U.S. patent application number 16/562223 was filed with the patent office on 2020-02-27 for systems and methods for high throughput analysis of conformation in biological entities.
The applicant listed for this patent is Biodesy, Inc.. Invention is credited to Dar Bahatt, Louis J. Dietz, Ray Hebert, Joshua Salafsky.
Application Number | 20200064354 16/562223 |
Document ID | / |
Family ID | 54930217 |
Filed Date | 2020-02-27 |
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United States Patent
Application |
20200064354 |
Kind Code |
A1 |
Salafsky; Joshua ; et
al. |
February 27, 2020 |
SYSTEMS AND METHODS FOR HIGH THROUGHPUT ANALYSIS OF CONFORMATION IN
BIOLOGICAL ENTITIES
Abstract
Methods, devices, and systems are disclosed for performing high
throughput analysis of conformational change in biological
molecules or other biological entities using surface-selective
nonlinear optical detection techniques.
Inventors: |
Salafsky; Joshua; (San
Francisco, CA) ; Dietz; Louis J.; (Mountain View,
CA) ; Hebert; Ray; (Florence, OR) ; Bahatt;
Dar; (Foster City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biodesy, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
54930217 |
Appl. No.: |
16/562223 |
Filed: |
September 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15965577 |
Apr 27, 2018 |
10451630 |
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16562223 |
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14754465 |
Jun 29, 2015 |
9989534 |
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15965577 |
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62019285 |
Jun 30, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G02B 5/04 20130101; G01N
21/552 20130101; G02B 5/045 20130101; B01L 3/502715 20130101; B01L
2300/0636 20130101; G01N 33/6845 20130101; B01L 2300/0819 20130101;
B01L 3/5085 20130101; G01N 21/636 20130101; G01N 33/54373 20130101;
G01N 33/54366 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; B01L 3/00 20060101 B01L003/00; G01N 21/552 20060101
G01N021/552; G01N 21/63 20060101 G01N021/63; G01N 33/543 20060101
G01N033/543 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with the support of the United
States government under Grant Number IIP-1256619 from the National
Science Foundation.
Claims
1. A method for determining interactions between target molecules
and test compounds, comprising: a) contacting at least one target
molecule with at least one test compound, wherein the at least one
target molecule is labeled with a nonlinear-active label and is
tethered to at least one discrete region on an upper surface of a
planar substrate; b) illuminating the at least one target molecule
with one or more excitation light beams using total internal
reflection of the one or more excitation light beams from the upper
surface of the planar substrate, wherein the planar substrate
comprises an array of prisms integrated with a lower surface,
wherein the one or more excitation light beams are directed by an
entrance prism to a single discrete region that is adjacent to but
not directly above the entrance prism, wherein the one or more
reflected excitation light beams and a nonlinear optical signal
generated by the at least one labeled target molecule tethered to
the single discrete region are collected by an exit prism that is
also adjacent to but not directly underneath the single discrete
region, and wherein the entrance prism and the exit prism for the
single discrete region are different, non-unique elements of the
array of prisms; and c) determining if a conformational change was
induced in the at least one target molecule through contact with
the at least one test compound by detecting a change in a property
of the nonlinear optical signal; wherein determinations of
conformational change are performed at an average rate of at least
10 test entities tested per hour.
2. The method of claim 1, wherein the determinations of
conformational change are performed at an average rate of at least
100 test compounds tested per hour.
3. The method of claim 1, wherein the at least one target molecule
is tethered to each of two or more discrete regions.
4. The method of claim 1, wherein at least two different target
molecules are tethered to at least two different discrete
regions.
5. The method of claim 1, wherein the target molecules are selected
from the group consisting of cells, proteins, peptides, receptors,
enzymes, antibodies, DNA, RNA, oligonucleotides, small molecules,
and carbohydrates, or any combination thereof.
6. The method of claim 1, wherein the target molecules are drug
targets or portions thereof.
7. The method of claim 1, wherein each of at least four different
target molecules are tethered to at least four different discrete
regions.
8. The method of claim 1, wherein each discrete region comprises an
area of up to about 100 mm.sup.2 on the upper surface of the planar
substrate.
9. The method of claim 7, wherein each discrete region comprises a
supported lipid bilayer.
10. The method of claim 9, wherein the target molecules are
tethered to or embedded within the supported lipid bilayer.
11. The method of claim 1, wherein the at least one test compound
is selected from the group consisting of cells, proteins, peptides,
receptors, enzymes, antibodies, DNA, RNA, oligonucleotides, small
molecules, and carbohydrates, or any combination thereof.
12. The method of claim 1, wherein the test compound is a drug
candidate or portion thereof.
13. The method of claim 1, wherein the contacting step occurs in
solution and comprises utilizing a pre-programmed fluid dispensing
unit to dispense the at least one test compound.
14. The method of claim 1, wherein the contacting step comprises
contacting each of at least four different target molecules with a
different test compound.
15. The method of claim 1, wherein the contacting step comprises
serially contacting the at least one target molecule with at least
100 different test compounds.
16. The method of claim 1, wherein the contacting step comprises
serially contacting the at least one target molecule with at least
10,000 different test compounds.
17. The method of claim 1, wherein the one or more excitation light
beams are provided by one or more lasers.
18.-19. (canceled)
20. The method of claim 1, wherein the non-linear optical signal is
selected from the group consisting of second harmonic light, sum
frequency light, and difference frequency light.
21. The method of claim 1, further comprising moving said planar
substrate relative to the position of one or more external sources
of the one or more excitation light beams.
22. The method of claim 21, wherein each discrete region of two or
more discrete regions is optically coupled with an entrance prism
and a different exit prism that are both integrated with the lower
surface of the substrate.
23.-24. (canceled)
25. The method of claim 1, further comprising repeating the
illuminating and determining steps a plurality of times after said
contacting step, thereby determining conformational changes in the
at least one target molecule as a function of time.
26.-42. (canceled)
43. The method of claim 1, wherein the change in a property of the
nonlinear optical signal comprises a change in intensity,
wavelength, or polarization.
Description
CROSS-REFERENCE
[0001] This application is a Continuation Application of U.S.
application Ser. No. 15/965,577, filed Apr. 27, 2018, which is a
Continuation Application of U.S. application Ser. No. 14/754,465,
filed Jun. 29, 2015, which claims the benefit of U.S. Provisional
Application No. 62/019,285, filed Jun. 30, 2014, each of which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Over the past two decades, the advent of high throughput
experimentation has transformed the way life science and biomedical
research is carried out. A convergence of technologies in fields
such as genetic engineering, organic chemistry, materials science,
microfabrication and microelectronics has led to new technology
platforms (e.g. microarray technologies, microfluidic devices and
systems, bead-based combinatorial compound libraries and assay
systems, and microplate-based assay systems) to address
applications ranging from high throughput screening of compound
libraries for drug discovery to rapid whole genome sequencing.
[0004] Examples of high throughput screening systems for drug
discovery include microfluidics-based platform technologies for
running continuous-flow assays (e.g. receptor-ligand binding assays
and cell-based assays for identifying receptor agonists), and
microplate-based systems in which binding reactions, enzymatic
reactions, or cell-based assays are run in a microwell plate
format, and automated liquid-dispensing stations and plate-handling
robotics provide for automated sample preparation, assay, and
detection steps. The majority of existing high throughput platform
technologies for drug discovery utilize fluorescence-based optical
detection. Although fluorescence techniques provide for very high
detection sensitivity, and are generally much more environmentally
friendly than the more traditional, radioisotope-based approaches
that predominated in biological assay methodologies of two decades
ago, there are a number of drawbacks to the use of fluorescence.
Examples include: (i) the requirement for sophisticated light
sources, detectors, and optical systems, the performance of which
are often sensitive to misalignment or instrumental drift, and (ii)
photo-bleaching phenomena, which may result in degradation of
signal over time in samples subjected to repeat measurements.
[0005] Another, more serious limitation of existing high throughput
screening technologies stems from the growing awareness that a
number of potential therapeutic targets, e.g. potential cancer
therapeutic targets, that are attractive targets from a biological
perspective are intractable ("undruggable") from a chemical
standpoint because they are generally not amenable to conventional
drug discovery approaches. These protein targets typically possess
a relatively large contact area when interacting with other
proteins (i.e. through protein-protein interactions) or due to the
fact that they possess a ligand that binds with extremely high
affinity to the active site of the protein. In either case, finding
a conventional small molecule or biologic (protein) drug candidate
that will block the interaction (i.e. interfere with and/or obscure
the large contact area in the case of protein-protein interactions,
or displace the high affinity ligand) is extremely difficult.
Allosteric modulators for such "undruggable" targets offer an
attractive therapeutic solution. By definition, allosteric
molecules bind to a site other than a protein's active site,
thereby changing the protein's conformation with a concomitant
functional effect (e.g. activation of a receptor). Allosteric
modulation of target proteins has the added benefit of not having
to rely on inhibition or competition with the binding of the
natural ligand to the protein, which can result in unintended
clinical side effects. However, it has been difficult to identify
allosteric modulators using currently available conventional
techniques. For example, structural information obtained from X-ray
crystallography or NMR methods is often of limited value for drug
discovery purposes due to low throughput, low sensitivity, the
non-physiological conditions utilized, the size of the protein
amenable to the technique, and many other factors. What is needed,
therefore are high throughput techniques for screening collections
of candidate compounds to rapidly identify agents capable of, for
example, allosteric modulation of the target protein's
conformation.
[0006] As described more fully below, second harmonic generation
(SHG) is a nonlinear optical process which may be configured as
surface-selective detection technique that enables detection of
conformational change in proteins and other biological targets (as
described previously, for example, in U.S. Pat. No. 6,953,694, and
U.S. patent application Ser. No. 13/838,491). In order to deploy
SHG-based detection of conformational change in a high throughput
format, it may be advantageous to design novel mechanisms for
rapid, precise, and interchangeable positioning of substrates
(comprising the biological targets to be analyzed) with respect to
the optical system used to deliver excitation light, which at the
same time ensure that efficient optical coupling between the
excitation light and the substrate surface is maintained. One
preferred format for high throughput optical interrogation of
biological samples is the glass-bottomed microwell plate.
[0007] The systems and methods disclosed herein provide mechanisms
for coupling the high intensity excitation light required for SHG
and other nonlinear optical techniques to a substrate, e.g. the
glass substrate in a glass-bottomed microwell plate, by means of
total internal reflection in a manner that is compatible with the
requirements for a high throughput analysis system.
SUMMARY OF THE INVENTION
[0008] Disclosed herein are methods for determining interactions
between biological entities and test entities, comprising: (a)
contacting each of at least four biological entities with at least
one test entity; (b) illuminating each of the at least four
biological entities with one or more excitation light beams using a
surface selective optical technique; and (c) determining whether or
not a conformational change was induced in each of the at least
four biological entities through contact with the at least one test
entity; wherein determinations of conformational change are
performed at an average rate of at least 10 test entities tested
per hour. In some embodiments, the determinations of conformational
change are performed at an average rate of at least 100 test
entities tested per hour.
[0009] In some embodiments, the at least four biological entities
are the same. In some embodiments, the at least four biological
entities are different. In some embodiments, the biological
entities are selected from the group consisting of cells, proteins,
peptides, receptors, enzymes, antibodies, DNA, RNA,
oligonucleotides, small molecules, and carbohydrates, or any
combination thereof. In some embodiments, the biological entities
are drug targets or portions thereof.
[0010] In some embodiments, each of the at least four biological
entities are situated in a different discrete region on a
substrate. In some embodiments, each discrete region comprises an
area of up to about 100 mm.sup.2 on a substrate surface. In some
embodiments, each discrete region comprises a supported lipid
bilayer. In some embodiments, the biological entities are tethered
to or embedded within the supported lipid bilayer.
[0011] In some embodiments, the at least one test entity is
selected from the group consisting of cells, proteins, peptides,
receptors, enzymes, antibodies, DNA, RNA, oligonucleotides, small
molecules, and carbohydrates, or any combination thereof. In some
embodiments, the test entity is a drug candidate or portion
thereof.
[0012] In some embodiments, the contacting step occurs in solution
and comprises utilizing a pre-programmed fluid dispensing unit to
dispense the at least one test entity. In some embodiments, the
contacting step comprises contacting each of the at least four
biological entities with a different test entity. In some
embodiments, the contacting step comprises serially contacting each
of the at least four biological entities with at least 100
different test entities. In some embodiments, the contacting step
comprises serially contacting each of the at least four biological
entities with at least 10,000 different test entities.
[0013] In some embodiments, the one or more excitation light beams
are provided by one or more lasers. In some embodiments, the
surface selective optical detection technique comprises the use of
total internal reflection of at least one excitation light beam
from a planar substrate surface.
[0014] In some embodiments, the determining step further comprises
analyzing a non-linear optical signal. In some embodiments, the
non-linear optical signal is selected from the group consisting of
second harmonic light, sum frequency light, and difference
frequency light.
[0015] In some embodiments, the methods further comprise moving
said substrate relative to the position of one or more external
sources of the one or more excitation light beams. In some
embodiments, each discrete region is optically coupled with an
entrance prism and a different exit prism that are both integrated
with a bottom surface of the substrate. In some embodiments, the
illuminating step comprises directing incident excitation light
onto an entrance prism positioned adjacent to, but not directly
below, each of the discrete regions. In some embodiments, the
methods further comprise collecting non-linear optical signals
generated at each of the discrete regions upon illumination using
an exit prism positioned adjacent to, but not directly below, each
of the discrete regions, and directing said non-linear optical
signals to a detector. In some embodiments, the methods further
comprise repeating the illuminating and determining steps a
plurality of times after said contacting step, thereby determining
conformational changes in each of the at least four biological
entities as a function of time.
[0016] Also disclosed herein are devices comprising: (a) a
substrate, the substrate comprising: (i) an M.times.N array of
discrete regions formed on a surface of the substrate, wherein M is
the number of rows of discrete regions and N is the number of
columns of discrete regions in the array, and each discrete region
is configured for containing a biological entity, and (ii) an
R.times.S array of prisms integrated with the substrate and
optically coupled to the discrete regions, wherein R is the number
of rows of prisms and S is the number of columns of prisms in the
array; wherein R=M+2 and S=N, or R=M and S=N+2.
[0017] In some embodiments, each of the discrete regions is
optically coupled with at least one input prism and at least one
output prism, and wherein the input prism and the output prism are
spatially distinct. In some embodiments, M=8 and N=12. In some
embodiments, M=16 and N=24. In some embodiments, M=32 and N=48. In
some embodiments, M is greater than 4 and N is greater than 4. In
some embodiments, each discrete region comprises a supported lipid
bilayer or is configured to facilitate the formation of a supported
lipid bilayer. In some embodiments, the devices further comprise a
well-forming component bonded to a top surface of the substrate in
order to isolate each discrete region in a separate well. In some
embodiments, each of the discrete regions comprises an area of up
to about 100 mm.sup.2. In some embodiments, each discrete region or
well is located directly above a single prism of the array of
prisms integrated with the substrate. In some embodiments, the
substrate is composed of glass, fused-silica, or plastic.
[0018] Disclosed herein are injection molding processes for
fabricating a prism array part from a plastic, the process
comprising the use of two or more mold ejection devices to apply
uniform pressure to the prism array part during a mold release
step.
[0019] In some embodiments, the plastic is selected from the group
consisting of cyclic olefin copolymer (COC), cyclic olefin polymer
(COP), and acrylic. In some embodiments, the two or more mold
ejection devices impact the prism array part only in regions where
the optical performance of the part is non-critical. In some
embodiments, the two or more mold ejection devices comprise an
array of m.times.n blade-like ejector features. In some
embodiments, m is greater than 2 and less than 20. In some
embodiments, n is greater than 2 and less than 20.
INCORPORATION BY REFERENCE
[0020] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] 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:
[0022] FIG. 1A provides a schematic illustration of the energy
level diagrams for fluorescence (an absorption process).
[0023] FIG. 1B provides a schematic illustration of the energy
level diagrams for second harmonic generation (a two photon
scattering process).
[0024] FIG. 2 provides a schematic illustration of a conformational
change in a protein (labeled with a nonlinear-active tag) which is
induced by binding of a ligand, and its impact on the distance
and/or orientation of a nonlinear-active label relative to an
optical interface to which the protein is attached.
[0025] FIGS. 3A-D show data that demonstrate the detection of
spermine- or spermidine-induced conformational changes in
alpha-synuclein using second harmonic light generation. FIG. 3A
shows the SHG response in real time upon exposure of
alpha-synuclein to 5 mM spermine. The arrow denotes spermine
addition. The change in SHG intensity is normalized to the value
just prior to injection. FIG. 3B shows the dose response curve
(plotted on a log scale) for spermine-induced conformational change
in alpha-synuclein as measured by SHG (the change in SHG
concentration is quantified as percent shift). FIG. 3C shows the
SHG response in real time upon exposure of alpha-synuclein to 3 mM
spermidine. The arrow denotes spermidine addition. The change in
SHG intensity is normalized to the value just prior to injection.
FIG. 3D shows the dose response curve (plotted on a log scale) for
spermidine-induced conformational change in alpha-synuclein as
measured by SHG (the change in SHG concentration is quantified as
percent shift). Error bars=SEM. N=3.
[0026] FIG. 4 illustrates one example of the system architecture
for a high throughput analysis system for determining
conformational change in biological molecules or other biological
entities based on nonlinear optical detection.
[0027] FIG. 5 shows a schematic for one example of an optical setup
used for analysis of conformational change in biological molecules
using nonlinear optical detection.
[0028] FIG. 6 shows a photograph of an optical setup used for
analysis of conformational change in biological molecules using
nonlinear optical detection.
[0029] FIG. 7 shows a schematic illustration depicting the use of a
prism to direct excitation light at an appropriate incident angle
such that the excitation light undergoes total internal reflection
at the top surface of a substrate. The two dashed lines to the
right of the prism indicate the optical path of the reflected
excitation light and the nonlinear optical signal generated at the
substrate surface when nonlinear-active species are tethered to the
surface. The substrate is optionally connected to the actuator of
an X-Y translation stage for re-positioning between measurements.
The curved lines between the top surface of the prism and the lower
surface of the substrate indicate the presence a thin layer (not to
scale) of index-matching fluid used to ensure high optical coupling
efficiency between the prism and substrate.
[0030] FIGS. 8A-C show different views of one exemplary design
concept for a system that uses a continuously recirculating flow of
index-matching fluid to provide high optical coupling efficiency
between the prism (attached to the optical instrument in this
example) and the substrate (configured as the transparent bottom of
a microwell plate in this example). The substrate (microwell plate)
is free to translate relative to the prism while a continuous flow
of index-matching fluid provided by the indicated fluid channels
ensures good optical coupling of excitation light with the
substrate. FIG. 8A: top-front axonometric view. FIG. 8B: top-rear
axonometric view. FIG. 8C: bottom-front axonometric view.
[0031] FIG. 9 shows a schematic illustration depicting the use of a
layer of index-matching elastomeric material attached or adjacent
to the lower surface of a transparent substrate (configured in a
microwell plate format in this example) to ensure high optical
coupling efficiency between a prism and the upper surface of the
substrate. In some embodiments of this approach, the upper surface
of the prism is slightly domed to focus the compression force when
bringing the microwell plate and prism into contact, thereby
reducing or eliminating the formation of air gaps between the prism
and elastomeric material.
[0032] FIGS. 10A-B illustrate a microwell plate with integrated
prism array for providing good optical coupling of the excitation
light to the top surface of the substrate. In this approach, the
prism indicated in the schematic illustrations of FIGS. 4, 5, 7,
and 9 are replaced by the prism array attached to the underside of
the substrate. FIG. 10A: top axonometric view. FIG. 10B: bottom
axonometric view.
[0033] FIGS. 10C-D show exploded views of the microwell plate
device shown in FIGS. 10A-B. FIG. 10C: top axonometric view. FIG.
10D: bottom axonometric view.
[0034] FIG. 11 illustrates the incident and exit light paths for
coupling the excitation light to the substrate surface via total
internal reflection using the design concept illustrated in FIGS.
10A-B.
[0035] FIG. 12 shows a photograph of a prototype for the prism
array design concept illustrated in FIGS. 10A-B.
[0036] FIGS. 13A-C show one example of a prism array design
according to the present disclosure. FIG. 13A: top view. FIG. 13B:
front view. FIG. 13C: right side view.
[0037] FIG. 14 shows a crossed-polarizer image of a prism array
fabricated using a first mold design and first injection molding
process. Note the high level of stress-induced birefringence
observed.
[0038] FIG. 15 shows data for the SHG signal intensity measured at
different positions in a microwell plate device incorporating the
prism array design illustrated in FIGS. 13A-C and FIG. 14. SHG
signal intensity was measured for different rows (different traces)
as a function of well column number.
[0039] FIGS. 16A-C show an example of an improved prism array
design according to the present disclosure. FIG. 16A: top view.
FIG. 16B: front view. FIG. 16C: right side view.
[0040] FIG. 17 shows a crossed-polarizer image of a prism array
fabricated using an improved mold design and optimized injection
molding process. Note that the level of stress-induced
birefringence observed is significantly reduced compared to that
shown in FIG. 14.
[0041] FIG. 18 shows data for the SHG signal intensity measured at
different positions in a microwell plate device incorporating the
prism array design illustrated in FIGS. 16A-C and FIG. 17. SHG
signal intensity was measured for different rows (different traces)
as a function of well column number. Note that the level of SHG
signal intensity is higher and more uniform across the microwell
plate compared to that shown in FIG. 15.
[0042] FIG. 19 shows a cut-away version of the mold tool used to
fabricate the prism array part illustrated in FIGS. 16A-C.
[0043] FIG. 20 illustrates the array of blade-like ejection
features used in the mold tool for providing uniform pressure on
the prism array part during release from the mold.
[0044] FIG. 21 illustrates a computer system that may be configured
to control the operation of the systems disclosed herein.
[0045] FIG. 22 is a block diagram illustrating a first example
architecture of a computer system that can be used in connection
with example embodiments of the present invention.
[0046] FIG. 23 is a diagram showing one embodiment of a network
with a plurality of computer systems, a plurality of cell phones
and personal data assistants, and Network Attached Storage
(NAS).
[0047] FIG. 24 is a block diagram of a multiprocessor computer
system using a shared virtual address memory space in accordance
with an example embodiment.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The systems and methods disclosed herein relate to high
throughput analysis of conformation in biological entities. In
addition, the systems and methods described are equally suitable
for high throughput analysis of orientation or conformational
change. In some aspects of the present disclosure, systems and
methods are described for determining orientation, conformation, or
changes in orientation or conformation of biological entities in
response to contacting the biological entities with one or more
test entities. As used herein, determining orientation,
conformation, or changes thereof may involve measurement of a
nonlinear optical signal which is related to and/or proportional to
the average orientation of a nonlinear-active label or tag. As used
herein, "high throughput" refers to the ability to perform rapid
analysis of conformation for a large plurality of biological
entities optionally contacted with one or more test entities, or to
the ability to perform rapid analysis of conformation for one or
more biological entities optionally contacted with a large
plurality of test entities, or to any combination of these two
modalities. In general, the systems and methods disclosed rely on
the use of second harmonic generation (SHG) or related nonlinear
optical techniques for detection of orientation, conformation, or
conformational change, as described previously, for example, in
U.S. Pat. No. 6,953,694, and U.S. patent application Ser. No.
13/838,491.
Detection of Conformation Using Second Harmonic Generation
[0049] Second harmonic generation, in contrast to the more widely
used fluorescence-based techniques, is a nonlinear optical process,
in which two photons of the same excitation wavelength or frequency
interact with a nonlinear material and are re-emitted as a single
photon having twice the energy, i.e. twice the frequency and half
the wavelength, of the excitation photons (FIGS. 1A-1B). Second
harmonic generation only occurs in nonlinear materials lacking
inversion symmetry (i.e. in non-centrosymmetric materials) and
requires a high intensity excitation light source. It is a special
case of sum frequency generation and is related to other nonlinear
optical phenomena such as difference frequency generation.
[0050] Second harmonic generation and other nonlinear optical
techniques can be configured as surface-selective detection
techniques because of their dependence on the orientation of the
nonlinear-active species. Tethering of the nonlinear-active species
to a surface, for example, can instill an overall degree of
orientation that is absent when molecules are free to undergo
rotational diffusion in solution. An equation commonly used to
model the orientation-dependence of nonlinear-active species at an
interface is:
.chi..sup.(2)=N.sub.s<.alpha..sup.(2)>
where .chi..sup.(2) is the nonlinear susceptibility, N.sub.s is the
total number of nonlinear-active molecules per unit area at the
interface, and <.alpha..sup.(2)> is the average orientation
of the nonlinear hyperpolarizability (.alpha..sup.(2)) of these
molecules. The intensity of SHG is proportional to the square of
the nonlinear susceptibility, and is thus dependent on both the
number of oriented nonlinear-active species at the interface and to
changes in their average orientation.
[0051] Second harmonic generation and other nonlinear optical
techniques may be rendered additionally surface selective through
the use of total internal reflection as the mode for delivery of
the excitation light to the optical interface on which
nonlinear-active species have been immobilized. Total internal
reflection of the incident excitation light creates an "evanescent
wave" at the interface, which may be used to selectively excite
only nonlinear-active labels that are in close proximity to the
surface, i.e. within the spatial decay distance of the evanescent
wave, which is typically on the order of tens of nanometers. Total
internal reflection may also be used to excite fluorescence in a
surface-selective manner, for example to excite a fluorescence
donor attached to the optical interface, which then transfers
energy to a suitable acceptor molecule via a fluorescence resonance
energy transfer (FRET) mechanism. In the present disclosure, the
evanescent wave generated by means of total internal reflection of
the excitation light is preferentially used to excite a
nonlinear-active label or molecule. The the efficiency of exciting
nonlinear active species will depend strongly on both their average
orientation and on their proximity to the interface.
[0052] This surface selective property of SHG and other nonlinear
optical techniques can be exploited to detect conformational change
in biological molecules immobilized at interfaces. For example,
conformational change in a receptor molecule due to binding of a
ligand, might be detected using a nonlinear-active label or moiety
wherein the label is attached to or associated with the receptor
such that the conformational change leads to a change in the
orientation or distance of the label with respect to the interface
(FIG. 2), and thus to a change in a physical property of the
nonlinear optical signal. Until recently, the use of
surface-selective nonlinear optical techniques has been confined
mainly to applications in physics and chemistry, since relatively
few biological samples are intrinsically non-linearly active.
Recently, the use of second harmonic active labels ("SHG labels")
has been introduced, allowing virtually any molecule or particle to
be rendered highly non-linear active. The first example of this was
demonstrated by labeling the protein cytochrome c with an oxazole
dye and detecting the protein conjugate at an air-water interface
with second harmonic generation [Salafsky, J., "`SHG-labels` for
Detection of Molecules by Second Harmonic Generation", Chem. Phys.
Lett. 342(5-6):485-491 (2001)]. Examples of SHG data that
demonstrate the detection of spermine- or spermidine-induced
conformational changes in alpha-synuclein are shown in FIGS.
3A-3D.
[0053] Surface-selective nonlinear optical techniques are also
coherent techniques, meaning that the fundamental and nonlinear
optical light beams have wave fronts that propagate through space
with well-defined spatial and phase relationships. The use of
surface-selective nonlinear optical detection techniques for
analysis of conformation of biological molecules or other
biological entities has a number of inherent advantages over other
optical approaches, including: i) sensitive and direct dependence
of the nonlinear signal on the orientation and/or dipole moment(s)
of the nonlinear-active species, thereby conferring sensitivity to
conformational change; (ii) higher signal-to-noise (lower
background) than fluorescence-based detection since the nonlinear
optical signal is generated only at surfaces that create a
non-centrosymmetric system, i.e. the technique inherently has a
very narrow "depth-of-field"; (iii) as a result of the narrow
"depth of field", the technique is useful when measurements must be
performed in the presence of a overlaying solution, e.g. where a
binding process might be obviated or disturbed by a separation or
rinse step. This aspect of the technique may be particularly useful
for performing equilibrium binding measurements, which require the
presence of bulk species, or kinetics measurements where the
measurements are made over a defined period of time; (iv) the
technique exhibits lower photo-bleaching and heating effects than
those that occur in fluorescence, due to the facts that the
two-photon absorption cross-section is typically much lower than
the one-photon absorption cross-section for a given molecule, and
that SHG (and sum frequency generation or difference frequency
generation) involves scattering, not absorption; (v) minimal
collection optics are required and higher signal to noise is
expected since the fundamental and nonlinear optical beams (e.g.,
second harmonic light) have well-defined incoming and outgoing
directions with respect to the interface. This is particularly
advantageous compared to fluorescence-based detection, as
fluorescence emission is isotropic and there may also be a large
fluorescence background component to detected signals arising from
out-of-focal plane fluorescent species.
High Throughput Systems and Methods
[0054] Systems and methods are disclosed herein for implementing
high throughput analysis of conformation in biological entities
based on the use of second harmonic generation or related nonlinear
optical detection techniques. As used herein, "high throughput" is
a relative term used in comparison to structural measurements
performed using traditional techniques such as NMR or X-ray
crystallography. As will be described in more detail below, the
SHG-based methods and systems disclosed herein are capable of
performing structural determinations at a rate that is at least an
order-of-magnitude faster than these conventional techniques.
[0055] In one aspect, this disclosure provides a method for high
throughput detection of conformation or conformational change in
one or more biological entities, the method comprising (i) labeling
one or more target biological entities, e.g. protein molecules,
with a nonlinear-active label or tag, (ii) immobilizing the one or
more labeled target biological entities at one or more discrete
regions of a planar substrate surface, wherein the substrate
surface further comprises an optical interface, (iii) sequentially
exposing each discrete region to excitation light by changing the
position of the substrate relative to an external light source,
(iv) collecting a nonlinear optical signal emitted from each
discrete region as it is exposed to excitation light, and (v)
processing said nonlinear optical signal to determine an
orientation, conformation, or conformational change of each of the
one or more biological entities. In another aspect, the method
further comprises (vi) contacting each of the one or more
biological entities with one or more test entities following the
first exposure to excitation light, (vii) subsequently re-exposing
each discrete region to excitation light one or more times, (viii)
collecting a nonlinear optical signal from each discrete region as
it is exposed to excitation light, and (ix) processing said
nonlinear optical signals to determine whether or not a change in
orientation or conformation has occurred in the one or more
biological entities as a result of contacting with said one or more
test entities. In one aspect of the method, nonlinear optical
signals are detected only once following contact of the one or more
biological entities with one or more test entities (i.e. endpoint
assay mode), and then used to determine whether or not
conformational change has occurred. In another aspect, nonlinear
optical signals are collected repeatedly and at defined time
intervals following contact of the one of more biological entities
with one or more test entities (i.e. kinetics mode), and then used
to determine the kinetics of conformational change in the one or
more biological entities. In a preferred aspect of the method, each
discrete region of the substrate comprises a supported lipid
bilayer structure, and biological entities are immobilized in each
discrete region by means of tethering to or embedding in the lipid
bilayer. In another preferred aspect of the method, the excitation
light is delivered to the substrate surface, i.e. the optical
interface, by means of total internal reflection, and the nonlinear
optical signals emitted from the discrete regions of the substrate
surface are collected along the same optical axis as the reflected
excitation light.
[0056] In order to implement high throughput analysis of
conformation or conformational change using nonlinear optical
detection, the systems described herein require several components
(illustrated schematically in FIG. 4), including (i) at least one
suitable excitation light source and optics for delivering the at
least one excitation light beam to an optical interface, (ii) an
interchangeable substrate comprising the optical interface, to
which one or more biological entities have been tethered or
immobilized in discrete regions of the substrate, (iii) a
high-precision translation stage for positioning the substrate
relative to the at least one excitation light source, and (iv)
optics for collecting nonlinear optical signals generated as a
result of illuminating each of the discrete regions of the
substrate with excitation light and delivering said nonlinear
signals to a detector, and (v) a processor for analyzing the
nonlinear optical signal data received from the detector and
determining conformation or conformational change for the one or
more biological entities immobilized on the substrate. In some
aspects, the systems and methods disclosed herein further comprise
the use of (vi) a programmable fluid-dispensing system for
delivering test entities to each of the discrete regions of the
substrate, and (vii) the use of plate-handling robotics for
automated positioning and replacement of substrates at the
interface with the optical system.
[0057] The methods and systems disclosed herein may be configured
for analysis of a single biological entity contacted with a
plurality of test entities, or for analysis of a plurality of
biological entities contacted with a single test entity, or any
combination thereof. When contacting one or more biological
entities with a plurality of test entities, the contacting step may
be performed sequentially, i.e. by exposing the immobilized
biological entity to a single test entity for a specified period of
time, followed by an optional rinse step to remove the test entity
solution and regenerate the immobilized biological entity prior to
introducing to the next test entity, or the contacting step may be
performed in parallel, i.e. by having a plurality of discrete
regions comprising the same immobilized biological entity, and
exposing the biological entity in each of the plurality of discrete
regions to a different test entity. The methods and systems
disclosed herein may be configured to perform analysis of
conformational change in at least one biological entity, at least
two biological entities, at least four biological entities, at
least six biological entities, at least eight biological entities,
at least ten biological entities, at least fifteen biological
entities, or at least twenty biological entities. In some aspects,
methods and systems disclosed herein may be configured to perform
analysis of conformational change in at most twenty biological
entities, at most fifteen biological entities, at most ten
biological entities, at most eight biological entities, at most six
biological entities, at most four biological entities, at most two
biological entities, or at most one biological entity. Similarly,
the methods and systems disclosed herein may be configured to
perform analysis of conformational change upon exposure of the one
or more biological entities to at least 1 test entity, at least 5
test entities, at least 10 test entities, at least 50 test
entities, at least 100 test entities, at least 500 test entities,
at least 1,000 test entities, at least 5,000 test entities, at
least 10,000 test entities, or at least 100,000 test entities. In
some aspects, the methods and systems disclosed herein may be
configured to perform analysis of conformational change upon
exposure of the one or more biological test entities to at most
100,000 test entities, at most 10,000 test entities, at most 5,000
test entities, at most 1,000 test entities, at most 500 test
entities, at most 100 test entities, at most 50 test entities, at
most 10 test entities, at most 5 test entities, or at most 1 test
entity.
Biological Entities and Test Entities
[0058] As used herein, the phrase "biological entities" comprises
but is not limited to cells, proteins, peptides, receptors,
enzymes, antibodies, DNA, RNA, biological molecules,
oligonucleotides, solvents, small molecules, synthetic molecules,
carbohydrates, or any combination thereof. Similarly, the phrase
"test entities" also comprises but is not limited to cells,
proteins, peptides, receptors, enzymes, antibodies, DNA, RNA,
biological molecules, oligonucleotides, solvents, small molecules,
synthetic molecules, carbohydrates, or any combination thereof. In
some aspects, biological entities may comprise drug targets, or
portions thereof, while test entities may comprise drug candidates,
or portions thereof.
Nonlinear-Active Labels and Labeling Techniques
[0059] As noted above, most biological molecules are not
intrinsically nonlinear-active. Exceptions include collagen, a
structural protein that is found in most structural or load-bearing
tissues. SHG microscopy has been used extensively in studies of
collagen-containing structures, for example, the cornea. Other
biological molecules or entities must be rendered nonlinear-active
by means of introducing a nonlinear-active moiety such as a tag or
label. A label for use in the present invention refers to a
nonlinear-active moiety, tag, molecule, or particle which can be
bound, either covalently or non-covalently to a molecule, particle
or phase (e.g., a lipid bilayer) in order to render the resulting
system more nonlinear optical active. Labels can be employed in the
case where the molecule, particle or phase (e.g., lipid bilayer) is
not nonlinear active to render the system nonlinear-active, or with
a system that is already nonlinear-active to add an extra
characterization parameter into the system. Exogenous labels can be
pre-attached to the molecules, particles, or other biological
entities, and any unbound or unreacted labels separated from the
labeled entities before use in the methods described herein. In a
specific aspect of the methods disclosed herein, the
nonlinear-active moiety is attached to the target molecule or
biological entity in vitro prior to immobilizing the target
molecules or biological entities in discrete regions of the
substrate surface. The labeling of biological molecules or other
biological entities with nonlinear-active labels allows a direct
optical means of detecting interactions between the labeled
biological molecule or entity and another molecule or entity (i.e.
the test entity) in cases where the interaction results in a change
in orientation or conformation of the biological molecule or entity
using a surface-selective nonlinear optical technique.
[0060] In alternative aspects of the methods and systems described
herein, at least two distinguishable nonlinear-active labels are
used. The orientation of the attached two or more distinguishable
labels would then be chosen to facilitate well defined directions
of the emanating coherent nonlinear light beam. The two or more
distinguishable labels can be used in assays where multiple
fundamental light beams at one or more frequencies, incident with
one or more polarization directions relative to the optical
interface are used, with the resulting emanation of at least two
nonlinear light beams.
[0061] Examples of nonlinear-active tags or labels include, but are
not limited to, the compounds listed in Table 1, and their
derivatives.
TABLE-US-00001 TABLE 1 Examples of Nonlinear-Active Tags
2-aryl-5-(4- Hemicyanines Polyimides pyridyl)oxazole 2-(4-pyridyl)-
lndandione-1,3- Polymethacrylates cyclo- pyidinium betaine
alkano[d]oxazoles 5-aryl-2-(4- lndodicarbocyanines PyMPO
pyridyl)oxazole (pyridyloxazole) 7-Hydroxycoumarin-3- Melamines
PyMPO, succinimidyl carboxylic acid, ester (1-(3- succinimidyl
(succinimidyloxy- ester carbonyl)benzyl)-4-(5- (4-PyMPO, maleimide
Azo dyes Merocyanines Stilbazims Benzooxazoles
Methoxyphenyl)oxazol- Stilbenes 2-yl)pyridinium bromide)
Bithiophenes Methylene blue Stryryl-based dyes Cyanines Oxazole or
oxadizole Sulphonyl-substituted molecules azobenzenes Dapoxyl
Oxonols Thiophenes carboxylic acid, succinimidyl ester
Diaminobenzene Perylenes Tricyanovinyl aniline compounds
Diazostilbenes Phenothiazine-stilbazole Tricyanovinyl azo
Fluoresceins Polyenes
[0062] In evaluating whether a species may be nonlinear-active, the
following characteristics can indicate the potential for nonlinear
activity: a large difference dipole moment (difference in dipole
moment between the ground and excited states of the molecule), a
large Stokes shift in fluorescence, or an aromatic or conjugated
bonding character. In further evaluating such a species, an
experimenter can use a simple technique known to those skilled in
the art to confirm the nonlinear activity, for example, through
detection of SHG from an air-water interface on which the
nonlinear-active species has been distributed. Once a suitable
nonlinear-active species bas been selected for the experiment at
hand, the species can be conjugated, if desired, to a biological
molecule or entity for use in the surface-selective nonlinear
optical methods and systems disclosed herein.
[0063] The following reference and references therein describe
techniques available for creating a labeled biological entity from
a synthetic dye and many other molecules: Greg T. Hermanson,
Bioconjugate Techniques, Academic Press, New York, 1996.
[0064] In a specific aspect of the methods and systems disclosed,
metal nanoparticles and assemblies thereof are modified to create
biological nonlinear-active labels. The following references
describe the modification of metal nanoparticles and assemblies: J.
P. Novak and D. L. Feldheim, "Assembly of Phenylacetylene-Bridged
Silver and Gold Nanoparticle Arrays", J. Am. Chem. Soc.
122:3979-3980 (2000); J. P. Novak, et al., "Nonlinear Optical
Properties of Molecularly Bridged Gold Nanoparticle Arrays", J. Am.
Chem. Soc. 122:12029-12030 (2000); Vance, F. W., Lemon, B. I., and
Hupp, J. T., "Enormous Hyper-Rayleigh Scattering from
Nanocrystalline Gold Particle Suspensions", J. Phys. Chem. B
102:10091-93 (1999).
[0065] In yet another aspect of the methods and systems disclosed
herein, the nonlinear activity of the system can also be
manipulated through the introduction of nonlinear analogues to
molecular beacons, that is, molecular beacon probes that have been
modified to incorporate a nonlinear-active label (or modulator
thereof) instead of fluorophores and quenchers. These nonlinear
optical analogues of molecular beacons are referred to herein as
molecular beacon analogues (MB analogues or MBA). The MB analogues
to be used in the described methods and systems can be synthesized
according to procedures known to one of ordinary skill in the
art.
Types of Biological Interactions Detected
[0066] The methods and systems disclosed herein provide for
detection of a variety of interactions between biological entities,
or between biological entities and test entities, depending on the
choice of biological entities, test entities, and non-linear active
labeling technique employed. In one aspect, the present disclosure
provides for the qualitative detection of binding events, e.g. the
binding of a ligand to a receptor, as indicated by the resulting
conformational change induced in the receptor. In another aspect,
the present disclosure provides for quantitative analysis of
binding events, e.g. the binding of a ligand to a receptor, by
performing replicate measurements using different concentrations of
the ligand molecule and generating a dose-response curve using the
percent change in maximal conformational change observed.
Similarly, other aspects of the present disclosure may provide
methods for qualitative or quantitative measurements of
enzyme-inhibitor interactions, antibody-antigen interactions, the
formation of complexes of biological macromolecules, or
interactions of receptors with allosteric modulators.
[0067] In other specific embodiments, MB analogues can be used
according to the methods disclosed herein as hybridization probes
that can detect the presence of complementary nucleic acid target
without having to separate probe-target hybrids from excess probes
as in solution-phase hybridization assays, and without the need to
label the targets oligonucleotides. MB analogue probes can also be
used for the detection of RNAs within living cells, for monitoring
the synthesis of specific nucleic acids in sample aliquots drawn
from bioreactors, and for the construction of self-reporting
oligonucleotide arrays. They can be used to perform homogeneous
one-well assays for the identification of single-nucleotide
variations in DNA and for the detection of pathogens or cells
immobilized to surfaces for interfacial detection.
[0068] Interactions between biological entities or biological and
test entities (e.g. binding reactions, conformational changes,
etc.) can be correlated through the methods presently disclosed to
the following measurable nonlinear signal parameters: (i) the
intensity of the nonlinear light, (ii) the wavelength or spectrum
of the nonlinear light, (iii) the polarization of the nonlinear
light, (iv) the time-course of (i), (ii), or (iii), and/or vi) one
or more combinations of (i), (ii), (iii), and (iv).
Laser Light Sources and Excitation Optical System
[0069] FIG. 5 illustrates one aspect of the methods and systems
disclosed herein wherein second harmonic light is generated by
reflecting incident fundamental excitation light from the surface
of a substrate comprising the sample interface (or optical
interface). FIG. 6 shows a photograph of one example of a suitable
optical setup. A laser provides the fundamental light necessary to
generate second harmonic light at the sample interface. Typically
this will be a picosecond or femtosecond laser, either wavelength
tunable or not tunable, and commercially available (e.g. a Ti:
Sapphire femtosecond laser or fiber laser system). Light at the
fundamental frequency (w) exits the laser and its polarization is
selected using, for example a half-wave plate appropriate to the
frequency and intensity of the light (e.g., available from Melles
Griot, Oriel, or Newport Corp.). The beam then passes through a
harmonic separator designed to pass the fundamental light but block
nonlinear light (e.g. second harmonic light). This filter is used
to prevent back-reflection of the second harmonic beam into the
laser cavity which can cause disturbances in the lasing properties.
A combination of mirrors and lenses are then used to steer and
shape the beam prior to reflection from a final mirror that directs
the beam via a prism to impinge at a specific location and with a
specific angle .theta. on the substrate surface such that it
undergoes total internal reflection at the substrate surface. One
of the mirrors in the optical path can be scanned if required using
a galvanometer-controlled mirror scanner, a rotating polygonal
mirror scanner, a Bragg diffractor, acousto-optic deflector, or
other means known in the art to allow control of a mirror's
position. The substrate comprising the optical interface and
nonlinear-active sample surface can be mounted on an x-y
translation stage (computer controlled) to select a specific
location on the substrate surface for generation of the second
harmonic beam. In some aspects of the methods and systems presently
described, it is desirable to scan or rotate one mirror in order to
slightly vary the angle of incidence for total internal reflection,
and thereby maximize the nonlinear optical signal emitted from the
discrete regions of the substrate surface without substantially
changing the position of the illuminating excitation light spot. In
some aspects, two (or more) lasers having different fundamental
frequencies may be used to generate sum frequency or difference
frequency light at the optical interface on which the non-linear
active sample is immobilized.
Substrate Formats, Optical Interface, and Total Internal
Reflection
[0070] As described above, the systems and methods of the present
disclosure utilize a planar substrate for immobilization of one or
more biological entities on a top surface of the substrate, wherein
the top substrate surface further comprises the optical interface
(or sample interface) used for exciting nonlinear optical signals.
The substrate can be glass, silica, fused-silica, plastic, or any
other solid material that is transparent to the fundamental and
second harmonic light beams, and that supports total internal
reflection at the substrate/sample interface when the excitation
light is incident at an appropriate angle. In some aspects of the
invention, the discrete regions within which biological entities
are contained are configured as one-dimensional or two-dimensional
arrays, and are separated from one another by means of a
hydrophobic coating or thin metal layer. In other aspects, the
discrete regions may comprise indents in the substrate surface. In
still other aspects, the discrete regions may be separated from
each other by means of a well-forming component such that the
substrate forms the bottom of a microwell plate (or microplate),
and each individual discrete region forms the bottom of one well in
the microwell plate. In one aspect of the present disclosure, the
well-forming component separates the top surface of the substrate
into 96 separate wells. In another aspect, the well-forming
component separates the top surface of the substrate into 384
wells. In yet another aspect, the well-forming component separates
the top surface of the substrate into 1,536 wells. In all of these
aspects, the substrate, whether configured in a planar array,
indented array, or microwell plate format, may comprise a
disposable or consumable device or cartridge that interfaces with
other optical and mechanical components of the high throughput
system.
[0071] The methods and systems disclosed herein further comprise
specifying the number of discrete regions or wells into which the
substrate surface is divided, irrespective of how separation is
maintained between discrete regions or wells. Having larger numbers
of discrete regions or wells on a substrate may be advantageous in
terms of increasing the sample analysis throughput of the method or
system. In one aspect of the present disclosure, the number of
discrete regions or wells per substrate is between 10 and 1,600. In
other aspects, the number of discrete regions or wells is at least
10, at least 20, at least 50, at least 100, at least 200, at least
300, at least 400, at least 500, at least 750, at least 1,000, at
least 1,250, at least 1,500, or at least 1,600. In yet other
aspects of the disclosed methods and systems, the number of
discrete regions or wells is at most 1,600, at most 1,500, at most
1,000, at most 750, at most 500, at most 400, at most 300, at most
200, at most 100, at most 50, at most 20, or at most 10. In a
preferred aspect, the number of discrete regions or wells is 96. In
another preferred aspect, the number of discrete regions or wells
is 384. In yet another preferred aspect, the number of discrete
regions or wells is 1,536. Those of skill in the art will
appreciate that the number of discrete regions or wells may fall
within any range bounded by any of these values (e.g. from about 12
to about 1,400).
[0072] The methods and systems disclosed herein also comprise
specifying the surface area of the discrete regions or wells into
which the substrate surface is divided, irrespective of how
separation is maintained between discrete regions or wells. Having
discrete regions or wells of larger area may facilitate
ease-of-access and manipulation of the associated biological
entities in some cases, whereas having discrete regions or wells of
smaller area may be advantageous in terms of reducing assay reagent
volume requirements and increasing the sample analysis throughput
of the method or system. In one aspect of the present disclosure,
the surface area of the discrete regions or wells is between 1
mm.sup.2 and 100 mm.sup.2. In other aspects, the area of the
discrete regions or wells is at least 1 mm.sup.2, at least 2.5
mm.sup.2, at least 5 mm.sup.2, at least 10 mm.sup.2, at least 20
mm.sup.2, at least 30 mm.sup.2, at least 40 mm.sup.2, at least 50
mm.sup.2, at least 75 mm.sup.2, or at least 100 mm.sup.2. In yet
other aspects of the disclosed methods and systems, the area of the
discrete regions or wells is at most 100 mm.sup.2, at most 75
mm.sup.2, at most 50 mm.sup.2, at most 40 mm.sup.2, at most 30
mm.sup.2, at most 20 mm.sup.2, at most 10 mm.sup.2, at most 5
mm.sup.2, at most 2.5 mm.sup.2, or at most 1 mm.sup.2. In a
preferred aspect, the area of discrete regions or wells is about 35
mm.sup.2. In another preferred aspect, the area of the discrete
regions or wells is about 8.6 mm.sup.2. Those of skill in the art
will appreciate that the area of the discrete regions or wells may
fall within any range bounded by any of these values (e.g. from
about 2 mm.sup.2 to about 95 mm.sup.2).
[0073] Discrete regions of the substrate surface are sequentially
exposed to (illuminated with) excitation light by re-positioning
the substrate relative to the excitation light source. Total
internal reflection of the incident excitation light creates an
"evanescent wave" at the sample interface, which excites the
nonlinear-active label and results in generation of second harmonic
light (or in some aspects, sum frequency or difference frequency
light). Because the intensity of the evanescent wave, and hence the
intensity of the nonlinear optical signals generated, is dependent
on the incident angle of the excitation light beam, precise
orientation of the substrate plane with respect to the optical axis
of the excitation beam and efficient optical coupling of the beam
to the substrate is critical for achieving optimal SHG signal
across the array of discrete regions. In some aspects of the
present disclosure, total internal reflection is achieved by means
of a single reflection of the excitation light from the substrate
surface. In other aspects, the substrate may be configured as a
waveguide such that the excitation light undergoes multiple total
internal reflections as it propagates along the waveguide. In yet
other aspects, the substrate may be configured as a zero-mode
waveguide, wherein an evanescent field is created by means of
nanofabricated structures.
[0074] Efficient optical coupling between the excitation light beam
and the substrate in an optical setup such as the one illustrated
in FIGS. 5 and 7 would typically be achieved by use of an
index-matching fluid such as mineral oil, mixtures of mineral oil
and hydrogenated terphenyls, perfluorocarbon fluids, glycerin,
glycerol, or similar fluids having a refractive index near 1.5,
wherein the index-matching fluid is wicked between the prism and
the lower surface of the substrate. Since a static, bubble-free
film of index-matching fluid is likely to be disrupted during fast
re-positioning of the substrate, the systems and methods disclosed
herein include alternative approaches for creating efficient
optical coupling of the excitation beam to the substrate in high
throughput systems.
[0075] FIG. 8 illustrates one aspect of a high throughput system of
the present disclosure in which a continuously recirculating stream
of index-matching fluid is used to maintain efficient optical
coupling between the prism, which is mounted as part of the optical
system, and the substrate, which is configured in a microwell plate
format (e.g. a glass bottom microplate format) and is free to
translate relative to the prism. The continuous flow of
index-matching fluid in this case ensures that the thin film of
fluid between the prism and substrate is never disrupted as the two
components move relative to each other, i.e. any small bubbles or
discontinuities in the thin layer of fluid will be eliminated or
pushed out from the gap between the prism and substrate by means of
the fluid flow. Index-matching fluid is introduced into the gap via
the two fluid channels indicated in the drawing, and may be
collected in a suitable reservoir or sump, from which it may be
recirculated using a small pump. In an alternative implementation
of the same concept, instead of the line-contact between substrate
and prism indicated in FIG. 8, point contact between a single
discrete region and a cylindrical total internal reflection (TIR)
probe would be utilized, where the index-matching fluid would flow
up through a center fluid channel, and then down over the sides of
the cylindrical TIR probe to be collected in a suitable reservoir
or sump.
[0076] FIG. 9 illustrates another aspect of a high throughput
system of the present disclosure, in which a thin layer of
index-matching elastomeric material is used in place of
index-matching fluid to maintain efficient optical coupling between
the prism and substrate. In this case, the substrate is again
packaged in a microwell plate format (e.g. a glass bottom
microplate format), but with a thin layer of an index-matching
elastomeric material attached to or adjacent to the lower surface
of the substrate, such that when placed in contact with the upper
surface of the prism, the elastomer fills the gap between prism and
substrate and provides for efficient optical coupling. Examples of
elastomeric materials that may be used include, but are not limited
to silicones having a refractive index of about 1.4. In one aspect
of the present disclosure, the refractive index of the elastomeric
material is between about 1.35 and about 1.6. In other aspects, the
index of refraction is about 1.6 or less, about 1.55 or less, about
1.5 or less, about 1.45 or less, about 1.4 or less, or about 1.35
or less. In yet other aspects, the index of refraction is at least
about 1.35, at least about 1.4, at least about 1.45, at least about
1.5, at least about 1.55, or at least about 1.6. Those of skill in
the art will appreciate that the index of refraction of the
elastomeric layer may fall within any range bounded by any of these
values (e.g. from about 1.4 to about 1.6). In one aspect of this
approach, the thickness of the layer of elastomeric material is
between about 0.1 mm and 2 mm. In other aspects, the thickness of
the elastomeric layer is at least 0.1 mm, at least 0.2 mm, at least
0.4 mm, at least 0.6 mm, at least 0.8 mm, at least 1.0 mm, at least
1.2 mm, at least 1.4 mm, at least 1.6 mm, at least 1.8 mm, or at
least 2.0 mm. In another aspect of this approach, the thickness of
the elastomeric layer is at most 2.0 mm, at most 1.8 mm, at most
1.6 mm, at most 1.4 mm, at most 1.2 mm, at most 1.0 mm, at most 0.8
mm, at most 0.6 mm, at most 0.4 mm, at most 0.2 mm, or at most 0.1
mm. Those of skill in the art will appreciate that the thickness of
the elastomeric layer my fall within any range bounded by any of
these values (e.g. from about 0.1 mm to about 1.5 mm). In another
aspect of this approach, the upper surface of the prism has a
partially-cylindrical ridge or is domed (FIG. 9) to focus the
compression force and provide better contact between substrate,
elastomeric layer, and prism surface. This approach may also
require the use of a third axis of translation for positioning of
the substrate, i.e. between excitation and detection steps, the
substrate (microwell plate) would be raised slightly to eliminate
contact between the elastomeric layer and the prism prior to
re-positioning the substrate to the location of the next discrete
region to be analyzed.
[0077] FIGS. 10A-D illustrate a preferred aspect of a high
throughput system of the present disclosure, in which an array of
prisms or gratings is integrated with the lower surface of the
substrate (packaged in a microwell plate format) and used to
replace the fixed prism, thereby eliminating the need for
index-matching fluids or elastomeric layers entirely. The array of
prisms (or gratings) is aligned with the array of discrete regions
or wells on the upper surface of the substrate in such a way that
incident excitation light is directed by an "entrance prism"
("entrance grating") to a discrete region or well that is adjacent
to but not directly above the entrance prism (entrance grating), at
an angle of incidence that enables total internal reflection of the
excitation light beam from the sample interface (see FIG. 11), and
such that the reflected excitation beam, and nonlinear-optical
signals generated at the illuminated discrete region, are collected
by an "exit prism" ("exit grating") that is again offset from
(adjacent to but not directly underneath) the discrete region under
interrogation, and wherein the entrance prism and exit prism
(entrance grating and exit grating) for each discrete region are
different, non-unique elements of the array.
[0078] In general, for an array of discrete regions comprising M
rows.times.N columns of individual features, the corresponding
prism or grating array will have M+2 rows.times.N columns or N+2
columns.times.M rows of individual prisms or gratings. In some
embodiments, M may have a value of at least 2, at least 4, at least
6, at least 8, at least 12, at least 14, at least 16, at least 18,
at least 20, at least 25, at least 30, at least 35, at least 40, at
least 45, or at least 50 rows. In some embodiments, M may have a
value of at most 50, at most 45, at most 40, at most 35, at most
30, at most 25, at most 20, at most 18, at most 16, at most 14, at
most 12, at most 10, at most 8, at most 6, at most 4, or at most 2
rows. Similarly, in some embodiments, N may have a value of at
least 2, at least 4, at least 6, at least 8, at least 12, at least
14, at least 16, at least 18, at least 20, at least 25, at least
30, at least 35, at least 40, at least 45, or at least 50 columns.
In some embodiments, N may have a value of at most 50, at most 45,
at most 40, at most 35, at most 30, at most 25, at most 20, at most
18, at most 16, at most 14, at most 12, at most 10, at most 8, at
most 6, at most 4, or at most 2 columns. As will be apparent to
those of skill in the art, M and N may have the same value or
different values, and may have any value within the range specified
above, for example, M=15 and N=45.
[0079] The geometry and dimensions of the individual prisms or
gratings, including the thickness of the prism or grating array
layer, are optimized to ensure that incident light undergoes total
internal reflection at the selected discrete region of the
substrate, and nonlinear optical signals generated at the selected
discrete region are collected, with high optical coupling
efficiency, independently of the position of substrate (microwell
plate) relative to the excitation light beam. FIG. 12 shows a
photograph of a prism array prototype. The prism or grating arrays
may be fabricated by a variety of techniques known to those of
skill in the art, for example, in a preferred aspect, they may be
injection molded from smooth flowing, low birefringence materials
such as cyclic olefin copolymer (COC) or cyclic olefin polymer
(COP), acrylic, polyester, or similar polymers. In some aspects,
the prism or grating array may be fabricated as a separate
component, and subsequently integrated with the lower surface of
the substrate. In other aspects, the prism or grating array may be
fabricated as an integral feature of substrate itself.
Immobilization Chemistries
[0080] As disclosed herein, substrates in any of the formats
described above are further configured for immobilization of
biological entities within the specified discrete regions.
Immobilization of biological molecules or cells may be accomplished
by a variety of techniques known to those of skill in the art, for
example, through the use of aminopropyl silane chemistries to
functionalize glass or fused-silica surfaces with amine functional
groups, followed by covalent coupling using amine-reactive
conjugation chemistries, either directly with the biological
molecule of interest, or via an intermediate spacer or linker
molecule. Non-specific adsorption may also be used directly or
indirectly, e.g. through the use of BSA-NHS
(BSA-N-hydroxysuccinimide) by first attaching a molecular layer of
BSA to the surface and then activating it with N,N'-disuccinimidyl
carbonate. The activated lysine, aspartate or glutamate residues on
the BSA react with surface amines on proteins.
[0081] In a preferred aspect of the present disclosure, biological
molecules may be immobilized on the surface by means of tethering
to or embedding in "supported lipid bilayers", the latter
comprising small patches of lipid bilayer confined to a silicon or
glass surface by means of hydrophobic and electrostatic
interactions, where the bilayer is "floating" above the substrate
surface on a thin layer of aqueous buffer. Supported phospholipid
bilayers can also be prepared with or without membrane proteins or
other membrane-associated components as described, for example, in
Salafsky et al., "Architecture and Function of Membrane Proteins in
Planar Supported Bilayers: A Study with Photosynthetic Reaction
Centers", Biochemistry 35 (47): 14773-14781 (1996); Gennis, R.,
Biomembranes, Springer-Verlag, 1989; Kalb et al., "Formation of
Supported Planar Bilayers by Fusion of Vesicles to Supported
Phospholipid Monolayers", Biochimica Biophysica Acta. 1103:307-316
(1992); and Brian et al. "Allogeneic Stimulation of Cytotoxic
T-cells by Supported Planar Membranes", PNAS-Biological Sciences
81(19): 6159-6163 (1984), relevant portions of which are
incorporated herein by reference. Supported phospholipid bilayers
are well known in the art and there are numerous techniques
available for their fabrication. Supported bilayers should
typically be submerged in aqueous solution to prevent their
destruction when exposed to air.
Collection Optics and Detector
[0082] FIG. 5 further illustrates the collection optics and
detector used to detect nonlinear optical signals generated upon
sequential illumination of the discrete regions of the substrate.
Because surface-selective nonlinear optical techniques are coherent
techniques, meaning that the fundamental and nonlinear optical
light beams have wave fronts that propagate through space with
well-defined spatial and phase relationships, minimal collection
optics are required. Emitted nonlinear optical signals are
collected by means of a prism (or the integrated prism or grating
array of the microplate device described above) and directed via a
dichroic reflector and mirror to the detector. Additional optical
components, e.g. lenses, optical bandpass filters, mirrors, etc.
are optionally used to further shape, steer, and/or filter the beam
prior to reaching the detector. A variety of different
photodetectors may be used, including but not limited to
photodiodes, avalanche photodiodes, photomultipliers, CMOS sensors,
or CCD devices.
X-Y Translation Stage
[0083] As illustrated in FIG. 4, implementation of the high
throughput systems disclosed herein ideally utilizes a high
precision X-Y (or in some cases, an X-Y-Z) translation stage for
re-positioning the substrate (in any of the formats described
above) in relation to the excitation light beam. Suitable
translation stages are commercially available from a number of
vendors, for example, Parker Hannifin. Precision translation stage
systems typically comprise a combination of several components
including, but not limited to, linear actuators, optical encoders,
servo and/or stepper motors, and motor controllers or drive units.
High precision and repeatability of stage movement is required for
the systems and methods disclosed herein in order to ensure
accurate measurements of nonlinear optical signals when
interspersing repeated steps of optical detection and/or
liquid-dispensing. Also, as the size of the focal spot for the
excitation light [20-200 microns in diameter or on a side is
substantially smaller than the size of the discrete regions on the
substrate, in some aspects of the present disclosure, it may also
be desirable to return to a slightly different position within a
given discrete region when making replicate measurements, or to
slowly scan the excitation beam across a portion of the discrete
region over the course of a single measurement, thereby eliminating
potential concerns regarding the photo-bleaching effects of long
exposures or prior exposures.
[0084] Consequently, the methods and systems disclosed herein
further comprise specifying the precision with which the
translation stage is capable of positioning a substrate in relation
to the excitation light beam. In one aspect of the present
disclosure, the precision of the translation stage is between about
1 um and about 10 um. In other aspects, the precision of the
translation stage is about 10 um or less, about 9 um or less, about
8 um or less, about 7 um or less, about 6 um or less, about 5 um or
less, about 4 um or less, about 3 um or less, about 2 um or less,
or about 1 um or less. Those of skill in the art will appreciate
that the precision of the translation stage may fall within any
range bounded by any of these values (e.g. from about 1.5 um to
about 7.5 um).
Fluid Dispensing System
[0085] As illustrated in FIG. 4, some embodiments of the high
throughput systems disclosed herein further comprise an automated,
programmable fluid-dispensing (or liquid-dispensing) system for use
in contacting the biological or target entities immobilized on the
substrate surface with test entities (or test compounds), the
latter typically being dispensed in solutions comprising aqueous
buffers with or without the addition of a small organic solvent
component, e.g. dimethylsulfoxide (DMSO). Suitable automated,
programmable fluid-dispensing systems are commercially available
from a number of vendors, e.g. Beckman Coulter, Perkin Elmer,
Tecan, Velocity 11, and many others. In a preferred aspect of the
systems and methods disclosed herein, the fluid-dispensing system
further comprises a multichannel dispense head, e.g. a 4 channel, 8
channel, 16 channel, 96 channel, or 384 channel dispense head, for
simultaneous delivery of programmable volumes of liquid (e.g.
ranging from about 1 microliter to several milliliters) to multiple
wells or locations on the substrate.
Plate-Handling Robotics
[0086] In other aspects of the high throughput systems disclosed
herein, the system further comprises a microplate-handling (or
plate-handling) robotic system (FIG. 4) for automated replacement
and positioning of substrates (in any of the formats described
above) in relation to the optical excitation and detection optics,
or for optionally moving substrates between the optical instrument
and the fluid-dispensing system. Suitable automated, programmable
microplate-handling robotic systems are commercially available from
a number of vendors, including Beckman Coulter, Perkin Elemer,
Tecan, Velocity 11, and many others. In a preferred aspect of the
systems and methods disclosed herein, the automated
microplate-handling robotic system is configured to move
collections of microwell plates comprising immobilized biological
entities and/or aliquots of test compounds to and from refrigerated
storage units.
Processor/Controller and Constraint-Based Scheduling Algorithm
[0087] In another aspect of the present disclosure, the high
throughput systems disclosed further comprise a processor (or
controller, or computer system) (FIG. 4) configured to run system
software which controls the various subsystems described
(excitation and detection optical systems, X-Y (or X-Y-Z)
translation stage, fluid-dispensing system, and plate-handling
robotics) and synchronizes the different operational steps involved
in performing high throughput conformational analysis. In addition
to handling the data acquisition process, i.e. collection of output
electronic signals from the detector that correspond to the
nonlinear optical signals associated with conformational change,
the processor or controller is also typically configured to store
the data, perform data processing and display functions (including
determination of whether or not changes in orientation or
conformation have occurred for the biological entities, or
combinations of biological and test entities, that have been
tested), and operate a graphical user interface for interactive
control by an operator. The processor or controller may also be
networked with other processors, or connected to the internet for
communication with other instruments and computers at remote
locations.
[0088] Typical input parameters for the processor/controller may
include set-up parameters such as the total number of microwell
plates to be analyzed; the number of wells per plate; the number of
times excitation and detection steps are to be performed for each
discrete region of the substrate or well of the microplate (e.g. to
specify endpoint assay or kinetic assay modes); the total
timecourse over which kinetic data should be collected for each
discrete region or well; the order, timing, and volume of test
compound solutions to be delivered to each discrete region or well;
the dwell time for collection and integration of nonlinear optical
signals; the name(s) of output data files; and any of a number of
system set-up and control parameters known to those skilled in the
art.
[0089] In a preferred aspect of the present disclosure, the
processor or controller is further configured to perform system
throughput optimization by means of executing a constraint-based
scheduling algorithm. This algorithm utilizes system set-up
parameters as described above to determine an optimal sequence of
interspersed excitation/detection and liquid-dispensing steps for
discrete regions or wells that may or may not be adjacent to each
other, such that the overall throughput of the system, in terms of
number of biological entities and/or test entities analyzed per
hour, is maximized. Optimization of system operational steps is an
important aspect of achieving high throughput analysis. In some
aspects of the disclosed methods and systems, the average
throughput of the analysis system may range from about 10 test
entities tested per hour to about 1,000 test entities tested per
hour. In some aspects, the average throughput of the analysis
system may be at least 10 test entities tested per hour, at least
25 test entities tested per hour, at least 50 test entities tested
per hour, at least 75 test entities tested per hour, at least 100
test entities tested per hour, at least 200 test entities tested
per hour, at least 400 test entities tested per hour, at least 600
test entities tested per hour, at least 800 test entities tested
per hour, or at least 1,000 test entities tested per hour. In other
aspects, the average throughput of the analysis system may be at
most 1,000 test entities tested per hour, at most 800 test entities
tested per hour, at most 600 test entities tested per hour, at most
400 test entities tested per hour, at most 200 test entities tested
per hour, at most 100 test entities tested per hour, at most 75
test entities tested per hour, at most 50 test entities tested per
hour, at most 25 test entities tested per hour, or at most 10 test
entities tested per hour.
Computer Systems and Networks
[0090] In various embodiments, the methods and systems of the
invention may further comprise software programs installed on
computer systems and use thereof. Accordingly, as noted above,
computerized control of the various subsystems and synchronization
of the different operational steps involved in performing high
throughput conformational analysis, including data analysis and
display, are within the bounds of the invention.
[0091] The computer system 500 illustrated in FIG. 21 may be
understood as a logical apparatus that can read instructions from
media 511 and/or a network port 505, which can optionally be
connected to server 509 having fixed media 512. The system, such as
shown in FIG. 21 can include a CPU 501, disk drives 503, optional
input devices such as keyboard 515 and/or mouse 516 and optional
monitor 507. Data communication can be achieved through the
indicated communication medium to a server at a local or a remote
location. The communication medium can include any means of
transmitting and/or receiving data. For example, the communication
medium can be a network connection, a wireless connection or an
internet connection. Such a connection can provide for
communication over the World Wide Web. It is envisioned that data
relating to the present disclosure can be transmitted over such
networks or connections for reception and/or review by a party 522
as illustrated in FIG. 21.
[0092] FIG. 22 is a block diagram illustrating a first example
architecture of a computer system 100 that can be used in
connection with example embodiments of the present invention. As
depicted in FIG. 22, the example computer system can include a
processor 102 for processing instructions. Non-limiting examples of
processors include: the Intel Xeon.TM. processor, the AMD
Opteron.TM. processor, the Samsung 32-bit RISC ARM 1176JZ(F)-S
v1.0.TM. processor, the ARM Cortex-A8 Samsung S5PC100.TM.
processor, the ARM Cortex-A8 Apple A4.TM. processor, the Marvell
PXA 930.TM. processor, or a functionally-equivalent processor.
Multiple threads of execution can be used for parallel processing.
In some embodiments, multiple processors or processors with
multiple cores can also be used, whether in a single computer
system, in a cluster, or distributed across systems over a network
comprising a plurality of computers, cell phones, and/or personal
data assistant devices.
[0093] As illustrated in FIG. 22, a high speed cache 104 can be
connected to, or incorporated in, the processor 102 to provide a
high speed memory for instructions or data that have been recently,
or are frequently, used by processor 102. The processor 102 is
connected to a north bridge 106 by a processor bus 108. The north
bridge 106 is connected to random access memory (RAM) 110 by a
memory bus 112 and manages access to the RAM 110 by the processor
102. The north bridge 106 is also connected to a south bridge 114
by a chipset bus 116. The south bridge 114 is, in turn, connected
to a peripheral bus 118. The peripheral bus can be, for example,
PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge
and south bridge are often referred to as a processor chipset and
manage data transfer between the processor, RAM, and peripheral
components on the peripheral bus 118. In some alternative
architectures, the functionality of the north bridge can be
incorporated into the processor instead of using a separate north
bridge chip.
[0094] In some embodiments, system 100 can include an accelerator
card 122 attached to the peripheral bus 118. The accelerator can
include field programmable gate arrays (FPGAs) or other hardware
for accelerating certain processing. For example, an accelerator
can be used for adaptive data restructuring or to evaluate
algebraic expressions used in extended set processing.
[0095] Software and data are stored in external storage 124 and can
be loaded into RAM 110 and/or cache 104 for use by the processor.
The system 100 includes an operating system for managing system
resources; non-limiting examples of operating systems include:
Linux, Windows, MacOS.TM., BlackBerry OS.TM., iOS.TM., and other
functionally-equivalent operating systems, as well as application
software running on top of the operating system for managing data
storage and optimization in accordance with example embodiments of
the present invention.
[0096] In this example, system 100 also includes network interface
cards (NICs) 120 and 121 connected to the peripheral bus for
providing network interfaces to external storage, such as Network
Attached Storage (NAS) and other computer systems that can be used
for distributed parallel processing.
[0097] FIG. 23 is a diagram showing a network 200 with a plurality
of computer systems 202a, and 202b, a plurality of cell phones and
personal data assistants 202c, and Network Attached Storage (NAS)
204a, and 204b. In example embodiments, systems 202a, 202b, and
202c can manage data storage and optimize data access for data
stored in Network Attached Storage (NAS) 204a and 204b. A
mathematical model can be used for the data and be evaluated using
distributed parallel processing across computer systems 202a, and
202b, and cell phone and personal data assistant systems 202c.
Computer systems 202a, and 202b, and cell phone and personal data
assistant systems 202c can also provide parallel processing for
adaptive data restructuring of the data stored in Network Attached
Storage (NAS) 204a and 204b. FIG. 23 illustrates an example only,
and a wide variety of other computer architectures and systems can
be used in conjunction with the various embodiments of the present
invention. For example, a blade server can be used to provide
parallel processing. Processor blades can be connected through a
back plane to provide parallel processing. Storage can also be
connected to the back plane or as Network Attached Storage (NAS)
through a separate network interface.
[0098] In some example embodiments, processors can maintain
separate memory spaces and transmit data through network
interfaces, back plane or other connectors for parallel processing
by other processors. In other embodiments, some or all of the
processors can use a shared virtual address memory space.
[0099] FIG. 24 is a block diagram of a multiprocessor computer
system 300 using a shared virtual address memory space in
accordance with an example embodiment. The system includes a
plurality of processors 302a-f that can access a shared memory
subsystem 304. The system incorporates a plurality of programmable
hardware memory algorithm processors (MAPs) 306a-f in the memory
subsystem 304. Each MAP 306a-f can comprise a memory 308a-f and one
or more field programmable gate arrays (FPGAs) 310a-f. The MAP
provides a configurable functional unit and particular algorithms
or portions of algorithms can be provided to the FPGAs 310a-f for
processing in close coordination with a respective processor. For
example, the MAPs can be used to evaluate algebraic expressions
regarding the data model and to perform adaptive data restructuring
in example embodiments. In this example, each MAP is globally
accessible by all of the processors for these purposes. In one
configuration, each MAP can use Direct Memory Access (DMA) to
access an associated memory 308a-f, allowing it to execute tasks
independently of, and asynchronously from, the respective
microprocessor 302a-f. In this configuration, a MAP can feed
results directly to another MAP for pipelining and parallel
execution of algorithms.
[0100] The above computer architectures and systems are examples
only, and a wide variety of other computer, cell phone, and
personal data assistant architectures and systems can be used in
connection with example embodiments, including systems using any
combination of general processors, co-processors, FPGAs and other
programmable logic devices, system on chips (SOCs), application
specific integrated circuits (ASICs), and other processing and
logic elements. In some embodiments, all or part of the computer
system can be implemented in software or hardware. Any variety of
data storage media can be used in connection with example
embodiments, including random access memory, hard drives, flash
memory, tape drives, disk arrays, Network Attached Storage (NAS)
and other local or distributed data storage devices and
systems.
[0101] In example embodiments, the computer system can be
implemented using software modules executing on any of the above or
other computer architectures and systems. In other embodiments, the
functions of the system can be implemented partially or completely
in firmware, programmable logic devices such as field programmable
gate arrays (FPGAs) as referenced in FIG. 24, system on chips
(SOCs), application specific integrated circuits (ASICs), or other
processing and logic elements. For example, the Set Processor and
Optimizer can be implemented with hardware acceleration through the
use of a hardware accelerator card, such as accelerator card 122
illustrated in FIG. 22.
EXAMPLE 1
Illustrative
[0102] G-protein coupled receptors (e.g. serotonin, dopamine,
glutamate, chemokine, and histamine receptors) are one class of
proteins that undergo a conformational change when activated by a
ligand, and are thus amenable to study using the present invention.
GPCRs that are not intrinsically nonlinear-active may be labeled
using a nonlinear active label, and the conformation change is
detected via a change in the orientation of the nonlinear active
label. One or more labeled GPCR proteins can be attached to a
surface, for example by embedding the protein molecules in an array
of supported lipid bilayer structures on a glass substrate, where
each bilayer structure in the array contains a single species of
GPCR molecule. In a preferred embodiment of the present invention,
each supported lipid bilayer structure is confined within an
individual well of a microplate. The conformation change that
results when binding of a ligand activates a GPCR receptor causes a
change in the orientation of the label with respect to the optical
interface on which the molecules are immobilized, and thus a change
in properties of the nonlinear optical beams (e.g., second harmonic
light) such as intensity, wavelength or polarization.
[0103] In a screening experiment, a microwell plate containing the
immobilized GPCRs is positioned on the translation stage mechanism
of the high throughput analysis system and moved into position for
measuring a signal in a first well. A background signal can be
measured for the one or more GPCR samples prior to exposure of the
immobilized GPCR molecules to a test compound by measuring the
nonlinear optical signal from the first well, repositioning the
microwell plate to a second well to repeat the background
measurement, and so forth. Repeat measurements of nonlinear optical
signals are then made for each well at one (endpoint assay mode) or
more (kinetic measurement mode) defined time points following the
addition of the test compound, and analyzed to determine if the
test compound induced conformational change in the one or more GPCR
species.
[0104] In some cases, binding of a test compound to a GPCR molecule
may lead to a change in measured nonlinear optical properties even
though the GPCR is not activated by the test compound. For example,
this can be due to an interaction between the test compound and the
GPCR molecule in the bound complex which alters the orientation of
the attached label with respect to the receptor molecule, rather
than a change in the conformation of the receptor molecule. A
control can be performed, if desired, to assign measured changes in
nonlinear optical properties to binding or activation of the
receptor, for example, by using a compound which is known to bind
to a given GPCR receptor but not to produce a conformational
change. If necessary, the position of the label on the GPCR can be
altered by changing the conjugation chemistry of the label and/or
genetically modifying the receptor to introduce new labeling sites,
in order to ensure that observed changes in nonlinear optical
signal correlate to receptor activation or conformational
change.
[0105] In the example described above, each of the GPCR molecules
immobilized in the array of lipid bilayer structures on the glass
substrate (and further separated by means of the wells in which
they reside) are subjected to measurement and analysis by means of
repositioning the glass substrate (microplate) with respect to the
excitation light beam through the use of the precision X-Y
translation stage, while maintaining efficient optical coupling via
the recirculating index-matching fluid, index-matching elastomer,
or prism grating designs disclosed above.
[0106] Typically, the dispensing of test compound solutions into
the wells of the microplate will be performed by a programmable,
automated liquid-dispensing unit that is integrated with the
optical instrument system. Each of the GPCR species immobilized in
the wells of the microplate may be exposed to the same test
compound, or each may be exposed to a different test compound. In
some aspects of the present invention, the high throughput analysis
system will further comprise robotics for moving microwell plates
comprising the GPCR samples to be analyzed from an external storage
system into a "home" position for the translation stage, or for
replacing GPCR-containing microplates for which analysis has been
completed with new sample plates. In other aspects of the present
invention, the high throughput analysis system will further
comprise additional robotics for moving standard microplates
containing the collection of test compounds into and out of a
"home" position for the liquid-dispensing unit.
[0107] In a preferred aspect of the present invention, a computer
system (i.e. a processor or controller) is configured to run
software for (i) controlling all programmable components of the
high throughput analysis system, (ii) synchronizing the operational
steps of moving microwell plates into and out of position, taking
background optical measurements, dispensing test compound
solutions, and repeating the optical measurements for use in
end-point or kinetic mode assays, (iii) storing and processing the
nonlinear optical signal data received from the detector, and (iv)
optimizing the overall throughput of analysis (e.g. in terms of
number of GPCR sample/test compound combinations analyzed per hour)
by using the input system setup parameters (e.g. number of
microwell plates to be analysed, number of wells per plate, number
of test compounds to be tested, endpoint versus kinetic measurement
mode, etc.) to calculate an optimal order for interspersing
background measurement, test compound dispensing, and repeat
measurement steps for the different wells on each microplate.
EXAMPLE 2
Mold Design & Process for Fabricating Prism Arrays
[0108] A mold was created to fabricate the original "skip-prism"
design shown in FIGS. 13A-13C, with the injection molding process
performed by a specialty optical molding house (Apollo Optical,
Rochester, N.Y.). The resulting parts show stress birefringence
(see the crossed-polarizer image in FIG. 14), and the measured SHG
signal is adversely affected by this birefringence (FIG. 15), with
the signal dropping along the length of the prism array.
[0109] In order to reduce or eliminate the birefringence effect on
SHG signal, we experimented with the type of plastic used (COP,
COC, acrylic, etc), different molding processing conditions
(temperature, pressure, flow rate, cycle time, etc), and
post-molding annealing. We were able to find materials and process
parameters that reduced the birefringence modestly, but those
conditions also tended to cause the plastic part to adhere tightly
to the mold and fracture upon release from the mold. (Note: this
mold contained ejection devices that pushed only on the vent and
gate ends of the part). Annealing the part reduced stress
birefringence significantly, but caused excessive mechanical
warping of the part thereby making it unusable.
[0110] Injection molding flow simulations indicated that stress was
created primarily at the transition from the planar gate to the
prism-structured part (results not shown). The simulation results
also predicted that the part could be made longer than required and
then trimmed to length, thereby mechanically removing the stressed
regions of the molded part and resulting in a prism array of the
same final length as in the original design.
[0111] The results of the mold flow simulation were used to design
a new mold that includes several significant changes: [0112] 1. The
overall length of the part has been increased to allow birefringent
transition zones to be trimmed off. [0113] 2. Gate and vent
features were made longer to reduce shear stress in the plastic.
[0114] 3. Mold eject features were added along the length and width
of the part to facilitate release from the mold and reduce the
chance of fracturing the part during release. [0115] 4. "Glue
bumps" were added to the planar side of the array to control the
thickness of the glue layer when the prism array is laminated to a
glass-bottom microplate, and to improve glue adhesion during
temperature variation.
[0116] The new part design is shown in FIGS. 16A-16C, including the
gate (left) and vent (right) features. FIG. 19 shows a cut-away
version of the mold tool. Note that there is a 6.times.3 array of
"ejector blade" devices (i.e. "ejection devices") that are used
during mold release to apply more uniform pressure to the part
during release (FIG. 20). Additional ejection features (not shown)
are also used in the gate and vent regions. Generally,
optical-quality molded parts do not use ejector features at all
since ejectors impact on the surface of the part and can create
blemishes. Since our prism array design has some regions that are
not optically addressed, we were able to arrange the ejector blades
to impact the part only in those regions where optical performance
of the part is non-critical.
[0117] In general, the larger the number of blade-like ejector
features in an m.times.n array of ejection devices, the more
uniform the pressure exerted on the part during mold release. In
some embodiments, m and/or n will have a value of at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 11, at least 12, at least 13,
at least 14, at least 15, at least 16, at least 17, at least 18, at
least 19, or at least 20. In some embodiments, m and/or n will have
a value of at most 20, at most 19, at most 18, at most 17, at most
16, at most 15, at most 14, at most 13, at most 12, at most 11, at
most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at
most 4, at most 3, or at most 2. The values of m and n may the same
or may be different, and may include any combination of values
within the above-specified range.
[0118] The new mold design, specifically the ejection features,
allowed the injection molding vendor to experiment with different
pressure and temperature profiles which would have caused
release-fracture in the original mold. These conditions effectively
de-stress the plastic during the molding process, but cause the
part to adhere more tightly to the optical mold surface. The
ejector features distributed across the length and width of the
part allow the part to be ejected from the mold without fracturing.
Prism parts made with new mold have negligible stress
birefringence, as shown in the cross-polarizer image in FIG. 17.
SHG data also shows a much more uniform signal response along the
length of the prism array (FIG. 18). Residual birefringence is
still detectable, but the extra length of the part allows us to
optimize the trim locations to cut off the transition regions where
there is the most birefringence (data not shown).
[0119] 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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