 5 nsec, 0.01
.mu.sec, 0.05 .mu.sec, 0.1 .mu.sec, 0.5 .mu.sec, 1 .mu.sec, 5
.mu.sec, 0.01 msec, 0.05 msec, 0.1 msec, 0.5 msec, 1 msec, 5 msec,
10 msec, 50 msec, 100 msec, 0.5 sec, 1 sec, 5 sec, 10 sec, or 60
sec.
[0197] In some embodiments the detector detects the signal at the
substrate. In some embodiments the detector will detect the signal
in the solution. In some embodiments, the detector will detect
signal in both the solution and at the substrate.
[0198] In some embodiments the detector system is capable of
detecting electrical, electrochemical, magnetic, mechanical,
acoustic, or electromagnetic (light) signals.
[0199] Where the detector is capable of detecting optical signals,
the detector can be, for example a photomultiplier tube (PMT), a
CMOS sensor, or a (CCD) sensor. In some embodiments, the detector
comprises a fiber-optic sensor.
[0200] In some embodiments, the system comprising the detector is
capable of sensitive fluorescent measurements including synchronous
fluorimetry, polarized fluorescent measurements, laser induced
fluorescence, fluorescence decay, and time resolved
fluorescence.
[0201] In some embodiments, the system comprises a light source,
for example, for excitation of fluorescence. The light source is
generally optically coupled to the substrate, for example with one
or more lenses or waveguides. The light source can provide a single
wavelength, e.g. a laser, or a band of wavelengths.
[0202] FIG. 7 shows a block diagram of the components of systems of
the present invention. The system comprises of (i) reaction chamber
which includes the microarray substrate, probes, analytes, and
solution, (ii) heating and cooling modules and temperature sensor,
(iii) temperature controller, and (iv) detector which is connected
to an analysis block, where the latter is a part of a computing
system.
[0203] FIG. 8 shows an example of a real-time microarray system
where real-time binding of BHQ2 quencher-labeled cDNA molecules
were detected using a fluorescent laser-scanning microscope. The
substrate in this example was a transparent glass slides and the
probes were 25 bp Cy5-labeled oligonucleotides. The light source
(laser) and detector were both located on the back of the
substrate.
[0204] In some embodiments, the system comprises an instrument that
has a detector assembly, and a computing system, where the
instrument can accept a sub-assembly. The sub assembly comprises a
chamber that will hold the fluid volume and the solid substrate
having a surface and a plurality of probes. The sub-assembly can be
loaded into the instrument in order to monitor the reaction during
the binding event, and after saturation.
[0205] In some embodiments, the system comprises: an assay assembly
comprising means to engage a microarray and means to perform an
assay on a surface of the microarray; and a detector assembly
comprising means to detect signals measured at multiple time points
from each of a plurality of spots on the microarray during the
performance of the assay.
[0206] In some embodiments, the means to perform the assay comprise
a compartment wherein the surface of the microarray comprises a
floor of the compartment and means to deliver reagents and analytes
into the compartment. Any method can be used to seal the microarray
to the compartment including using adhesives and gaskets to seal
the fluid. Any method can be used to deliver reagents and analytes
including using syringes, pipettes, tubing, and capillaries.
[0207] In some embodiments, the system comprises a means of
controlling the temperature. Control of temperature can be
important to allow control of binding reaction rates, e.g. by
controlling stringency. The temperature can be controlled by
controlling the temperature at any place within the system
including controlling the temperature of the fluid or the
temperature of the solid substrate. Any means can be used for
controlling the temperature including resistive heaters, Peltier
devices, infrared heaters, fluid or gas flow. The temperatures can
be the same or different for solution or substrate or different
parts of each. Ideally the temperature is consistently controlled
within the binding region. In some embodiments the temperature is
controlled to within about 0.01, 0.05, 0.1, 0.5, or 1.degree.
C.
[0208] In some embodiments the system is capable of changing the
temperature during the binding reaction. In some embodiments, the
temperature can be rapidly changed during the binding reaction. In
some embodiments, the system is capable of changing the temperature
at a rate of temperature change corresponding to a change of
1.degree. C. in less than about 0.01 msec, 0.1 msec, 0.5 msec, 1
msec, 5 msec, 10 msec, 50 msec, 100 msec, 0.5 sec, 1 sec, 10 sec,
or 60 sec.
[0209] In other embodiments the temperature is changed slowly,
gradually ramping the temperature over the course of the binding
reaction.
[0210] One exemplary embodiment of changing the temperature during
the binding reaction involves a change in temperature to change the
binding stringency and probability. Most bindings in affinity-based
biosensors are a strong function of temperature, thus by changing
temperature we can alter the stringency and observe the capturing
the new capturing process with a new set of capturing
probabilities.
[0211] In some embodiments, the system is capable of measuring
temperature in one or multiple locations in the solution or on the
solid substrate. The temperature can be measured by any means
including by thermometer, thermocouple, or thermochromic shift.
[0212] In some embodiments the system comprises a feedback loop for
temp control wherein the measured temperature is used as an input
to the system in order to more accurately control temperature.
[0213] In some embodiments, the system comprises an apparatus to
add or remove material from the fluid volume. In some embodiments,
the system can add or remove a liquid from the fluid volume. In
some embodiments, the system is capable of adding or removing
material from the fluid volume in order to change the:
concentration, pH, stringency, ionic strength, or to add or remove
a competitive binding agent. In some embodiments, the system is
capable of changing the volume of the fluid volume during the
reaction.
[0214] One exemplary embodiment of adding material to the fluid
volume during the binding reaction comprises the addition of
incubation buffer. The incubation buffer is the buffer in which the
analytes are residing. By adding the incubation buffer, the
concentration of analytes in the system will decrease and therefore
the binding probability and kinetic of binding will both decrease.
Furthermore, if the reaction has already reached equilibrium, the
addition of the buffer will cause the system to move another
equilibrium state in time.
[0215] Another exemplary embodiment of adding material to the fluid
volume during the binding reaction is adding a competing binding
species. The competing species can be of the same nature of the
analyte but in general they are molecules which have affinity to
capturing probes. For DNA microarrays for example, the competing
species can be synthesized DNA oligo-nucleotides with partially or
completely complementary sequence to the capturing probes. In
immunoassays, the competing species are antigens.
[0216] In some embodiments the system comprises elements to apply
an electric potential to the fluid volume to electrically change
the stringency of the medium. In some embodiments, the system will
provide an electrical stimulus to the capturing region using an
electrode structure which is placed in proximity of the capturing
region. If the analyte is an electro-active species and/or ion, the
electrical stimulus can apply an electrostatic force of the
analyte. In certain embodiments, this electrostatic force is
adjusted to apply force on the bonds between analyte and capturing
probe. If the force is applied to detach the molecule, the affinity
of the analyte-probe interaction is reduced and thus the stringency
of the bond is evaluated. The electrical stimulus is generally a DC
and/or time-varying electrical potentials. Their amplitude can be
between 1 mV to 10V, but typically between 10 mV to 100 mV. The
frequency of time-varying signal can be between 1 Hz to 1000 MHz,
in some embodiments, the frequency of the time-varying signal is
between 100 Hz to 100 kHz. The use of electric potential to control
stringency is described in U.S. Pat. No. 6,048,690.
[0217] In some embodiments the system comprises a computing system
for analyzing the detected signals. In some embodiments, the system
is capable of transferring time point data sets to the computing
system wherein each time point data set corresponds to detected
signal at a time point, and the computing system is capable of
analyzing the time point data sets, in order to determine a
property related to the analyte and probe. The methods of the
current invention can, in some cases, generate more data, sometimes
significantly more data than for conventional microarrays. Thus a
computer system and software that can store and manipulate the data
(for instance, images taken at time points) can be essential
components of the system. The data can be analyzed in real-time, as
the reaction unfolds, or may be stored for later access.
[0218] The information corresponding to detected signal at each
time point can be single values such as signal amplitude, or can be
more complex information, for instance, where each set of signal
information corresponds to an image of a region containing signal
intensity values at multiple places within an addressable
location.
[0219] The property related to analyte and/or probe can be, for
example, analyte concentration, binding strength, or competitive
binding, and cross-hybridization.
[0220] In some embodiments the computing system uses the algorithms
described above for determining concentration and/or
cross-hybridization.
[0221] One aspect of the invention is software for use in
characterizing binding between analyte and probe. In one
embodiment, the software carries out the steps of i) accessing
stored images taken at different time points, ii) performing image
processing to determine the location of the spots and convert the
data to a collection of time series (one for each spot)
representing the temporal behavior of the signal intensity for each
spot, and iii) for each spot on the array determining whether a
reaction has happened (this is often done by comparing with control
spots on the array). Optionally, the software can perform the steps
of iv) determining whether the reaction at each spot involves the
binding of a single analyte or multiple analytes (if, for example,
cross-hybridization is occurring), v) estimating the reaction rates
using statistical system identification methods. Examples of
statistical system identification methods include methods such as
Prony's method. In the case that step iv) is used, (multiple
bindings per spot), the reaction rate of each binding is
determined, and vi) using the reaction rates to estimate the
unknown quantity of interest (analyte concentration, binding
strength, etc.) using, for example optimal Bayesian methods.
[0222] In some embodiments, the system will have software for
interfacing with the instrument, for example allowing the user to
display information in real-time and allowing for user to interact
with the reaction (i.e., add reagents, change the temperature,
change the pH, dilution, etc.).
Uses
[0223] Where the probe and analyte are nucleic acids, the present
invention provides methods of expression monitoring and generic
difference screening. The term expression monitoring is used to
refer to the determination of levels of expression of particular,
typically preselected, genes. The invention allows for many genes,
e.g. 10, 100, 1,000, 10,000, 100,000 or more genes to be analyzed
at once. Nucleic acid samples are hybridized to the arrays and the
resulting hybridization signal as a function of time provides an
indication of the level of expression of each gene of interest. In
some embodiments, the array has a high degree of probe redundancy
(multiple probes per gene) the expression monitoring methods
provide accurate measurement and do not require comparison to a
reference nucleic acid.
[0224] In another embodiment, this invention provides generic
difference screening methods, that identify differences in the
abundance (concentration) of particular nucleic acids in two or
more nucleic acid samples. The generic difference screening methods
involve hybridizing two or more nucleic acid samples to the same
oligonucleotide array, or to different oligonucleotide arrays
having the same oligonucleotide probe composition, and optionally
the same oligonucleotide spatial distribution. The resulting
hybridizations are then compared allowing determination which
nucleic acids differ in abundance (concentration) between the two
or more samples.
[0225] Where the concentrations of the nucleic acids comprising the
samples reflects transcription levels genes in a sample from which
the nucleic acids are derived, the generic difference screening
methods permit identification of differences in transcription (and
by implication in expression) of the nucleic acids comprising the
two or more samples. The differentially (e.g., over- or under)
expressed nucleic acids thus identified can be used (e.g., as
probes) to determine and/or isolate those genes whose expression
levels differs between the two or more samples.
[0226] The expression monitoring and difference screening methods
of this invention may be used in a wide variety of circumstances
including detection of disease, identification of differential gene
expression between two samples (e.g., a pathological as compared to
a healthy sample), screening for compositions that upregulate or
downregulate the expression of particular genes, and so forth.
EXAMPLES
Example 1
[0227] FIG. 10 shows the layout of a 6.times.6 DNA microarray.
Three different DNA probes (1, 2, and Control) with three different
concentrations (2 .mu.M, 10 .mu.M, and 20 .mu.M) are spotted and
immobilized on the surface as illustrated. The probes contain a
single Cy3 fluorescent molecule at the 5' end. The DNA targets in
this experiment contain a quencher molecule. The analyte binding in
this system results in quenching of fluorescent molecules in
certain spots. FIG. 11 shows a few samples of the real-time
measurements of the microarray experiment wherein the control
targets are added to the system. As illustrated in FIG. 11, the
spots are quenched due to analyte binding.
[0228] FIGS. 12-15 each show data for 4 different spots with
similar oligonucletide capturing probes. The target DNA analyte is
introduced in the system at time zero and quenching (reduction of
signal) occurs only when binding happens. For FIG. 12, the light
intensity coefficient of variation was about 15%, however the
estimated time constant rate from real-time measurements had only
4.4% variations. For FIG. 13 the light intensity coefficient of
variation was about 15%, however the estimated time constant rate
from real-time measurements had only 2.1% variations. For FIG. 14
the light intensity coefficient of variation was about 22%, however
the estimated time constant rate from real-time measurements had
only 6% variations. For FIG. 15 the light intensity coefficient of
variation was about 22%, however the estimated time constant rate
from real-time measurements had only a 4.8% variation.
[0229] In FIG. 16, the signals measured during two real-time
experiments wherein target 2 is applied to the microarray, first at
2 ng and then at 0.2 ng, are shown. The measured light intensities
at the corresponding probe spots decay over time as the targets to
the probes bind and the quenchers come in close proximity to the
fluorescent labels attached to the end of the probes. The rate of
the decay, which can be estimated by a curve fitting technique, is
proportional to the amount of the target present. The time constant
of the measured process is defined as the inverse of the rate of
decay. The ratio of the time constants of the two processes is 10,
which is precisely the ratio of the amounts of targets applied in
the two experiments.
Example 2
[0230] This example provides a derivation of an algorithm, and the
use of the algorithm to determine analyte concentration from a
real-time binding data. The derivation proceeds as follows:
[0231] Assume that the hybridization process starts at t=0, and
consider discrete time intervals of the length .DELTA.t. Consider
the change in the number of bound target molecules during the time
interval (i.DELTA.t, (i+1).DELTA.t). We can write
n.sub.b(i+1)-n.sub.b(i)[n.sub.t-n.sub.b(i)]p.sub.b(i).DELTA.t-n.sub.b(i)-
p.sub.r(i).DELTA.t,
where n.sub.t denotes the total number of target molecules,
n.sub.b(i) and n.sub.b(i+1) are the numbers of bound target
molecules at t=i.DELTA.t and t=(i+1).DELTA.t, respectively, and
where p.sub.b(i) and p.sub.r(i) denote the probabilities of a
target molecule binding to and releasing from a capturing probe
during the i.sup.th time interval, respectively. Hence,
n b ( i + 1 ) - n b ( i ) .DELTA. t = [ n t - n b ( i ) ] p b ( i )
- n b ( i ) p r ( i ) . ( 1 ) ##EQU00018##
[0232] It is reasonable to assume that the probability of the
target release does not change between time intervals, i.e.,
p.sub.r(i)=p.sub.r, for all i. On the other hand, the probability
of forming a target-probe pair depends on the availability of the
probes on the surface of the array. If we denote the number of
probes in a spot by n.sub.p, then we can model this probability
as
p b ( i ) = ( 1 - n b ( i ) n p ) - p b = n p - n b ( i ) n p p b ,
( 2 ) ##EQU00019##
where p.sub.b denotes the probability of forming a target-probe
pair assuming an unlimited abundance of probes.
[0233] By combining (1) and (2) and letting .DELTA.t.fwdarw.0, we
arrive to
n b t = ( n t - n b ) n p - n b n p p p - n b p r = n t p b - [ ( 1
+ n t n p ) p b + p r ] n b + p b n p n b 2 ( 3 ) ##EQU00020##
[0234] Note that in (3), only n.sub.b=n.sub.b(t), while all other
quantities are constant parameters, albeit unknown. Before
proceeding any further, we will find it useful to denote
.alpha. = ( 1 + n t n p ) p b + p r , .beta. = n t p b , .gamma. =
p b n p . ( 4 ) ##EQU00021##
[0235] Clearly, from (4),
p b = .beta. n t , n p = p b .gamma. , p r = .alpha. - ( 1 + n t n
p ) p b . ##EQU00022##
[0236] Using (4), we can write (3) as
n b t = .beta. - .alpha. n b + .gamma. n b 2 = .gamma. ( n b -
.lamda. 1 ) ( n b - .lamda. 2 ) , ( 5 ) ##EQU00023##
where .lamda..sub.1 and .lamda..sub.2 are introduced for
convenience and denote the roots of
.beta.-.alpha.n.sub.b+.gamma.n.sub.b.sup.2=0.
[0237] Note that .gamma.=.beta./(.lamda..sub.1.lamda..sub.2). The
solution to (5) is found as
n b ( t ) = .lamda. 1 + .lamda. 1 ( .lamda. 1 - .lamda. 2 ) .lamda.
2 e .beta. ( 1 .lamda. 1 1 .lamda. 2 ) t - .lamda. 1 .
##EQU00024##
[0238] We should point out that (3) describes the change in the
amount of target molecules, n.sub.b, captured by the probes in a
single probe spot of the microarray. Similar equations, possibly
with different values of the parameters n.sub.p, n.sub.t, p.sub.b,
and p.sub.r, hold for other spots and other targets.
Estimating Parameters of the Model
[0239] The following is an outline of a procedure for estimation of
the parameters. Ultimately, by observing the hybridization process,
we would like to obtain n.sub.t, n.sub.p, p.sub.b, and p.sub.r.
However, we do not always have direct access to n.sub.b(t) in (6),
but rather to y.sub.b(t)=kn.sub.b(t), where k denotes a
transduction coefficient. In particular, we observe
y b ( t ) = .lamda. 1 * + .lamda. 1 * ( .lamda. 1 * - .lamda. 2 * )
.lamda. 2 * e .beta. ( 1 .lamda. 1 * 1 .lamda. 2 * ) t - .lamda. 1
, ( 7 ) ##EQU00025##
where .lamda..sub.1*=k.lamda..sub.1, .lamda..sub.2*=k.lamda..sub.2,
and .beta.*=k.beta..
[0240] For convenience, we also introduce
.gamma. * = .beta. * .lamda. 1 * .lamda. 2 * = .gamma. k , .alpha.
* = .gamma. * ( .lamda. 1 * + .lamda. 2 * ) = .alpha. . ( 8 )
##EQU00026##
[0241] From (5), it follows that
.beta. * = y b t | t = 0 . ( 9 ) ##EQU00027##
[0242] Assume, without a loss of generality, that .lamda..sub.1* is
the smaller and .lamda..sub.2* the larger of the two, i.e.,
.lamda..sub.1*=min(.lamda..sub.1, .lamda..sub.2), and
.lamda..sub.2*=max(.lamda..sub.1,.lamda..sub.2). From (7), we find
the steady-state of y.sub.b(t),
.lamda..sub.1*=lim y.sub.b(t),t.fwdarw..infin.. (10)
[0243] So, from (9) and (10) we can determine .beta.* and
.lamda..sub.1*, two out of the three parameters in (7). To find the
remaining one, .lamda..sub.2*, one needs to fit the curve (7) to
the experimental data.
[0244] Having determined .beta.*, .lamda..sub.1*, and
.lamda..sub.2*, we use (8) to obtain .alpha.* and .gamma.*. Then,
we should use (4) to obtain pb, pr, n.sub.p, and n.sub.t from
.alpha.*, .beta.*, and .gamma.*. However, (4) gives us only 3
equations while there are 4 unknowns that need to be determined.
Therefore, we need at least 2 different experiments to find all of
the desired parameters. Assume that the arrays and the conditions
in the two experiments are the same except for the target amounts
applied. Denote the target amounts by n.sub.t, and n.sub.t; on the
other hand, p.sub.b and p.sub.r remain the same in the two
experiments. Let the first experiment yield .alpha..sub.1*,
.beta..sub.1*, and .gamma..sub.1*, and the second one yield
.alpha..sub.2*, .beta..sub.2*, and .gamma..sub.2* (we note that
.gamma..sub.1*=.gamma..sub.2*). Then it can be shown that
p b = .beta. 1 * .gamma. 1 * - .beta. 2 * .gamma. 2 * .alpha. 1 * -
.alpha. 2 * , and ( 11 ) p r = .alpha. 1 * - p b - .beta. 1 *
.gamma. 1 * p b . ( 12 ) ##EQU00028##
[0245] Moreover,
n p = p b k .gamma. 1 * , and ( 13 ) n t 1 = .beta. 1 * .gamma. 1 *
p b 2 n p , n t 2 = .beta. 2 * .gamma. 2 * p * b n p . ( 14 )
##EQU00029##
[0246] We note that quantities (13)-(14) are known within the
transduction coefficient k, where k=y.sub.b(0)/n.sub.p. To find k
and thus unambiguously quantify n.sub.p, n.sub.t1, and n.sub.t2, we
need to perform a calibration experiment (i.e., an experiment with
a known amount of targets n.sub.t).
Experimental Example
[0247] Here we describe the experiments designed to test the
validity of the proposed model and demonstrate the parameter
estimation procedure. To this end, two DNA microarray experiments
are performed. The custom 8-by-9 arrays contain 25mer probes
printed in 3 different probe densities. The targets are Ambion mRNA
Spikes, applied to the arrays with different concentrations. The
concentrations used in the two experiments are 80 ng/50 .mu.l and
16 ng/50 .mu.l. The signal measured in the first experiment, where
80 ng of the target is applied to the array, is shown in FIG. 17.
The smooth line shown in the same figure represents the fit
obtained according to (7). In the second experiment, 16 ng of the
target is applied to the array. The measured signal, and the
corresponding fit obtained according to (7), are both shown in FIG.
18.
[0248] Applying (11)-(14), we obtain
p.sub.h=1.9.times.10.sup.-3,p.sub.r=2.99.times.10.sup.-5.
[0249] Furthermore, we find that
n.sub.t1/n.sub.t2=.beta.*.sub.1/.beta.*.sub.2=3.75 (15)
[0250] Note that the above ratio is relatively close to its true
value, 80/16=5. Finally, assuming that one of the experiments is
used for calibration, we find that the value of the transduction
coefficient is k=4.1.times.10.sup.-4, and that the number of probe
molecules in the observed probe spots is
n.sub.p=1.6.times.10.sup.-11.
Example 3
[0251] This example shows how the methods and systems of the
present invention can be used for measurement of gene expression.
Real-time microarray technology can measure, for example,
expression level differences for different cell types or tissues,
distinct developmental stages, cancerous versus normal cells or
tissues, treated versus untreated cells or tissues, mutant versus
wild-type cells, tissues or organisms.
[0252] The gene expression profiles of inflorescences of the
Arabidopsis thaliana floral homeotic mutants apetala1, apetala2,
apetala3, pistillata, and agamous can be compared with that of
wild-type plants. By combining the data sets from the individual
mutant/wild type comparisons, it is possible to identify a large
number of genes that are, within flowers, predicted to be
specifically or at least predominantly expressed in one type of
floral organ. For each sample, floral buds from approximately 50
plants are collected, and RNA is isolated from 100 mg of tissue
with the RNeasy RNA Isolation Kit (Qiagen). To prepare labeled
target material from those samples, an in vitro transcription
amplification method followed by aminoallyl-UTP-mediated labeling
is used. In brief, first and second strand cDNA is synthesized from
3 .mu.g of total RNA using a polyA-primer with a T7 promoter
sequence. Then in vitro transcription is performed using the
Megascript T7 kit (Amb ion), in the presence of aminoallyl-UTP and
of a reduced amount of UTP, to incorporate the modified aaUTP into
the aRNA during the transcription process. Finally, dye molecules
(Cy3 Mono-Reactive Dye, Cy5 Mono-Reactive Dye, Amersham) are
coupled to the amplified RNA and the dye-labeled RNA is fragmented
before hybridization. These and similar protocols are well
established in the microarray field and are well known to those
skilled in the art, and commercial kits are available for cDNA
synthesis, in vitro transcription amplification, and amino-allyl
labeling, such as the Amino Allyl MessageAmp.TM. II aRNA
Amplification Kit, from Ambion. Aminoallyl UTP contains a reactive
primary amino group on the C5 position of uracil that can be
chemically coupled to N-hydroxysuccinimidyl ester-derivatized
reactive dyes (NHS ester dyes), in a simple efficient reaction.
[0253] Amine-reactive quenchers are commercially available, for
example QSY.RTM. 9 carboxylic acid, succinimidyl ester, from
Invitrogen. The real-time microarray technology is used with
minimal modifications to the sample preparation and labeling
procedures: namely, with the simple substitution of the QSY-9 ester
for the Cy-dye ester. In this case, total RNA samples are prepared
as described above. The purified total RNA is then amplified using
the Amino Allyl MessageAmp.TM. II aRNA Amplification Kit, from
Ambion, and the resulting aRNA is labeled with QSY9. This labeled
RNA population is then used in hybridization with Arabidopsis
real-time microarrays. Such arrays consist of an arranged
collection of probes that correspond to all (or a subset of) the
genes in the Arabidopsis genome. The oligonucleotide probes are
labeled with a fluorescent moiety (such as Cy3) and printed by
contact deposition onto CodeLink slides (GE Healthcare), which are
processed and blocked after printing following manufacturer's
instructions.
Example 4
[0254] This example relates to using an algorithm to measure
cross-hybridization. A variety of different techniques to recover
the signal, including, but not limited to, total least squares,
ESPRIT, and Prony's method, (see Dowling et. al. IEEE Trans. on
Antennas and Propag., vol. 42, no. 5, 1994 and van der Veen et al.
Proc. of the IEEE, 81(9):1277-1308, 1993).
[0255] In this example we study the performance of one such
algorithm in simulation and illustrate the results in FIG. 19. In
particular, we consider the so-called total least squares (TLS)
algorithm in the situation where two target analytes bind to the
same probe spot--one due to hybridization, and the other due to
cross-hybridization. Parameters of the system (probabilities of
hybridization, cross-hybridization, release, etc.) are chosen so as
to mimic realistic experimental scenarios. The probability of
hybridization is assumed to be 5 times greater than the probability
of cross-hybridization (i.e., p.sub.h/p.sub.c=5). The number of
hybridizing target is $n.sub.h=10.sup.9$, while the number of
cross-hybridizing molecules is varied. In FIG. 19, we plot the
relative mean-square error of estimating n.sub.h (averaged over
many realizations of noise) as a function of the ratio
n.sub.h/n.sub.c. The simulation results indicate potentially
successful suppression of cross-hybridization over 3 orders of
magnitude of n.sub.h/n.sub.c.
[0256] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
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