U.S. patent application number 10/681770 was filed with the patent office on 2004-04-15 for method of analyzing multiple samples simultaneously by detecting absorption and systems for use in such a method.
This patent application is currently assigned to Iowa State University Research Foundation, Inc.. Invention is credited to Gong, Xiaoyi, Yeung, Edward S..
Application Number | 20040070763 10/681770 |
Document ID | / |
Family ID | 32072673 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040070763 |
Kind Code |
A1 |
Yeung, Edward S. ; et
al. |
April 15, 2004 |
Method of analyzing multiple samples simultaneously by detecting
absorption and systems for use in such a method
Abstract
The present invention provides a method of analyzing multiple
samples simultaneously by absorption detection. The method
comprises: (i) providing a planar array of multiple containers,
each of which contains a sample comprising at least one absorbing
species, (ii) irradiating the planar array of multiple containers
with a light source comprising or consisting essentially of at
least one wavelength of light that is absorbed by one or more of
the absorbing species, the absorption of which is to be detected,
and (iii) detecting absorption of light by one or more of the
absorbing species with a detection means that is in line with the
light source and is positioned in line with and parallel to the
planar array of multiple containers at a distance of at least about
10 times a cross-sectional distance of a container in the planar
array of multiple containers measured orthogonally to the plane of
the planar array of multiple containers. The detection of
absorption of light by a sample in the planar array of multiple
containers indicates the presence of an absorbing species in the
sample. The method can further comprise: (iv) measuring the amount
of absorption of light detected in (iii) for an absorbing species
in a sample. The measurement of the amount of absorption of light
detected in (iii) indicates the amount of the absorbing species in
the sample. Also provided by the present invention is a system for
use in the above method. The system comprises: (i) a light source
comprising or consisting essentially of at least one wavelength of
light that is absorbed by one or more absorbing species, the
absorption of which is to be detected, (ii) a planar array of
multiple containers, into each of which can be placed a sample
comprising at least one absorbing species, and (iii) a detection
means that is in line with the light source and is positioned in
line with and parallel to the planar array of multiple containers
at a distance of at least about 10 times a cross-sectional distance
of a container in the planar array of multiple containers measured
orthogonally to the plane of the planar array of multiple
containers.
Inventors: |
Yeung, Edward S.; (Ames,
IA) ; Gong, Xiaoyi; (Edison, NJ) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
Iowa State University Research
Foundation, Inc.
Ames
IA
|
Family ID: |
32072673 |
Appl. No.: |
10/681770 |
Filed: |
October 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10681770 |
Oct 7, 2003 |
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10070531 |
Jun 5, 2002 |
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10070531 |
Jun 5, 2002 |
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PCT/US00/20447 |
Jul 28, 2000 |
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60153263 |
Sep 9, 1999 |
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Current U.S.
Class: |
356/436 |
Current CPC
Class: |
B01J 2219/00522
20130101; G01N 27/44782 20130101; G01N 21/253 20130101; B01J
2219/00707 20130101; G01N 30/95 20130101; G01N 21/31 20130101; G01N
30/74 20130101; B01J 2219/00659 20130101; G01N 30/74 20130101; B01J
2219/00585 20130101; G01N 27/44721 20130101; G01N 30/466
20130101 |
Class at
Publication: |
356/436 |
International
Class: |
G01N 021/00 |
Goverment Interests
[0002] This invention was made with government support under
Contract No. W-7405-Eng-82 awarded by the U.S. Department of
Energy. Therefore, the Government may have certain rights to this
invention.
Claims
What is claimed is:
1. A parallel capillary electrophoresis system for separating and
analyzing the components of multiple chemical samples, said system
comprising a bundle of capillary tubes arrayed to have at least
portions of the tubes extending generally parallel to one another
in a first plane, each tube being adapted for the flow of a fluid
sample therethrough, a power source for applying a potential
difference between inlet end portions and outlet end portions of
the tubes to cause an electrical current to flow through the
contents of the capillary tubes at a level sufficient to cause
separation in said fluid samples, a light source for emitting light
to pass through said capillary tube portions, a photodetector
comprising a linear array of photodetector elements for receiving
light passing through said capillary tubes, the light passing
through each of said capillary tube portions illuminating several
photodetector elements, each said photodetector element generating
a pixel signal corresponding to the light received by said
photodetector element, an analog to digital converter converting
each of the pixel signals into a digital value corresponding to the
light received by one of the photodetector elements, and a
processor receiving the digital values and generating a plurality
of output signals corresponding thereto, each output signal being a
function of at least two digital values corresponding to the light
received by two photodetector elements, respectively, so that the
output signals correspond to the light passing through the bundle
of capillary tubes; wherein the processor generates output signals
such that each output signal is a function of at least two digital
values corresponding to the light passing substantially
concurrently through two photodetector elements, respectively.
2. The system as set forth in claim 1 wherein the processor selects
one digital value and averages the selected digital value with at
least a second digital value to generate averaged values and
wherein the output signals are a function of the averaged
values.
3. The system as set forth in claim 2 wherein the selected digital
value and the second digital value correspond to pixel signals from
contiguous photodetector elements.
4. The system as set forth in claim 1 wherein each pixel signal is
converted into a sequence of digital values and wherein the
processor provides output signals which are a function of the
sequence of digital values.
5. The system as set forth in claim 1 wherein the at least two
digital values are selected to minimize short time fluctuations or
other noise of the pixel signals to generate an improved signal to
noise ratio of the pixel signals.
6. A parallel capillary electrophoresis system for separating and
analyzing the components of multiple chemical samples, said system
comprising a bundle of capillary tubes arrayed to have at least
portions of the tubes extending generally parallel to one another
in a first plane, each tube being adapted for the flow of a fluid
sample therethrough, a power source for applying a potential
difference between inlet end portions and outlet end portions of
the tubes to cause an electrical current to flow through the
contents of the capillary tubes at a level sufficient to cause
separation in said fluid samples, a light source for emitting light
to pass through said capillary tube portions, a photodetector
comprising a linear array of photodetector elements for receiving
light passing through said capillary tubes, the light passing
through each said capillary tube portions illuminating several
photodetector elements, each said photodetector element generating
a pixel signal corresponding to the light received by said
photodetector element, an analog to digital converter converting
each of the pixel signals into a digital value corresponding to the
light received by one of the photodetector elements, and a
processor receiving the digital values and generating a plurality
of output signals corresponding thereto, each output signal being a
function of at least two digital values corresponding to the light
received by two photodetector elements, respectively, so that the
output signals correspond to the light passing through the bundle
of capillary tubes; wherein the processor selects one peak digital
value and averages the selected digital value with four digital
values which correspond to pixel signals from photodetector
elements adjacent to the photodetector element corresponding to the
selected peak digital value and wherein the output signals are a
function of the averaged values.
7. The system as set forth in claim 6 wherein the selected peak
digital value corresponds to the light passing through one
capillary tube portion.
8. A parallel capillary electrophoresis system for separating and
analyzing the components of multiple chemical samples, said system
comprising a bundle of capillary tubes arrayed to have at least
portions of the tubes extending generally parallel to one another
in a first plane, each tube being adapted for the flow of a fluid
sample therethrough, a power source for applying a potential
difference between inlet end portions and outlet end portions of
the tubes to cause an electrical current to flow through the
contents of the capillary tubes at a level sufficient to cause
separation in said fluid samples, a light source for emitting light
to pass through said capillary tube portions, a photodetector
comprising a linear array of photodetector elements for receiving
light passing through said capillary tubes, the light passing
through each of said capillary tube portions illuminating several
photodetector elements, each of said photodetector elements
generating a pixel signal corresponding to the light received by
said photodetector element, an analog to digital converter
converting each of the pixel signals into a digital value
corresponding to the light received by one of the photodetector
elements, and a processor receiving the digital values and
generating a plurality of output signals corresponding thereto,
each output signal being a function of at least two digital values
corresponding to the light received by two photodetector elements,
respectively, so that the output signals correspond to the light
passing through the bundle of capillary tubes; wherein the
processor selects one peak digital value and averages the selected
digital value with at least a second digital value to generate
averaged values and wherein the output signals are a function of
the averaged values, and further comprising a display receiving the
output signals and generating an electropherogram corresponding
thereto.
9. A parallel capillary electrophoresis system for separating and
analyzing the components of multiple chemical samples, said system
comprising: a bundle of capillary tubes arrayed to have at least
portions of the tubes extending generally parallel to one another
in a first plane, each tube being adapted for the flow of a fluid
sample therethrough; a power source for applying a potential
difference between inlet end portions and outlet end portions of
the tubes to cause an electrical current to flow through the
contents of the capillary tubes at a level sufficient to cause
separation in said fluid samples; a light source for emitting light
to pass through said capillary tube portions; a photodetector
comprising a linear array of photodetector elements for receiving
light passing through said capillary tubes, said linear array being
positioned non-parallel to the first plane, the light passing
through each of said capillary tube portions illuminating several
photodetector elements, each said photodetector element generating
a pixel signal corresponding to the light received by said
photodetector element; an analog to digital converter converting
each of the pixel signals into a digital value corresponding to the
light received by one of the photodetector elements; and a
processor receiving the digital values and generating a plurality
of output signals corresponding thereto, each output signal being a
function of at least two digital values corresponding to the light
received by two photodetector elements, respectively, so that the
output signals correspond to the light passing through the bundle
of capillary tubes; wherein each pixel signal is converted into a
sequence of digital values and the output signals are a function of
an average over time of the sequence of digital values.
10. A parallel capillary electrophoresis system for separating and
analyzing the components of multiple chemical samples, said system
comprising a bundle of capillary tubes arrayed to have at least
portions of the tubes extending generally parallel to one another
in a first plane, each tube being adapted for the flow of a fluid
sample therethrough, a power source for applying a potential
difference between inlet end portions and outlet end portions of
the tubes to cause an electrical current to flow through the
contents of the capillary tubes at a level sufficient to cause
separation in said fluid samples, a light source for emitting light
to pass through said capillary tube portions, a photodetector
comprising a linear array of photodetector elements for receiving
light passing through said capillary tubes, the light passing
through each said capillary tube portions illuminating several
photodetector elements, each said photodetector element generating
a pixel signal corresponding to the light received by said
photodetector element, an analog to digital converter converting
each of the pixel signals into a digital value corresponding to the
light received by one of the photodetector elements, and a
processor receiving the digital values and generating a plurality
of output signals corresponding thereto, each output signal being a
function of at least two digital values corresponding to the light
received by two photodetector elements, respectively, so that the
output signals correspond to the light passing through the bundle
of capillary tubes; wherein the at least two digital values are
selected to minimize long time drifts of the pixel signals to
generate a substantially flat baseline of the pixel signals.
11. A method of processing a plurality of pixel signals, each
generated by one element of an array of photodetector elements
illuminated by light passing through a bundle of capillary tubes
during a multiplexed capillary electrophoresis process, said method
comprising: converting each of the pixel element signals into a
digital value corresponding to the light received by one of the
photodetector elements; selecting, for each capillary tube, at
least two digital values corresponding to the light received by two
photodetector elements; and generating output signals corresponding
to the light passing through the bundle of capillary tubes, each
output signal being a function of the selected digital values,
wherein each output signal is a function of at least two digital
values corresponding to the light passing substantially
concurrently through two photodetector elements, respectively.
12. The method as set forth in claim 11 comprising selecting one
digital value and averaging the selected digital value with at
least a second digital value to generate averaged values and
wherein the output signals are a function of the averaged
values.
13. The method as set forth in claim 12 wherein the selected
digital value and the second digital value correspond to pixel
signals from contiguous photodetector elements.
14. The method as set forth in claim 11 wherein each pixel signal
is converted into a sequence of digital values and wherein the
output signals are a function of the sequence of digital
values.
15. The method as set forth in claim 11 wherein the at least two
digital values are selected to minimize short time fluctuations or
other noise of the pixel signals to generate an improved signal to
noise ratio of the pixel signals.
16. A method of processing a plurality of pixel signals, each
generated by one element of an array of photodetector elements
illuminated by light passing through a bundle of capillary tubes
during a multiplexed capillary electrophoresis process, said method
comprising: converting each of the pixel element signals into a
digital value corresponding to the light received by one of the
photodetector elements; selecting, for each capillary tube, at
least two digital values corresponding to the light received by two
photodetector elements; generating output signals corresponding to
the light passing through the bundle of capillary tubes, each
output signal being a function of the selected digital values; and
selecting one peak digital value and averaging the selected digital
value with four digital values, which correspond to pixel signals
from photodetector elements adjacent to the photodetector element
corresponding to the selected peak digital value and wherein the
output signals are a function of the averaged values.
17. The method as set forth in claim 16 wherein the selected peak
digital value corresponds to the light passing through one
capillary tube portion.
18. A method of processing a plurality of pixel signals, each
generated by one element of an array of photodetector elements
illuminated by light passing through a bundle of capillary tubes
during a multiplexed capillary electrophoresis process, said method
comprising: converting each of the pixel element signals into a
digital value corresponding to the light received by one of the
photodetector elements; selecting, for each capillary tube, at
least two digital values corresponding to the light received by two
photodetector elements; generating output signals corresponding to
the light passing through the bundle of capillary tubes, each
output signal being a function of the selected digital values; and
selecting one peak digital value and averaging the selected digital
value with at least a second digital value to generate averaged
values and wherein the output signals are a function of the
averaged values, and further comprising displaying an
electropherogram corresponding to the output signals.
19. A method of processing a plurality of pixel signals, each
generated by one element of an array of photodetector elements
illuminated by light passing through a bundle of capillary tubes
during a multiplexed capillary electrophoresis process, said bundle
of capillary tubes arrayed to have at least portions of the tubes
extending generally parallel to one another in a first plane, said
method comprising: positioning the array of photodetector elements
non-parallel to the first plane; converting each pixel signal into
a sequence of digital values; a digital value corresponding to the
light received by one of the photodetector elements; selecting, for
each capillary tube, at least two digital values corresponding to
the light received by two photodetector elements; and generating
output signals corresponding to the light passing through the
bundle of capillary tubes, wherein the output signals are a
function of an average over time of the sequence of digital
values.
20. A method of processing a plurality of pixel signals, each
generated by one element of an array of photodetector elements
illuminated by light passing through a bundle of capillary tubes
during a multiplexed capillary electrophoresis process, said method
comprising: converting each of the pixel element signals into a
digital value corresponding to the light received by one of the
photodetector elements; selecting, for each capillary tube, at
least two digital values corresponding to the light received by two
photodetector elements, wherein the at least two digital values are
selected to minimize long time drifts of the pixel signals to
generate a substantially flat baseline of the pixel signals; and
generating output signals corresponding to the light passing
through the bundle of capillary tubes, each output signal being a
function of the selected digital values.
21. A parallel capillary electrophoresis system for separating and
analyzing the components of multiple chemical samples, said system
comprising: a bundle of capillary tubes arrayed to have at least
portions of the tubes extending generally parallel to one another
in a first plane, each tube being adapted for the flow of a fluid
sample therethrough; a power source for applying a potential
difference between inlet end portions and outlet end portions of
the tubes to cause an electrical current to flow through the
contents of the capillary tubes at a level sufficient to cause
separation in said fluid samples; a light source for emitting light
to pass through said capillary tube portions; a photodetector
comprising a linear array of photodetector elements for receiving
light passing through said capillary tubes, said linear array being
positioned non-parallel to the first plane, the light passing
through each of said capillary tube portions illuminating several
photodetector elements, each of said photodetector element
generating a pixel signal corresponding to the light received by
said photodetector element; an analog to digital converter
converting each of the pixel signals into a digital value
corresponding to the light received by one of the photodetector
elements; and a processor receiving the digital values and
generating a plurality of output signals corresponding thereto,
each output signal being a function of at least two digital values
corresponding to the light received by two photodetector elements,
respectively, so that the output signals correspond to the light
passing through the bundle of capillary tubes.
22. A system as set forth in claim 21 wherein the linear array is
positioned generally perpendicular to the first plane.
23. A system as set forth in claim 21 wherein the processor
generates output signals such that each output signal is a function
of at least two digital values corresponding to the light passing
substantially concurrently through two photodetector elements,
respectively.
24. A system for use in analyzing multiple samples simultaneously
by absorption detection, which system comprises: (i) a planar array
of multiple containers, into each of which can be placed a sample,
(ii) a light source for emitting light to pass through the planar
array of multiple containers, (iii) a photodetector, which is in
line with the light source, is positioned in line with and parallel
to the planar array of multiple containers, and comprises a linear
array of photosensitive elements for receiving light passing
through the planar array of multiple containers, wherein, upon
illumination of a photosensitive element by light passing through
the planar array of multiple containers, a pixel signal
corresponding to the light received by the photosensitive element
is generated, (iv) an analog to digital converter, which converts
the pixel signal for each illuminated photosensitive element to a
digital value corresponding to the light received by the respective
photosensitive element, and (v) a processor, which receives the
digital values and generates a plurality of output signals
corresponding thereto, each output signal being a function of at
least two digital values corresponding to the light passing
substantially concurrently through two photosensitive elements.
25. The system of claim 24, wherein the processor selects one peak
digital value and averages the selected digital value with four
digital values, which correspond to pixel signals from
photosensitive elements adjacent to the photosensitive element
corresponding to the selected peak digital value, and wherein the
output signals are a function of the averaged values.
26. The system of claim 24, wherein the processor selects one peak
digital value and averages the selected digital value with at least
a second digital value to generate averaged values and wherein the
output signals are a function of the averaged values, and further
comprising a display receiving the output signals and generating an
electropherogram corresponding thereto.
27. The system of claim 24, wherein each pixel signal is converted
into a sequence of digital values and the output signals are a
function of an average over time of the sequence of digital
values.
28. The system of claim 24, wherein the at least two digital values
are selected to minimize long time drifts of the pixel signals to
generate a substantially flat baseline of the pixel signals.
29. A method of processing a plurality of pixel signals, each
generated by one element of an array of photosensitive elements
illuminated by light passing through a planar array of multiple
containers, said method comprising: converting each of the pixel
element signals into a digital value corresponding to the light
received by one of the photosensitive elements; selecting, for each
container, at least two digital values corresponding to the light
received by two photosensitive elements; and generating output
signals corresponding to the light passing through the planar array
of multiple containers, each output signal being a function of the
selected digital values, wherein each output signal is a function
of at least two digital values corresponding to the light passing
substantially concurrently through two photosensitive elements,
respectively.
30. A method of processing a plurality of pixel signals, each
generated by one element of an array of photosensitive elements
illuminated by light passing through a planar array of multiple
containers, said method comprising: converting each of the pixel
element signals into a digital value corresponding to the light
received by one of the photosensitive elements; selecting, for each
container, at least two digital values corresponding to the light
received by two photosensitive elements; generating output signals
corresponding to the light passing through the planar array of
multiple containers, each output signal being a function of the
selected digital values; and selecting one peak digital value and
averaging the selected digital value with four digital values,
which correspond to pixel signals from photosensitive elements
adjacent to the photosensitive element corresponding to the
selected peak digital value and wherein the output signals are a
function of the averaged values.
31. A method of processing a plurality of pixel signals, each
generated by one element of an array of photosensitive elements
illuminated by light passing through a planar array of multiple
containers, said method comprising: converting each of the pixel
element signals into a digital value corresponding to the light
received by one of the photosensitive elements; selecting, for each
container, at least two digital values corresponding to the light
received by two photosensitive elements; generating output signals
corresponding to the light passing through the planar array of
multiple containers, each output signal being a function of the
selected digital values; and selecting one peak digital value and
averaging the selected digital value with at least a second digital
value to generate averaged values and wherein the output signals
are a function of the averaged values, and further comprising
displaying an electropherogram corresponding to the output
signals.
32. A method of processing a plurality of pixel signals, each
generated by one element of an array of photosensitive elements
illuminated by light passing through a planar array of multiple
containers, said planar array of multiple containers arrayed to
have at least portions of the containers extending generally
parallel to one another in a first plane, said method comprising:
positioning the array of photosensitive elements non-parallel to
the first plane; converting each pixel signal into a sequence of
digital values; a digital value corresponding to the light received
by one of the photosensitive elements; selecting, for each
container, at least two digital values corresponding to the light
received by two photosensitive elements; and generating output
signals corresponding to the light passing through the planar array
of multiple containers, wherein the output signals are a function
of an average over time of the sequence of digital values.
33. A method of processing a plurality of pixel signals, each
generated by one element of an array of photosensitive elements
illuminated by light passing through a planar array of multiple
containers, said method comprising: converting each of the pixel
element signals into a digital value corresponding to the light
received by one of the photosensitive elements; selecting, for each
container, at least two digital values corresponding to the light
received by two photosensitive elements, wherein the at least two
digital values are selected to minimize long time drifts of the
pixel signals to generate a substantially flat baseline of the
pixel signals; and generating output signals corresponding to the
light passing through the planar array of multiple containers, each
output signal being a function of the selected digital values.
34. A system for use in analyzing multiple samples simultaneously
by absorption detection, which system comprises: (i) a planar array
of multiple containers, into each of which can be placed a sample,
(ii) a light source for emitting light to pass through the planar
array of multiple containers, (iii) a photodetector, which is in
line with the light source, is positioned in line with and parallel
to the planar array of multiple containers, and comprises a linear
array of photosensitive elements for receiving light passing
through the planar array of multiple containers, wherein, upon
illumination of a photosensitive element by light passing through
the planar array of multiple containers, a pixel signal
corresponding to the light received by the photosensitive element
is generated, (iv) an analog to digital converter, which converts
the pixel signal for each illuminated photosensitive element to a
digital value corresponding to the light received by the respective
photosensitive element, and (v) a processor, which receives the
digital values and generates a plurality of output signals
corresponding thereto, each output signal being a function of at
least two digital values corresponding to the light received by two
photosensitive elements, respectively, so that the output signals
correspond to the light passing through the planar array of
multiple containers.
Description
CROSS REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This patent application is a continuation of copending
application Ser. No. 10/070,531, filed Jun. 5, 2002, which is the
U.S. national phase of International Patent Application No.
PCT/US00/20447, filed Jul. 28, 2000, which claims the benefit of
U.S. Provisional Patent Application No. 60/153,263, filed Sep. 9,
1999.
FIELD OF THE INVENTION
[0003] This invention relates to a method of analyzing multiple
samples simultaneously by detecting absorption and systems for use
in such a method.
BACKGROUND OF THE INVENTION
[0004] The rapid development of biological and pharmaceutical
technology has posed a challenge for high-throughput analytical
methods. For example, current development of combinatorial
chemistry has made it possible to synthesize hundreds or even
thousands of compounds per day in one batch. Characterization and
analysis of such huge numbers of compounds have become the
bottleneck. Parallel processing (i.e., simultaneous multi-sample
analysis) is a natural way to increase the throughput. However, due
to limitations related to column size, pressure requirements,
detector and stationary phase material, it will be very difficult
to build a highly multiplexed high-performance liquid
chromatography (HPLC) system. The same goes for building a highly
multiplexed gas chromatography (GC) system.
[0005] High performance capillary electrophoresis (CE) has rapidly
become an important analytical tool for the separation of a large
variety of compounds, ranging from small inorganic ions to large
biological molecules. With attractive features such as rapid
analysis time, high separation efficiency, small sample size, and
low solvent consumption, CE is being increasingly used as an
alternative or complementary technique to HPLC. For example, the
use of capillary gel electrophoresis has greatly improved DNA
sequencing rates compared to conventional slab gel electrophoresis.
Part of the improvement in speed, however, has been offset by the
loss of the ability (inherent in slab gels) to accommodate multiple
lanes in a single run. Highly multiplexed capillary
electrophoresis, by making possible hundreds or even thousands of
parallel sequencing runs, represents an attractive approach to
overcoming the current throughput limitations of existing DNA
sequencing instrumentation. Such a system has been disclosed in
U.S. Pat. Nos. 5,582,705 (Yeung et al.), 5,695,626 (Yeung et al.),
and 5,741,411 (Yeung et al.). In this system, light-induced
fluorescence is exclusively employed as the detection method.
[0006] While fluorescence detection is suitable for DNA sequencing
applications because of its high sensitivity and special labeling
protocols, UV absorption detection has remained very useful because
of its ease of implementation and wide applicability, especially
for the deep-UV (200-220 nm) detection of organic and biologically
important compounds. A capillary isoelectric focusing system using
a two-dimensional CCD detector, in which one dimension represents
the capillary length and the other dimension records the absorption
spectrum, has been described by Wu and Pawliszyn, Analyst
(Cambridge), 120, 1567-1571 (1995). The system has been used for
two capillary tubes but is not easily adapted for three or more
capillary tubes because the system requires the capillary tubes to
be separated by space. Instead of providing wavelength resolution
in the second CCD dimension, isoelectric focusing in two capillary
tubes is simultaneously monitored. The use of optical fibers for
illumination, however, has led to low light intensities and poor UV
transmission. So, only visible wavelengths have been employed for
the detection of certain proteins. Because the CCD has a very small
electron well capacity (about 0.3 million electrons), the limit of
detection (LOD) of this system is limited by the high shot noise in
absorption detection. The use of the CCD produces an overwhelming
amount of data per exposure, limiting the data rate to one frame
every 15 seconds. Also, the imaging scheme utilized is not suitable
for densely packed capillary arrays because of the presence of
mechanical slits to restrict the light paths. Further, in order to
avoid cross-talk, only square capillaries can be used.
[0007] Photodiode arrays (PDA) are used in many commercial CE and
HPLC systems for providing absorption spectra of the analytes in
real time. Transmitted light from a single point in the flow stream
is dispersed by a grating and recorded across the linear array. A
capillary zone electrophoresis system using a photodiode array as
the imaging absorption detector has been described by Culbertson
and Jorgenson, Anal. Chem., 70, 2629-2638 (1998). Different
elements in the array are used to image different axial locations
in one capillary tube to follow the progress of the separation.
Because the PDA has a much larger electron well capacity (tens of
million electrons), it is superior to the CCD for absorption
detection. Time-correlated integration is applied to improve the
signal-to-noise ratio (S/N).
[0008] What is still needed is an absorption detection approach for
the simultaneous analysis of multiple systems. One such system is
shown in U.S. Pat. No. 5,900,934 (Gilby et al.). This system
includes a photodetector array comprising a plurality of
photosensitive elements connected to provide a serial output. The
elements are typically pixels of a photodiode array (PDA). The
elements are illuminated by a light source positioned to illuminate
at least a portion of the photodetector array. The light source may
be an AC or DC mercury lamp or other useable light source for
chromatography. An array of separation channels is disposed between
the light source and the photodetector array, each of the
separation channels having a lumen, a sample introduction end and a
detection region disposed opposite the sample introduction end. The
array is a multiple parallel capillary electrophoresis system. A
mask element having at least one aperture for each associated
separation channel is required. Each aperture corresponds to its
associated separation channel, thereby selectively permitting light
from the light source to pass through the lumen of its associated
separation channel. At least a portion of the light passing through
the lumen of the associated separation channel falls on a
respective photosensitive element of the photodetector array to
effect measurement of absorption of light by a sample introduced
into the sample introduction end of the associated separation
channel.
[0009] The system described by Gilby et al. has disadvantages
because it limits the amount of light impinging on the separation
channel, providing less than desirable light intensity to the PDA.
Further, aligning the apertures and the mask elements with the
separation channels, e.g., capillaries, is difficult for several
reasons. For example, positioning the capillaries with equal
separation there between is difficult as the capillaries generally
are not of equal dimension, e.g., diameter tolerances vary greatly.
Further, for example, the mask geometry does not provide identical
light paths, which leads to nonlinear response. Also, a mask can
produce stray light, which leads to poor detection limits, and does
not completely eliminate crosstalk from the adjacent capillaries,
since the light beams are diverging and cannot escape the detector
element. In addition, a mask can be difficult to manufacture, due
to the requirement of uniformity. Also, Gilby places the sample and
the PDA too close together, resulting in stray light, cross talk
and the inability to use the maximum pathlength of light.
[0010] Thus, in view of the disadvantages inherent to the methods
and systems in the art, there remains a need a method of analyzing
multiple samples simultaneously by absorption detection. It is an
object of the present invention to provide such a method. It is
another object of the present invention to provide a system for use
in such a method. These and other objects and advantages of the
present invention as well as additional inventive features will
become apparent to one of ordinary skill in the art from the
detailed description provided herein.
[0011] The present invention also addresses other disadvantages in
the art. For example, since the invention of the polymerase chain
reaction (PCR) in 1985 by Kary Mullis, the ultimate in sensitivity,
together with increasing ease in implementation, have placed this
technique in a central position in molecular biology research and
in clinical diagnosis (Rolf et al., PCR: Clinical Diagnostics and
Research, 1992, Springer-Verlag, Berlin and Heidelberg). In the
last ten years, PCR has stimulated numerous investigations in
genetic analysis, and is even being used to determine the genetic
basis of complex diseases (Sacki et al., Science 230: 1350 (1985)).
There is no need to reiterate the development of CE as a powerful
analytical tool in post-PCR analysis. A large amount of research
has been done to explore the advantages of CE over traditional slab
gel electrophoresis, including high-speed, high-resolution
restriction fragments analysis (Guttman et al., Anal. Chem. 62:
2348 (1992); Milofsky et al., Anal. Chem. 65: 153 (1993); Williams
et al., J. Chromatogr. A680: 525 (1994); Chang et al., J.
Chromatogr. B669: 113 (1995); Barron et al., Electrophoresis 16: 64
(1995); and Righetti et al., Anal. Biochem. 244: 195 (1997)),
high-speed, high-throughput DNA sequencing (Ruiz-Martinez et al.,
Anal. Chem. 65: 2851 (1993); Lu et al., J. Chromatogr. A680: 497
(1994); Lu et al., J. Chromatogr. A680: 503 (1994); Fung et al.,
Anal. Chem. 67: 1913 (1995); Zhang et al., Anal. Chem. 67: 4589
(1995); Carrilho et al., Anal. Chem. 68: 3305 (1996); and Kim et
al., J. Chromatogr. 781: 315 (1997)), rapid and precise DNA typing
and sizing (Baba et al., Electrophoresis 16: 1437 (1995); Noble,
Anal. Chem. 67: 613A (1995); Zhang et al., Anal. Chem. 68: 2927
(1996); Isenberg et al., Electrophoresis 17: 1505 (1996); Zhang et
al., J. Chromatogr. A768: 135 (1997); Butler et al.,
Electrophoresis 16: 974 (1995); and Wang et al., Anal. Chem. 67:
1197 (1995)), single-base mutation analysis (Marino et al.,
Electrophoresis 17: 1499 (1996); Arakawa et al., J. Chromatogr.:
A664: 89 (1994); Hebenbrock et al., Electrophoresis 16: 1429
(1995); Kuypers et al., J. Chromatogr.: B 675: 205 (1996); Cheng et
al., J. Cap. Elec. 2: 24 (1995); and Ren et al., Anal. Biochem.
245: 9 (1997)) and the analysis of disease causing genes (Lu et
al., Nature 368: 269 (1994); Felmlee et al., J. Cap. Elec. 2: 125
(1995); Gelfi et al., BioTechniques 19: 254 (1995); and Grossman et
al., Nucleic Acids Res. 22: 4527 (1994)). In particular, capillary
array electrophoresis, along with other micro-fabricated devices
(Ueno et al., Anal. Chem. 66: 1424 (1994); Takahashi et al., Anal.
Chem. 66: 1021 (1994); and Anazawa et al., Anal. Chem. 68: 2699
(1996)) are promising methods for the purpose of achieving
high-throughput DNA analysis. In this regard, single capillaries
have been utilized for DNA analysis (Guttman et al. (1992),
supra).
[0012] The conventional protocol for DNA analysis calls for
labeling with radionuclides or fluorescent tags before, during or
after size-based separation in slab gel electrophoresis or in
capillary gel electrophoresis (CGE). This derivatization process
involves expensive reagents and raises safety concerns for the
operator and for waste disposal because of the toxic nature of
these labeling reagents.
[0013] The present invention can be applied to genetic typing and
diagnosis based simply on UV absorption detection. The additive
contribution of each base pair to the total absorption signal
provides adequate detection sensitivity for analyzing most PCR
products. Not only is the use of specialized and potentially toxic
fluorescent labels eliminated, but also the complexity and cost of
the instrumentation are greatly reduced. The DNA analysis protocols
can, therefore, be designed to take advantage of high-throughput
capillary array gel electrophoresis and simple UV absorption
detection, based on the inherent spectral properties of the DNA
bases. UV absorption detection of DNA products reduces the cost of
analysis, since it does not require labeling.
[0014] Similarly, peptide mapping represents one of the most
powerful and successful tools available for the characterization of
proteins (Garnick et al., Anal. Chem. 60: 2546-2557 (1988); Borman,
Anal. Chem. 59: 969A-973A (1987)). Although less informative than
protein sequencing, it allows rapid analysis with simple
instrumentation. In peptide mapping, a sample protein is
selectively cleaved by enzymes or by chemical digestion (Tarr et
al., Anal. Biochem. 131: 99-107 (1983); Dong, Advances in
Chromatography 32: 22-51, Marcel Dekker, Inc.: New York (1992);
Geisow et al., Biochem. J. 161: 619-625 (1977); and Ward et al., J.
Chromatogr. 519: 199-216 (1990)). The peptide map then serves as a
unique fingerprint of the protein and can accurately reveal very
subtle differences among individual variants. Trypsin is by far the
most widely used proteolytic enzyme in peptide mapping. Its
desirable features are that cleavage at the C-terminal side of
lysine and arginine is generally quantitative under proper
conditions and that trypsin tolerates concentrations of urea as
high as 4 M (Dong (1992), supra). The disadvantage is that the
fragments formed may be too small, averaging 7-12 amino acid
residues, resulting in very complex tryptic maps. After tryptic
digestion, the digest is typically analyzed by various methods,
such as slab gel electrophoresis (Cleveland et al., J. Biol. Chem.
252: 1102-1106 (1977)), thin-layer chromatography (TLC) (Stephens,
Anal. Biochem. 84: 116-126 (1978)), HPLC (Hancock et al., Anal.
Biochem. 89: 203-212 (1978); Cox et al., Anal. Biochem. 154:
345-352 (1986); Fullmer et al., J. Biol. Chem. 254: 7208-7212
(1979); Vensel et al., J. Chromatogr. 266: 491-500 (1983);
Leadbeater et al., J. Chromatogr. 397: 435-443 (1987); Dong et al.,
J. Chromatogr. 499: 125-139 (1990); and Hartman et al., J.
Chromatogr. 360: 385-395 (1986), and capillary zone electrophoresis
(CZE) (Jorgenson et al., J. High Resolut.Chromatogr. Commun. 4:
230-231 (1981); Cobb et al., Anal. Chem. 61: 2226-2231 (1989);
Chang et al., Anal. Chem. 65: 2947-2951 (1993); Nashabeh et al., J.
Chromatogr. 536: 31-42 (1991); Ward et al., J. Chromatogr. 519:
199-216 (1990); Janini et al., J. Chromatogr. 848: 417-433 (1999);
Frenz et al., J. Chromatogr. 480: 379-391 (1989); and Grossman et
al., Anal. Chem. 61: 1186-1194 (1989)) to yield a peptide map.
Gradient reversed-phase HPLC is the most common form of peptide
mapping in use today (Leadbeater et al. (1987), supra; Dong et al.,
(1990), supra; and Hartman et al. (1986), supra).
[0015] In particular, CZE has received considerable attention as a
complementary method to reversed-phase liquid chromatography in
peptide mapping efforts (Jorgenson et al. (1981), supra; Cobb et
al. (1989), supra; Chang et al. (1993), supra; Nashabeh et al.
(1991), supra; Ward et al. (1990), supra; Janini et al. (1999),
supra; Frenz et al. (1989), supra; and Grossman et al. (1989),
supra). Separation of various peptides can be optimized through pH
adjustments. Through the addition of micelle-forming surfactants to
the running buffer, a dynamic partition mechanism (i.e.,
hydrophobicity) of peptide separation can also be established for
the neutral fragments. Although CE is quite efficient and fast for
analyzing peptide fragments, the complete separation of peptides in
a digest of high molecular mass proteins, for example, is not
possible by using a single buffer condition. Unlike HPLC, the
implementation of gradient separation in CE is not trivial (Whang
et al., Anal. Chem. 64: 502-506 (1992); and Chang et al., J.
Chromatogr. B 608: 65-72 (1992)).
[0016] Although these methods are useful for characterizing
proteins, there are still other problems, such as the relatively
large amount of sample required, long analysis time, and efficiency
of the derivatization reaction. Also, a typical map contains 20-150
peaks, all of which should ideally be totally resolved (Dong et al.
(1992), supra). Therefore, a high degree of column resolution and
system precision are required to reproduce accurately the maps,
preferably starting with subnanomolar quantities.
[0017] The present invention enables a peptide map to be obtained
that can serve as a unique fingerprint of the protein. Reliable
high-throughput analyses can be performed, for example, based on
multi-dimensional CE and a single prescribed experimental
protocol.
[0018] Combinatorial screening also has attracted much attention
recently because of its ability efficiently and reliably to zero in
and identify the best solution to a chemical or biochemical
question (Borman, C&E News, Mar. 8, 1999, pages 33-60). In
chemical synthesis, optimization of the reaction yield can be
achieved by simultaneously exploring all possible reaction
conditions, catalysts and reagents. In drug discovery, all related
structural variants of a given candidate can be tested against the
target. However, screening must be comprehensive so that there is
no chance of missing the best combination. This dictates having a
large number of experiments to cover many parameters and to extend
the range of each of these parameters. High throughput is a
requirement in order to produce a timely result. It is primarily
because of the advances in high-throughput technologies and
automation that combinatorial screening became practical. Still
needed are general and rugged analytical methodologies that can
keep up with the large number of reactions that can be performed in
any given time. Another issue is miniaturization of the entire
operation. This impacts the cost of reagents, proper disposal of
solvents, space for manipulation and storage, etc.
[0019] Currently, there are several parallel assays for screening
homogeneous catalysts. Modifications in UV absorption (Wagner et
al., Sicence 270: 1797-1800 (1995); Menger et al., J. Org. Chem.
63: 7578-7579 (1998)), fluorescence (Cooper et al., J. Am. Chem.
Soc. 120: 9971-9972 (1998); Shaughnessy et al., J. Am. Chem. Soc.
121: 2123-2132 (1999)), color (Lavastre et al., Chem. Int. Ed. 38:
3163-3165 (1999)) or temperature (Taylor et al., Science 280:
267-270 (1998); Reetz et al., Angew. Chem. Int. Ed. 37: 2647-2650
(1999)) induced by the catalytic reactions are indicators of
catalytic activity. In these approaches, although the relative
activity of the catalyst is determined quickly, no quantitative
information about the overall yield or the regioselectivity and
stereoselectivity of the process can be obtained. It is also
necessary that the product exhibit very different measurable
properties compared to the solvent or the reagents. Most of the
time, secondary screening is necessary. Mass spectrometry (MS)
(Orschel et al., Angew. Chem. Int. Ed. 38: 2791-2794 (1999)), which
also has been widely used to screen catalysts, can provide
selective detection. However, to address stereoselectivity, these
procedures still tend to be laborious (Reetz et al., Angew. Chem.
Int. Ed. 38: 1758-1761 (1999)). So far, MS is still a serial,
rather than a parallel, approach, although the analysis time is
reasonably short.
[0020] Separation-based techniques can solve the above problems.
Serial methods, which include HPLC and CE, have been used to
analyze asymmetric catalysis (Porte et al., J. Am. Chem. Soc. 120:
9180-9187 (1998); Ding et al., Angew. Chem. Int. Ed. 38: 497-501
(1999)) and alkylation reactions (Gaus et al., Biotech. &
Bioeng. 1998/1999 61: 169-177). The throughput that can be achieved
with serial separation schemes is low even with special techniques,
such as sequential sample injection (Roche et al., Anal. Chem. 69:
99-104 (1997)) and sample multiplexing (Woodbury et al., Anal.
Chem. 67: 885-890 (1995)). Multiplex HPLC is another interesting
approach (Gong et al., Anal. Chem. 71: 4989-4996 (1999)), but
achieving a high degree of multiplexing, such as 96 capillaries in
capillary array electrophoresis (CAE), is not trivial. Thin-layer
chromatography and gel electrophoresis, on the other hand, are
difficult to completely automate.
[0021] A highly successful format for combinatorial screening is
that of DNA chips (Southern, Electrophoresis 16: 1539-1542 (1995);
Chee et al., Science 274: 610-614 (1996); and Winzeler et al.,
Science 281: 1194-1197 (1998)). A comprehensive set of
oligonucleotides immobilized within a small area is used to
identify specific target sequences by hybridization.
Oligonucleotide chips also have been used to develop aptamers that
exhibit specific protein-nucleotide binding (Weiss et al., J.
Virol. 71: 8790-8797 (1997)). Such heterogeneous screening assays
have benefited from sensitive detection based on laser-induced
fluorescence (LIF), either by selective labeling or by selective
quenching. For homogeneous assays, the 96-well microtiter plate is
a popular format. Fluidic operations, plate readers and
autosamplers to interface to standard analytical instruments have
been developed for this format. When there is a color (absorption)
change or fluorescence change, detection and quantitation is
straightforward. In many situations, however, the reaction mixture
is complex and some degree of separation or purification is needed
before measurement. Multiple liquid chromatographs or single
instruments with several columns can in principle be used for
analysis of the reaction mixtures. Still, much higher throughput
and much smaller sample sizes, which means much smaller amounts of
reagents, are desirable. The present invention enables such higher
throughput and smaller sample sizes and does not require the
species of interest to be fluorescent.
BRIEF SUMMARY OF THE INVENTION
[0022] The present invention provides a method of analyzing
multiple samples simultaneously by absorption detection. The method
comprises:
[0023] (i) providing a planar array of multiple containers, each of
which contains a sample comprising at least one absorbing
species,
[0024] (ii) irradiating the planar array of multiple containers
with a light source comprising or consisting essentially of at
least one wavelength of light that is absorbed by one or more of
the absorbing species, the absorption of which is to be measured,
and
[0025] (iii) detecting absorption of light by one or more of the
absorbing species with a detection means that is in line with the
light source and is positioned in line with and parallel to the
planar array of multiple containers at a distance of at least about
10 times a cross-sectional distance of a container in the planar
array of multiple containers measured orthogonally to the plane of
the planar array of multiple containers. The detection of
absorbtion of light by a sample in the planar array of multiple
containers indicates the presence of an absorbing species in the
sample. The method can further comprise:
[0026] (iv) measuring the amount of absorption of light detected in
(iii) for an absorbing species in a sample. The measurement of the
amount of absorption of light detected in (iii) indicates the
amount of the absorbing species in the sample.
[0027] Also provided by the present invention is a system for use
in the above method. The system comprises:
[0028] (i) a light source comprising or consisting essentially of
at least one wavelength of light that is absorbed by one or more
absorbing species, the absorption of which is to be detected,
[0029] (ii) a planar array of multiple containers, into each of
which can be placed a sample comprising at least one absorbing
species, and
[0030] (iii) a detection means that is in line with the light
source and is positioned in line with and parallel to the planar
array of multiple containers at a distance of at least about 10
times a cross-sectional distance of a container in the planar array
of multiple containers measured orthogonally to the plane of the
planar array of multiple containers.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 is a diagram of a system for use in the present
inventive method.
[0032] FIG. 2A is a graph of counts vs. pixel number.
[0033] FIG. 2B is a graph of counts vs. pixel number.
[0034] FIG. 3 is a graph of absorbance vs. frame number.
[0035] FIG. 4 is a graph of light intensity (counts) vs. frame
number vs. value after subtraction (counts).
[0036] FIG. 5 is a graph of intensity vs. frame number.
[0037] FIG. 6 is the result of CZE separation of four visible dyes
in a 96 capillary array.
[0038] FIG. 7 is a graph of migration time vs. capillary
number.
[0039] FIG. 8 is a graph of peak area vs. capillary number.
[0040] FIG. 9 is a reconstructed two-dimensional electropherogram
for capillary array electrophoresis.
[0041] FIG. 10 is a set of extracted electropherograms for
capillary array electrophoresis.
[0042] FIG. 11 represents the peptide maps of three variants of
bovine .beta.-lactoglobulin [SEQ ID NO: 1].
[0043] FIG. 12 shows the results of the six-dimensional separations
(capillary vs. migration time) of tryptic digests of BLGA and BLGB
in the 96-capillary array.
[0044] FIG. 13 shows selected electropherograms of BLGA extracted
from the data in FIG. 12.
[0045] FIG. 14 shows selected electropherograms of BLGB extracted
from the data in FIG. 12.
[0046] FIGS. 15A-D show typical peptide maps of BLGA and BLGB at
four pH conditions (FIG. 15A at pH 9.3; FIG. 15B at pH 8.1; FIG.
15C at pH 5.0; and FIG. 15D at pH 2.5) for CZE using a single
capillary after tryptic digestion.
[0047] FIG. 16 shows MEKC peptide maps of BLGA (B and D) and BLGB
(A and C) obtained at two different MEKC conditions using a
nonionic surfactant (A and B) and/or the combination of nonionic
and anionic surfactant (C and D).
[0048] FIG. 17 shows the effect of Tween 20 concentration at pH 8.1
on the MEKC peptide maps for BLGB in terms of migration time
(min).
[0049] FIG. 18A is a graph of the ratio of the amount of NADH
(injected) to the amount of NAD (injected) vs. the results from
nine electrokinetic injections.
[0050] FIG. 18B is a graph of the ratio of the amount of NADH
(injected) to the amount of NAD (injected) vs. the results from
hydrodynamic nine injections.
[0051] FIG. 19 is a reconstructed absorption image of combinatorial
screening of enzyme activity in a 96 capillary array in which the
capillaries (1-96) are displayed from top to bottom and migration
time (0-33 min) is plotted from left to right.
[0052] FIG. 20 is an electropherogram of products after 180 min
reaction for different LDH concentrations at pH=7.
[0053] FIG. 21 is an electropherogram of products after 180 min
reaction for different pH at an LDH concentration of
5.times.10.sup.-9 M.
[0054] FIG. 22 is a graph of NADH conversion percentage vs. pH for
series 1-9 at 180 min incubation.
[0055] FIG. 23 is a graph of reaction percentage vs. pH for series
1-9 for 30 min of LDH catalysis.
[0056] FIG. 24 which is a graph of reaction percentage vs. pH value
for series 1-9 for 24 hr of LDH catalysis.
[0057] FIG. 25 shows the separation of two isomeric forms (A and B)
of the product from the reagents and the internal standard using
two different buffers (1a and 1b).
[0058] FIG. 26 shows a 96 capillary separation for the reaction
conditions for 1b in FIG. 25 and a hydrodynamic injection of 1 min,
in which the horizontal direction spans 88 capillaries (the
remaining 8 capillaries contained solvent only and were not
plotted) and the vertical direction represents time.
[0059] FIG. 27 is a 3-dimensional bar graph of yield vs. catalyst
vs. base.
[0060] FIG. 28 is a selectivity plot of two isomers produced in the
reactions, wherein P1/P2 is the ratio of the two isomers A and B,
respectively.
[0061] FIG. 29 is a line graph of fractional conversion vs. time
(hr) vs. base, for the reaction using Pd(PPh.sub.3).sub.4 as the
catalyst and various bases.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention provides a method of analyzing
multiple samples simultaneously by absorption detection. The
present invention utilizes an integrated approach toward achieving
automation, high speed, high accuracy and low cost, such as in the
context of multiplexed electrophoresis. The method can be used, for
example, in multicapillary array zone electrophoresis, micellar
electrokinetic chromatography, capillary electrochromatography, and
capillary gel electrophoresis. When a multicapillary array is used,
as much as 100 times, or even 1,000 times or greater, higher
analysis throughput can be achieved when compared to conventional
single-capillary electrophoresis. The system is at least about
100-fold more sensitive than the system of Wu and Pawliszyn.
[0063] The method comprises:
[0064] (i) providing a planar array of multiple containers, each of
which contains a sample comprising at least one absorbing
species,
[0065] (ii) irradiating the planar array of multiple containers
with a light source comprising or consisting essentially of at
least one wavelength of light that is absorbed by one or more of
the absorbing species, the absorption of which is to be detected,
and
[0066] (iii) detecting absorption of light by the one or more
absorbing species with a detection means that is in line with the
light source and is positioned in line with and parallel to the
planar array of multiple containers at a distance such that stray
light exiting the planar array of multiple containers disperses
prior to impinging upon the detection means. The amount of stray
light falls off inversely as the square of the distance between the
planar array and the detector increase. Thus, the light impinging
upon the detection means is substantially only that which is
transmitted through the multiple containers. In this manner, the
intensity of the outputs from the planar array of multiple
containers is the strongest and, therefore, the intensity of the
outputs from the detection means is also the strongest, thereby
making the determination of the intensity outputs from the
detection means for each container in the planar array of multiple
containers easy. The detection of absorption of light by a sample
in the planar array of multiple containers indicates the presence
of an absorbing species in the sample.
[0067] The method can further comprise (iv) measuring the amount of
absorption of light detected in (iii) for an absorbing species in a
sample. The measurement of the amount of absorption of light
detected in (iii) indicates the amount of the absorbing species in
the sample. Methods of measuring the amount of absorption of light
are known in the art. Basically, one measures the intensity of
light in the absence and presence of a sample. The logarithm of the
ratio is the absorbance (Beer-Lambert law). Preferably, the
distance between the planar array of multiple containers and the
detection means is at least about 10 times, at which distance the
stray light is less than about 1%, more preferably, at least about
100 times, a cross-sectional distance of a container in the planar
array of multiple containers measured orthogonally to the plane of
the planar array of multiple containers.
[0068] The distance between the light source and the planar array
of multiple containers is not critical to the practice of the
present invention. However, the shorter the distance between the
light source and the planar array of multiple containers, the more
light will be received by the planar array of multiple containers.
The greater the distance between the light source and the planar
array of multiple containers, the more uniform will be the light
received by the planar array of multiple containers. The more light
that the planar array of multiple containers receives, the more
sensitive will be the detection.
[0069] The position of the light source in relation to the planar
array of multiple containers also is not critical to the practice
of the present invention as long as the light source irradiates the
planar array of multiple containers. Other considerations are as
noted in the preceding paragraph.
[0070] Preferably, the distance between the planar array of
multiple containers and the detection means is at least about 10
times, more preferably, at least about 100 times, a cross-sectional
distance of a container in the planar array of multiple containers
measured orthogonally to the plane of the planar array of multiple
containers. Thus, the distance between the planar array of multiple
containers and the detection means is preferably from about 1 cm to
about 30 cm, more preferably from about 3 cm to about 30 cm, and
most preferably from about 10 cm to about 30 cm. When cylindrical
capillary tubes are used as the multiple containers, preferably the
distance is from about 1 cm to about 30 cm, more preferably from
about 3 cm to about 30 cm, and most preferably from about 10 cm to
about 30 cm.
[0071] By "multiple containers" is meant at least three or more,
preferably at least about 10, more preferably at least about 90,
and desirably as many as can be accommodated by the system
described herein. While the multiple containers can comprise any
suitable containers, desirably the multiple containers allow the
passage of light from the light source through the walls of the
containers facing the light source, through the samples in the
containers, and through the walls of the containers facing the
detection means. Thus, the walls of the containers are desirably
transparent, although, in some instances, the walls of the
containers can be translucent. In this regard, it is not necessary
for the entirety of the walls of the containers to allow the
passage of light from the light source as described above as long
as at least a portion of the walls of the containers allow the
passage of light from the light source such that the samples in the
containers are irradiated and the light that is not absorbed by the
absorbing species in the samples is detectable by the detection
means. Preferably, the multiple containers comprise cylindrical
capillary tubes. Preferably, the planar array of multiple
containers comprises at least about 10 capillary tubes, more
preferably at least about 90 capillary tubes, such as 96 capillary
tubes, and desirably as many as can be accommodated by the system
described herein.
[0072] The planar array desirably further comprises at least one
control container. However, if the light source is stable, a
control container is not necessary.
[0073] In general, the containers used in the planar array should
have smooth surfaces and uniformly thick walls and be made of a
material that is transparent over the range of wavelengths of light
absorbed by an absorbing species in a sample, the absorbance of
which is to be detected or measured. Preferred materials for
containers include, but are not limited to, plastics, quartz, fused
silica (in particular for capillary tubes) and glass. The
cross-section of a container is not critical to the present
inventive method. However, the smaller the cross section of the
container, the more useful is the container in highly multiplexed
applications as a greater number of containers can be used in a
smaller amount of space. Similarly, the thickness of the wall of
the container is not critical to the present inventive method. The
wall should be of sufficient thickness so as to maintain the
structural integrity of the container, yet not so thick as to
impede adversely the passage of light through the container. The
shape of the container also is not critical to the present
inventive method. The container can have any suitable shape.
Desirably, the shape of the container is conducive to being closely
packed and minimizes the generation of stray light by the
container.
[0074] A cylindrical capillary tube is a preferred container for
use in the context of the present invention. Capillary tubes are
commercially available from a number of sources, including
Polymicro Technologies, Inc., Phoenix, Ariz. The capillary tube is
preferably coated with a polymer, such as polyimide, so that it is
mechanically stable. The coating must be removed in the region to
be irradiated by the light source. An excimer laser can be used to
remove the polymer coating.
[0075] Preferably, the multiple containers in the planar array are
arranged substantially parallel to each other. Also preferably, the
multiple containers in the planar array are also arranged
substantially adjacent to each other. For example, when the
multiple containers are capillary tubes, the capillary tubes are
closely packed so as to be substantially contiguous along their
parallel lengths, leaving essentially no space between adjacent
capillaries. Substantially adjacent capillary tubes can be
physically touching each other along all or a portion of their
lengths, although slight inconsistencies in capillary wall diameter
or other features of the array can prevent them from being in
contact along their entire lengths. The planar array desirably is
rigidly mounted to reduce flicker noise.
[0076] If a large amount of heat is generated during the method,
particularly in the vicinity of the planar array of multiple
containers, cooling should be employed to dissipate the heat.
Excessive heat can lead to mechanical vibrations between adjacent
containers in the planar array of multiple containers (e.g., such
as in the case of closely packed capillary tubes), which, in turn,
can lead to excess noise. A laminar flow of nitrogen gas, such as
in parallel to the portion of the containers undergoing detection,
can be used for cooling.
[0077] The detection means can comprise any suitable means of
detecting absorption. Preferably, the detection means comprises a
plurality of absorption detection elements, such as a plurality of
photosensitive elements, which desirably are positioned in a linear
array, although a two-dimensional image array detector can be used.
Desirably, the detection means is parallel to and in line with the
linear array of multiple containers. The detection means desirably
is rigidly mounted to reduce flicker noise. In this regard, the
relative positions of cell components used in the system must be
fixed.
[0078] Preferably, a linear photodiode array (PDA) is used.
Desirably, the PDA incorporates a linear image sensor chip, a
driver/amplifier circuit and a temperature controller, which
desirably thermoelectrically cools the sensor chip to a temperature
from about 0.degree. C. to about -40.degree. C. Lowering the
temperature lowers the dark count and minimizes the temperature
drift, thus enabling reliable measurements to be made over a wide
dynamic range. The driver/amplifier circuit is desirably interfaced
to a computer via an I/O board, which preferably also serves as a
pulse generator to provide a master clock pulse and a master start
pulse, which are required by the linear image sensor. The PDA
records the image linearly--not two-dimensionally. Preferably, the
data acquired are written directly to the hard disk in real time.
Also, preferably, the signals from up to at least about ten
elements of the PDA are displayed in real time.
[0079] Alternatively, a charge-coupled device (CCD) or a
charge-injection device (CID) can be used. However, the CCD records
in two dimensions, which is less efficient, requiring more computer
memory, is slower, requires every location to be read (not a single
line like PDA). Furthermore, whereas a CCD has only 100,000
electrons in each location, each element in a PDA can store 59
million electrons per pixel per location; thus, given that
detection sensitivity is related to the square root of the number
of electrons that can be detected, a PDA is orders of magnitude
more sensitive than a CCD.
[0080] Preferably, the PDA comprises linearly aligned pixels, in
which case each container in the planar array of multiple
containers desirably is a capillary tube and each capillary tube
preferably is optically coupled to less than about ten pixels, more
preferably from about 7 to about 9 pixels, some of which are
coupled to the walls of the capillary and some of which are coupled
to any space between the walls of adjacent capillaries and at least
one of which is coupled to the lumen of the capillary. Thus, the
stray light caused by the walls of a capillary is dispersed prior
to striking the pixels and/or is confined to the pixels coupled to
the side walls and generally does not affect the signal produced by
the pixel coupled to the lumen of the capillary. While the ratio of
capillaries to optically coupled pixels is preferably less than
about 1:10, more preferably from about 1:7 to about 1:9, the ratio
of capillaries to optically coupled pixels need not be an integer
ratio.
[0081] If the detection means is a PDA comprising linearly aligned
pixels, step (iii) of the method can comprise selecting one pixel
from the middle group of pixels, i.e., the pixel detecting the
strongest light intensity and using that pixel to detect the
absorbance by the target species. When more than one pixel is
optically coupled to the interior of a container, it is desirable
to select only one to analyze to and to disregard the others.
Alternatively, only one pixel can be optically coupled to each
container, obviating the need to make a pixel selection, although
this is less preferred because of the need for critical optical
alignment. In large arrays with many containers, which desirably
are capillary tubes, it can be practical to use a higher ratio of
pixels to containers so as to accommodate inconsistencies and
variations in packing of the containers, and the width of the walls
of the containers; etc. Where higher ratios of pixels to
capillaries are used, more than one pixel can be optically coupled
to the lumen of a container. Each pixel that is coupled to the
lumen of a container will produce a signal having an intensity
directly proportional to the intensity of light detected. The pixel
producing the signal having the greatest intensity, i.e., the
"brightest" pixel, is advantageously selected.
[0082] Selection of the appropriate pixel from those that are
optically coupled to the interior portion of a container can be
conveniently done by way of a calibration step. Thus, the method of
the present invention can further comprise a calibration step,
which is performed prior to introducing the samples into the
containers. Alternatively, every nth capillary, e.g., every 10th
capillary, includes a control or blank sample, i.e., a control
container as indicated above.
[0083] The method can be carried out at ambient temperature, such
as room temperature, such as from about 20.degree. C. to about
30.degree. C., or as low as 0.degree. C. or as high as 80.degree.
C. However, if the method employs a PDA as the detection means as
is preferred, desirably the PDA has its own cooler for operation at
subzero temperatures, such as from about 0.degree. C. to about
-40.degree. C.
[0084] The light source preferably comprises or consists
essentially of a wavelength in the range from about 180 nm to about
1500 nm. Examples of suitable light sources include mercury (for
ultraviolet (UV) light absorption), tungsten (for visible light
absorption), iodine (for UV light absorption), zinc (for UV light
absorption), cadmium (for UV light absorption), xenon (for UV light
absorption), deuterium (for visible light absorption), and the
like. Desirably, the light source comprises or consists essentially
of a wavelength of light that will be absorbed by an absorbing
species, the absorption of which is to be detected. Which
wavelength of light will be absorbed by an absorbing species of
interest, i.e., an absorbing species, the absorption of which is to
be detected or measured in accordance with the present invention,
can be determined using a standard absorption spectrometer.
Alternatively, spectroscopic tables that provide such information
are available in the art, such as through the National Institute of
Science and Technology (NIST). Desirably, a maximally absorbed
wavelength of light is selected for a given absorbing species to be
detected or measured such that smaller amounts of the absorbing
species can be detected. Generally, the light source provides light
impinging on the planar array of multiple containers orthogonal to
the plane in which the planar array of multiple containers. The
light source can be a point source. Also preferably, the light
source has a power output of about 0.5 mW to about 50 mW. The light
source can be AC or DC, although DC is preferred. Any flicker noise
from the light source can be eliminated by using a double beam of
light.
[0085] The pathlength of light is critical to the sensitivity of
the present inventive method. The longer the pathlength of light
absorbed by a sample in a container, the larger the signal for the
sample. This is in accordance with Beer's Law, which states that
absorbance=constant (which is the spectral characteristic of an
absorbing species in a sample in a container, the absorbance of
which is to be detected or measured).times.pathlength of the
light.times.concentration of the absorbing species in a sample in a
container. A high constant and a long pathlength are desired.
[0086] An optical filter desirably is positioned between the planar
array of multiple containers and the detection means. The optical
filter selects at least one wavelength of light from the light
source that is absorbed by an absorbing species, the absorption of
which is to be detected. While an optical filter can be positioned
between the light source and the planar array of multiple
containers in addition to, or as an alternative to, an optical
filter positioned between the planar array of multiple containers
and the detection means, the placement of a single optical filter
between the light source and the planar array of multiple
containers is disadvantageous inasmuch as it does not block the
subsequent fluorescence by the sample from reaching the detection
means. In contrast, the placement of an optical filter between the
planar array of multiple containers and the detection means blocks
sample fluorescence from reaching the detection means.
[0087] A flat-field lens also desirably is positioned between the
planar array of multiple containers and the detection means. The
flat-field lens couples light that is not absorbed by the one or
more absorbing species in each sample in the planar array of
multiple containers with the detection means. While a lens that is
not a flat-field lens can be used in the context of the present
invention, it is disadvantageous inasmuch as it does not image the
entire field evenly. Consequently, the edges of the field are
distorted and the absorption of the containers in the planar array
of multiple containers positioned at the edges of the field of the
lens cannot be detected or measured. The lens inverts the image of
the planar array of multiple containers onto the face of the
detection means, which preferably is a PDA.
[0088] Desirably, the coupling of light by the flat-field lens is
shielded from the light source. This way, only the light from the
lens is focused on the detection means.
[0089] The detection limit of rhodamine 6G for each capillary in a
planar array of multiple capillaries is about 1.8.times.10.sup.-8
M. The cross-talk between adjacent capillaries in a planar array of
multiple capillaries is less than about 0.2%.
[0090] While the sample can be introduced into each capillary tube
in a planar array of multiple capillaries by any suitable method,
preferably the sample is introduced into the capillary tube by
pressure, gravity, vacuum, capillary or electrophoretic action.
[0091] A beam expander can be positioned between the light source
and the planar array of multiple containers. The beam expander can
alter the focused line of the light source so as to irradiate more
effectively the multiple containers. The beam, optionally, can be
altered or redirected, as with a mirror, filter or lens, prior to
contacting the array.
[0092] A collimating focusing lens can be positioned between the
light source and the planar array of multiple containers.
[0093] The above components are placed to eliminate substantially
and, desirably, completely, stray light. There are two kinds of
stray light. One kind of stray light is the glare that results from
the containers having side walls and interior lumens. The other
kind of stray light is that which is due to the presence of other
containers in the planary array of multiple containers. This kind
of stray light is referred to as "cross talk." Cross talk
essentially is the glare from other containers. Thus, there needs
to be sufficient distance between the sample and the flat-field
lens to eliminate substantially and, desirably completely, the two
kinds of glare. A distance of at least about 1/r.sup.2, i.e., the
rate of decrease of stray light as the distance r increases, or
1/d, in which r=radius and d=diameter, will eliminate most of the
glare from the containers. Glare can be assessed by measuring a
totally absorbing material in a container; if there is any light
that is detected, that light is due to glare.
[0094] Preferably, raw data sets are extracted into single-diode
electropherograms and analyzed by converting the transmitted light
intensities collected at the PDA to absorbance values.
Root-mean-squared noise in the electropherograms is obtained using
a section of baseline near one of the analyte peaks. A preferred
manner of collecting and analyzing data obtained in accordance with
the present invention is set forth in Example 1.
[0095] Mathematical smoothing can be used to reduce the noise
significantly, without distorting the signal. See, for example,
Example 1. In this regard, as high a data acquisition rate as
possible should be employed to provide more data points for
smoothing. Boxcar smoothing, such as 25 point boxcar smoothing, is
a preferred method of mathematical smoothing.
[0096] In view of the above, the present invention further provides
a system for use in the above method, preferred embodiments of
which are exemplified in the Examples and FIG. 1 set forth herein.
The system comprises:
[0097] (i) a light source comprising or consisting essentially of
at least one wavelength of light that is absorbed by one or more
absorbing species, the absorption of which is to be detected,
[0098] (ii) a planar array of multiple containers, into each of
which can be placed a sample comprising at least one absorbing
species, and
[0099] (iii) a detection means that is in line with the light
source and is positioned in line with and parallel to the planar
array of multiple containers at a distance such that stray light
exiting the planar array of multiple containers disperses prior to
impinging upon the detection means. Thus, the light impinging upon
the detection means is substantially only that which is transmitted
through the multiple containers. In this manner, the intensity of
the outputs from the planar array of multiple containers is the
strongest and, therefore, the intensity of the outputs from the
detection means is also the strongest, thereby making the
determination of the intensity outputs from the detection means for
each container in the planar array of multiple containers easy.
Preferably, the distance is at least about 10 times, more
preferably, at least about 100 times, a cross-sectional distance of
a container in the planar array of multiple containers measured
orthogonally to the plane of the planar array of multiple
containers. The detection of absorption of light by a sample in the
planar array of multiple containers indicates the presence of an
absorbing species in the sample.
[0100] As indicated above, the distance between the light source
and the planar array of multiple containers is not critical to the
practice of the present invention. However, the shorter the
distance between the light source and the planar array of multiple
containers, the more light will be received by the planar array of
multiple containers. The greater the distance between the light
source and the planar array of multiple containers, the more
uniform will be the light received by the planar array of multiple
containers. The more light that the planar array of multiple
containers receives, the more sensitive will be the detection.
[0101] The position of the light source in relation to the planar
array of multiple containers also is not critical to the practice
of the present invention as long as the light source irradiates the
planar array of multiple containers. Other considerations are as
noted in the preceding paragraph.
[0102] Preferably, the distance between the planar array of
multiple containers and the detection means is at least about 10
times, more preferably, at least about 100 times, a cross-sectional
distance of a container in the planar array of multiple containers
measured orthogonally to the plane of the planar array of multiple
containers. Thus, the distance between the planar array of multiple
containers and the detection means is preferably from about 1 cm to
about 30 cm, more preferably from about 3 cm to about 30 cm, and
most preferably from about 10 cm to about 30 cm. When capillary
tubes are used as the multiple containers, preferably the distance
is from about 1 cm to about 30 cm, more preferably from about 3 cm
to about 30 cm, and most preferably from about 10 cm to about 30
cm.
[0103] By "multiple containers" is meant at least three or more,
preferably at least about 10, more preferably at least about 90,
and desirably as many as can be accommodated by the system
described herein. While the multiple containers can comprise any
suitable containers, desirably the multiple containers allow the
passage of light from the light source through the walls of the
containers facing the light source, through the samples in the
containers, and through the walls of the containers facing the
detection means. Thus, the walls of the containers are desirably
transparent, although, in some instances, the walls of the
containers can be translucent. In this regard, it is not necessary
for the entirety of the walls of the containers to allow the
passage of light from the light source as described above as long
as at least a portion of the walls of the containers allow the
passage of light from the light source such that the samples in the
containers are irradiated and the light that is not absorbed by the
absorbing species in the samples is detectable by the detection
means. Preferably, the multiple containers comprises cylindrical
capillary tubes. If cylindrical capillary tubes are used,
preferably the distance between the detection means and the planar
array of multiple containers is at least about 10 times, more
preferably at least about 100 times, the diameter of a capillary
tube. Preferably, the planar array of multiple containers comprises
at least about 10 cylindrical capillary tubes, more preferably at
least about 90 cylindrical capillary tubes, such as 96 cylindrical
capillary tubes, and desirably as many as can be accommodated by
the system described herein.
[0104] The planar array desirably further comprises at least one
control container. However, if the light source is stable, a
control container is not necessary.
[0105] In general, the containers used in the planar array should
have smooth surfaces, uniformly thick walls, and be made of a
material that is transparent over the range of wavelengths of light
absorbed by an absorbing species in a sample, the absorbance of
which is to be detected or measured. Preferred materials for
containers include, but are not limited to, quartz, fused silica
(in particular for capillary tubes) and glass. The cross-section of
a container is not critical to the present inventive method.
However, the smaller the cross section of the container, the more
useful is the container in highly multiplexed applications as a
greater number of containers can be used in a smaller amount of
space. Similarly, the thickness of the wall of the container is not
critical to the present inventive method. The wall should be of
sufficient thickness so as to maintain the structural integrity of
the container, yet not so thick as to impede adversely the passage
of light through the container. The shape of the container also is
not critical to the present inventive method. The container can
have any suitable shape. Desirably, the shape of the container is
conducive to being closely packed and minimizes the generation of
stray light by the container.
[0106] A capillary tube is a preferred container for use in the
context of the present invention. Capillary tubes are commercially
available from a number of sources, including Polymicro
Technologies, Inc. The capillary tube is preferably coated with a
polymer, such as polyimide, so that it is mechanically stable. The
coating must be removed in the region to be irradiated by the light
source. An excimer laser can be used to remove the polymer
coating.
[0107] Preferably, the multiple containers in the planar array are
arranged substantially parallel to each other. Also preferably, the
multiple containers in the planar array are also arranged
substantially adjacent to each other. For example, when the
multiple containers are capillary tubes, the capillary tubes are
closely packed so as to be substantially contiguous along their
parallel lengths, leaving essentially no space between adjacent
capillaries. Substantially adjacent capillary tubes can be
physically touching each other along all or a portion of their
lengths, although slight inconsistencies in capillary wall diameter
or other features of the array can prevent them from being in
contact along their entire lengths. The planar array desirably is
rigidly mounted to reduce flicker noise.
[0108] If a large amount of heat is generated during use of the
system, particularly in the vicinity of the planar array of
multiple containers, the system desirably further comprises a
cooling means. Excessive heat can lead to mechanical vibrations
between adjacent containers in the planar array of multiple
containers (e.g., such as in the case of closely packed capillary
tubes), which, in turn, can lead to excess noise. A laminar flow of
nitrogen gas, such as in parallel to the portion of the containers
undergoing detection, can be used.
[0109] The detection means can comprise any suitable means of
detecting absorption. Preferably, the detection means comprises a
plurality of absorption detection elements, such as a plurality of
photosensitive elements, which desirably are positioned in a linear
array, although a two-dimensional image array detector can be used.
Desirably, the detection means is parallel to and in line with the
linear array of multiple containers. The detection means desirably
is rigidly mounted to reduce flicker noise.
[0110] Preferably, a linear photodiode array (PDA) is used.
Desirably, the PDA incorporates a linear image sensor chip, a
driver/amplifier circuit and a temperature controller, which
desirably thermoelectrically cools the sensor chip to a temperature
from about 0.degree. C. to about -40.degree. C. Lowering the
temperature lowers the dark count and minimizes the temperature
drift, thus enabling reliable measurements to be made over a wide
dynamic range. The driver/amplifier circuit is desirably interfaced
to a computer via an I/O board, which preferably also serves as a
pulse generator to provide a master clock pulse and a master start
pulse, which are required by the linear image sensor. The PDA
records the image linearly--not two-dimensionally. Preferably, the
data acquired are written directly to the hard disk in real time.
Also, preferably, the signals from up to at least about ten
elements of the PDA are displayed in real time.
[0111] Alternatively, a charge-coupled device (CCD) or a
charge-injection device (CID) can be used. However, the CCD records
in two dimensions, which is less efficient, requiring more computer
memory, is slower, requires every location to be read (not a single
line like PDA), and has a reduced electron capacity. Furthermore,
whereas a CCD has only 100,000 electrons in each location, each
element in a PDA can store 59 million electrons per pixel per
location; thus, given that detection sensitivity is related to the
square root of the number of electrons that can be detected, a PDA
is orders of magnitude more sensitive than a CCD.
[0112] Preferably, the PDA comprises linearly aligned pixels, in
which case each container in the planar array of multiple
containers desirably is a capillary tube and each capillary tube
preferably is optically coupled to less than about ten pixels, more
preferably from about 7 to about 9 pixels, some of which are
coupled to the walls of the capillary and some of which are coupled
to any space between the walls of adjacent capillaries and at least
one of which is coupled to the lumen of the capillary. Thus, the
stray light caused by the walls of a capillary is dispersed prior
to striking the pixels and/or is confined to the pixels coupled to
the side walls and generally does not affect the signal produced by
the pixel coupled to the lumen of the capillary. While the ratio of
capillaries to optically coupled pixels is preferably less than
about 1:10, more preferably from about 1:7 to about 1:9, the ratio
of capillaries to optically coupled pixels need not be an integer
ratio. Optical coupling of the capillaries and the pixels in this
manner renders the system extremely stable.
[0113] Given that noise will ultimately determine the minimum
baseline fluctuation level and, thus, the limit of detection (LOD)
of the system, as explained in Example 1, it is desirable to have
as high a photon count as possible. Preferably, at least about
300,000, more preferably, at least about 3 million, and most
preferably, at least about 30 million photons are used. In
addition, so as to allow for baseline drift due to uncontrollable
variables over the period of data acquisition, the diodes
preferably are only 85-90% saturated.
[0114] The light source preferably comprises or consists
essentially of a wavelength in the range from about 180 nm to about
1500 nm. Examples of suitable light sources include mercury,
tungsten, iodine, zinc, cadmium, xenon, deuterium, and the like.
Desirably, the light source comprises or consists essentially of a
wavelength of light that will be absorbed by an absorbing species,
the absorption of which is to be detected. Which wavelength of
light will be absorbed by an absorbing species of interest, i.e.,
an absorbing species, the absorption of which is to be detected or
measured in accordance with the present invention, can be
determined using a standard absorption spectrometer. Alternatively,
spectroscopic tables that provide such information are available in
the art, such as through NIST. Desirably, a maximally absorbed
wavelength of light is selected for a given absorbing species to be
detected or measured such that smaller amounts of the absorbing
species can be detected. Generally, the light source provides light
impinging on the planar array of multiple containers orthogonal to
the plane in which the planar array of multiple containers. The
light source can be a point source. Also preferably, the light
source has a power output of about 0.5 mW to about 50 mW. The light
source can be AC or DC, although DC is preferred. Any flicker noise
from the light source can be eliminated by using a double beam of
light.
[0115] Desirably, an optical filter is positioned between the
planar array of multiple containers and the detection means. The
optical filter selects at least one wavelength of light from the
light source that is absorbed by an absorbing species, the
absorption of which is to be detected. While an optical filter can
be positioned between the light source and the planar array of
multiple containers in addition to, or as an alternative to, an
optical filter positioned between the planar array of multiple
containers and the detection means, the placement of a single
optical filter between the light source and the planar array of
multiple containers is disadvantageous inasmuch as it does not
block the subsequent fluorescence by the sample from reaching the
detection means. In contrast, the placement of an optical filter
between the planar array of multiple containers and the detection
means blocks sample fluorescence from reaching the detection
means.
[0116] Also desirably, a flat-field lens is positioned between the
planar array of multiple containers and the detection means. The
flat-field lens couples light that is not absorbed by the one or
more absorbing species in each sample in the planar array of
multiple containers with the detection means. While a lens that is
not a flat-field lens can be used in the context of the present
invention, it is disadvantageous inasmuch as it does not image the
entire field evenly. Consequently, the edges of the field are
distorted and the absorption of the containers in the planar array
of multiple containers positioned at the edges of the field of the
lens cannot be detected or measured. The lens inverts the image of
the planar array of multiple containers onto the face of the
detection means, which preferably is a PDA.
[0117] Preferably, the system further comprises a shield that
shields the coupling of light by the flat-field lens from the light
source. This way, only the light from the lens is focused on the
detection means.
[0118] The detection limit for rhodamine 6G for each capillary in a
planar array of capillary tubes in the system is about
1.8.times.10.sup.-8 M. The cross-talk between adjacent capillaries
is less than about 0.2%.
[0119] If the system utilizes capillary tubes or the like, the
system further comprises a means to introduce the sample into the
capillary tube. Preferably, the sample is introduced into the
capillary tube by pressure, gravity, vacuum, capillary or
electrophoretic action.
[0120] The system can further comprise a beam expander between the
light source and the planar array of multiple containers. The beam
expander can alter the focused line of the light source so as to
irradiate more effectively the multiple containers. The beam,
optionally, can be altered or redirected, as with a mirror, filter
or lens, prior to contacting the array.
[0121] The system can further comprise a collimating focusing lens
between the light source and the planar array of multiple
containers.
[0122] The above components are placed to eliminate substantially
and, desirably completely, stray light as described above. Thus,
there needs to be sufficient distance between the sample and the
flat-field lens to eliminate substantially and, desirably
completely, the two kinds of glare. A distance of at least about
1/r.sup.2 or 1/d, in which r=radius and d=diameter, will eliminate
most of the glare from the containers.
[0123] Desirably, the above components are collectively placed in a
light-tight construct, such as a metal box attached to an optical
table. Also, desirably, the components are centered above the
optical table.
EXAMPLES
[0124] The present invention is further demonstrated by way of the
following examples, which serve to illustrate the present invention
but are not intended to limit its scope in any way.
[0125] Fluorescein (F), rhodamine 6G, 5(6)-carboxyfluorescein (5CF,
6CF), .beta.-lactoglobulin A and B (BLGA and BLGB),
L-1-tosylamide-2-phenylethy- l chloromethyl ketone (TPCK)-treated
trypsin, CHES (2-[N-cyclohexylamino]e- thanesulfonic acid), tricine
(N-tris[hydroxymethyl]methylglycine), Trizma.RTM..multidot.Base
(tris[hydroxymethyl]aminomethane), ammonium acetate, CaCl2,
poly(vinylpyrrolidone) (PVP), sodium pyruvate (99+%),
.beta.-nicotinamine adenine dinucleotide, reduced form
(.beta.-NADH), L-lactate dehydrogenase (LDH-5(M4) 98+% isoenzyme
suspension in 2.1 M (NH4)2SO4), and sodium dodecyl sulfate (SDS)
were purchased from Sigma Chemical Co. (St. Louis, Mo.). Both of
the solutions of .beta.-NAD+ and .beta.-NADH were freshly prepared
and kept in a refrigerator before the experiment. The NADH solution
was covered by black tape to prevent exposure to light. Tween 20
was purchased from Aldrich Chemical Co. (Milwaukee, Wis.).
2,7-diacetate,dichloro-fluorescein (DADCF) was obtained from Acros
(Fair Lawn, N.J.). Ethidium bromide (EtBr) was obtained from
Molecular Probes, Inc. (Eugene, Oreg.). 50-bp and 100-bp DNA
ladders were purchased from Life Technologies (Gaithersburg, Md.).
The sample solutions for CZE were prepared by dissolving the
appropriate amounts of these fluoresceins in 1.times.TBE (0.089 M
Tris, 0.089 M borate, and 0.002 M ethylene diamine tetraacetic acid
(EDTA) in water) buffer with 0.2% (w/w) PVP. For the MEKC
experiments, the analytes and buffer additives were purchased from
Aldrich (Milwaukee, Wis.), J. T. Baker (Phillipsburg, N.J.) and
Sigma Chemical Co. The running buffer was prepared by adding
appropriate aliquots of 1.0 M HCl, 250 mM Brij-S stock solutions,
acetonitrile and 2-propanol into water. The pH was adjusted to 2.4
using 0.1 M HCl or 0.1 M NaOH stock solution and confirmed by a pH
meter. 1.times.TBE buffer was prepared by dissolving pre-mixed TBE
buffer powder (Amresco, Solon, Ohio) in deionized water. The
coating matrix used in Example 2 was made by dissolving 2% (w/v) of
1,300,000 MW PVP into the buffer, shaking for 2 min and letting it
stand for 1 h to get rid of bubbles. Poly(ethylene oxide) (PEO) was
obtained from Aldrich Chemical (Milwaukee, Wis.). The sieving
matrix used in Example 2 was made by dissolving 2% (w/v) 600,000 MW
PEO into the buffer. The solution was stirred vigorously overnight
until all the material was dissolved and no bubbles could be
observed. All buffers for Example 3 were filtered through a
Corning.RTM. Filter System, 0.22-.mu.m cellulose acetate filters
(Corning, N.Y.) or .mu.Star LB.TM., 0.22-.mu.m cellulose acetate
non-pyrogenic filters (Coaster, Cambridge), and degassed prior to
use. The water used to prepare solutions in Example 3 was deionized
with a Milli-Q water purification system (Millipore, Worcester,
Mass.). Bacteria-free 0.2 ml 96-well preloaded plates were obtained
from Marsh Biomedical Products, Inc. (Rochester, N.Y.). Sodium
phosphate monobasic (NaH2PO4.H2O) was purchased from Fisher (Fair
Lawn, N.J.). All water used in Example 4 was purified by a
Millipore water purification system to make sure that there was no
enzyme contamination.
Example 1
[0126] This example demonstrates a multiplexed capillary
electrophoresis system that employs a single linear photodiode
array detector.
[0127] Ninety six fused-silica capillaries (75 .mu.m i.d., 150
.mu.m o.d.; Polymicro Technologies, Phoenix, Ariz.) with 35 cm
effective length and 55 cm total length were packed side by side.
An excimer laser beam was used to burn off the polyimide coating in
the same region of each capillary to provide a "window" for passage
of light from a light source through a sample to be introduced into
and contained within each capillary. At the ground, i.e., exit, end
of the capillary array, the capillaries were bundled together to
allow simultaneous buffer filling and rinsing. At the injection
end, the capillary array was spread out and mounted onto a copper
plate to form an 8.times.12 format with dimensions that fit into a
96-well microtiter plate for sample introduction. In addition, 96
gold-coated pins (Mill-Max Mfg. Corp., Oyster Bay, N.Y.) were
located next to the capillary tips to serve as individual
electrodes. Samples and buffer trays were moved and aligned under
the capillary inlets. This way, the capillary array was never
physically moved. A high-voltage DC power supply from Spellman
(Plainview, N.Y.) provided power for electrophoresis. All 96
electrodes were connected to the same power supply.
[0128] A light source, an optical, i.e., interference, filter, a
capillary array holder, a camera lens and a PDA detector were
placed in a light-tight metal box attached to an optical table. All
optical components were centered 12.6 cm above the optical table.
As the light source, a 12-V tungsten lamp or a 254 nm hand-held
mercury lamp (model E-09816-02; Cole-Parner, Vernon Hills, Ill.)
was used for visible or ultraviolet absorption detection,
respectively. A diagram of a system for use in the present
inventive method is shown in FIG. 1. In the case of the tungsten
lamp (with a filament length of 1.1 cm), the light was first
expanded through a cylindrical lens to cover uniformly the
"windows" of the entire array of capillary tubes, which had a
combined width of 1.5 cm. The hand-held mercury lamp proved to have
a long enough emission length (7 cm), thus no beam expander was
needed for illuminating the entire array. The transmitted light
from the capillary array passed through an interference filter
(Oriel, Stamford, Conn.) and a quartz lens (Nikon, Melville, N.Y.;
f.l.=105 mm; F#=4.5). The interference filter was employed to
define the absorption wavelength. An inverted image of the
capillary array, at a nominal magnification factor of 1.5, was
created by the quartz lens on the face of the PDA. The PDA (model
S5964, Hamamatsu, Bridgewater, N.J.) incorporated a linear image
sensor chip, a driver/amplifier circuit and a temperature
controller. The linear image sensor chip had 1,024 dodes, each of
which was 25 .mu.m in width and 2,500 .mu.m in height. The
temperature controller thermoelectrically cooled the sensor chip to
0.degree. C. to lower the dark count and to minimize temperature
drift, thus enabling reliable measurements to be made over a wide
dynamic range. The built-in driver/amplifier circuit was interfaced
to an IBM-compatible computer (233 MHz Pentium, Packard Bell) via a
National Instrument PCI E series multifunction 16-bit I/O board.
The I/O board also served as a pulse generator to provide a master
clock pulse and a master start pulse required by the linear image
sensor. All codes used to operate the PDA and to acquire the data
were written in-house using National Instruments Labview 4.1
software (Austin, Tex.). The distance between the plane defined by
the capillary array and the plane defined by the PDA detector
elements was 30 cm.
[0129] A very large amount of data were generated for each CE run
using the 1,024-element PDA detector. A run of one hour with a data
acquisition rate of 10 Hz generated 70 Mb of data. All the data
were, therefore, written directly to the hard disk in real time.
Signals from up to ten elements of the PDA could be displayed in
real time in the Labview program. Real-time monitoring of more
pixels was limited by the video speed of our computer. The raw data
sets were extracted into single-diode electropherogram data by
another in-house Labview program. Data treatment and analysis were
performed using Microsoft Excel 97 (Microsoft, Seattle, Wash.) and
GRAMS/32 5.05 (Galactic Industries, Salem, N.H.). The transmitted
light intensities collected at the PDA were converted to absorbance
values using the tenth capillary (buffer solution only) as a
continuous blank reference, i.e., a control. The root-mean-squared
(rms) noise in all of the electropherograms was obtained using a
section of baseline near one of the analyte peaks. This baseline
section was of about the same width as the peaks of interest.
[0130] For the capillary zone electrophoresis experiments, the
capillary array was first flushed with methanol and then water for
clean up. Buffer (pH 8.0, 1.times.TBE with 0.2% (w/w)
polyvinylpyrrolidone (PVP)) was filled into the capillary array
while the injection end was immersed into a buffer tray. After
buffer filling, the filling ends were immersed into a second buffer
tray. The analytes were put into a 96-well microtiter sample plate
(1 .mu.l/well) and injected at the cathode for 6 sec at 11 kV (100
V/cm). The running voltage was also 11 kV. For the CE experiments,
the capillaries were washed for 1 min with buffer between runs. For
the MEKC experiments, the capillary array was first flushed with
0.1 M HCl and then water. The buffer additive used was Brij-S made
by sulfonation of Brij-30 with chlorosulfonic acid (Ding et al.,
Anal. Chem. 70: 1859-1865 (1998)). The analytes were injected at
the cathode for 3 sec at 10 kV and run at the same voltage.
[0131] A typical 96-capillary array image obtained using a tungsten
lamp and the PDA is shown in FIG. 2A, which is a graph of counts
vs. pixel number and represents the image on the PDA of the entire
96-capillary array. As can be seen in FIG. 2B, which is a graph of
counts vs. pixel number and represents the image on the PDA of one
region of the 96-capillary array, the center of each capillary
corresponds to a `peak` (a center peak represented by (a)) in the
image. Between two adjacent capillaries, there is normally a
spacing that also creates a transmission `peak` (a spacing peak
represented by (b)). These `spacing peaks` are usually a bit
broader and have larger intensities (saturated in this case) than
the `center peaks` in this imaging system, as shown in FIG. 2B. If
the array packing is not even, two adjacent capillaries can overlap
each other so that the `spacing peak` between them is not observed.
Including the spacing, each capillary was imaged upon 9-11 diodes
in the PDA. The 96-capillary array covered 912 pixels in total. As
can be seen in FIG. 2B, between the `center peak` and the `spacing
peak,` there is a `valley` (represented by (c)), which corresponds
to the wall of the capillary. When the capillary array image was
well-focused onto the PDA, the intensities of these valleys became
minimized. This feature was used to produce the best focusing. The
diodes that corresponded to the center peaks were used as the
absorption detectors for the corresponding capillaries. Their
intensities are about 40% to 90% of the saturation value of the
diodes. To maintain the relatively uniform intensity distribution
over the capillary array, we found that the emission length of the
light source should be at least two times larger than the width of
the capillary array (1.5 cm). The hand-held mercury lamp (7-cm
emission length) that was used as the UV light source was long
enough for uniform illumination of the entire array. The tungsten
lamp used as the visible light source, however, had only a 1-cm
emission length, so a cylindrical lens needed to be added to
magnify the light source to meet the illumination requirement. FIG.
2A shows a 2.times. variation in optical throughput from the center
to the edge of the array. This means that the detection limit will
vary by {square root}{square root over (2)}X across the array. The
sensitivity (signal), however, will vary by 2.times. unless the
intensities are first ratioed to the blank (buffer) and a log scale
is used (Beer's Law). Given that the mercury lamp was placed very
far from the array, the intensity distribution was, thus, much more
uniform than that in FIG. 2A.
[0132] No mechanical slits were used to define the image. While the
cylindrical capillaries do refract light onto other diodes in the
array, the distance from the array to the camera lens was
maintained at a distance that was greater than the radius of
curvature. Each diode receives a low level of stray light averaged
over all capillaries. This sets the limit on the "valleys" in FIG.
2 but contributes negligibly to cross-talk. The rays of light
crossing the diameters of the capillaries will travel straight and
are properly imaged at the PDA. Sensitivity (absorption path
length) is, thus, also optimized. Given that there are no
mechanical slits and each capillary spans 9 pixels, the system is
extremely stable. No realignment or refocusing is needed, although
period checks of the alignment and focus, such as weekly checks,
should be performed.
[0133] A clear understanding of the noise sources for the array
detector is important, as the noise will ultimately determine the
minimum baseline fluctuation level and, thus, the LOD of the
system. Dark noise of the PDA can be attributed to dark current
shot noise, diode reset noise and circuit noise, which are not
dependent on the number of photoelectrons generated in the diodes
(i.e., the input light intensity). According to the data given by
the manufacturer, the dark noise (s.sub.d) of the PDA is about
3,200 electrons at 0.degree. C. Shot noise is generally defined as
the combined noise associated with the random generation of photons
from the excitation source and the random generation of
photoelectrons in the diode junction, and is equal to the square
root of the number of photoelectrons counted in each diode,
(n.sub.e).sup.1/2. The total rms noise level (s) of the PDA in the
absence of flicker noise (see below) can be expressed using the
equation:
s=s.sub.d+(n.sub.e).sup.1/2. (1)
[0134] Therefore, it is desirable to have as high a photon count as
possible. The electron well capacity of a diode is generally
proportional to the area of the sensing junction. A long but narrow
diode will maximize the dynamic range and the spatial resolution
(in one dimension) at the same time. This comes with an increase in
dark current such that cooling becomes mandatory.
[0135] For the PDA used in this work, the saturation charge for
each diode is about 25 pC, or 156 million electrons. This is almost
three times as high as the PDA used in previous work (Culbertson et
al., Anal. Chem. 70: 2629-2638 (1998)). In real absorption
detection, however, the diodes should only be 85-90% saturated to
allow room for baseline drift due to uncontrollable variables over
the period of data acquisition. The total rms noise for an 85%
saturated diode was calculated to be 14,700 electrons according to
Equation (1), given sd to be equal to 3,200 electrons. Conversion
of this value into absorbance units gave an absorbance noise limit
of 4.7.times.10.sup.-5.
[0136] Actual noise was measured from single-diode
electropherograms. The tungsten lamp was used as the light source
and was moved back and forth behind the capillary array to control
the input light intensity and, thus, the number of photoelectrons
generated at the diode junction. The measured rms noise level of
one diode is linearly proportional to the square root of the number
of photoelectrons generated at the diode junction. The intercept of
the linear regression of the curve can be related to the rms dark
noise according to Equation (1). The measured intercept value was
3,266 electrons, which was close to what was given by the
manufacturer, i.e., 3,200 electrons. Also, the measured slope,
0.92, was close to the theoretical value of 1. When the diode was
more than 25% saturated with the tungsten lamp as the light source,
the major noise source was shot noise. This could be attributed to
the relatively low dark noise of the thermoelectrically cooled PDA
and the superior stability of the battery-powered DC tungsten lamp
(thus a negligible flicker noise). When a PDA was used at room
temperature, the rms noise level was at least 5 times higher. The
measured rms noise of the diode at 85% saturation level can be
converted to an absorbance unit of 4.8.times.10.sup.-5, which is
close to the expected noise limit of 4.7.times.10.sup.-5. When the
hand-held mercury lamp was used, the average measured rms noise for
one diode was 9.0.times.10.sup.-5 at 85% saturation level, which
was about two times higher than the expected value. This is
believed to be due to the additional intensity flicker noise
associated with the mercury lamp.
[0137] Mathematical smoothing can reduce the noise significantly
without distorting the signal if properly used. To ensure that more
data points can be used for smoothing, without sacrificing temporal
response, a higher data acquisition rate needs to be employed. For
the PDA detector, data acquisition rate is limited by the
digitization rate and the exposure time. The A/D converter in our
system is capable of functioning at 25 kHz. So, 40 msec is the
minimum exposure time for each data point in the 1024 array. With
the tungsten or mercury lamp as the light source in this
experiment, a 40-msec exposure time was more than sufficient to
attain around 85% saturation level for all diodes. Normally, an
analyte peak is more than 10 sec in width, and 9 data points are
enough to represent a typical chromatographic or electrophoretic
peak. So, up to 25 data points (1 sec in time) can be used for
smoothing with little sacrifice of the width of the analyte peaks.
Different kinds of smoothing approaches were compared, and boxcar
smoothing proved to be the most efficient method to suppress the
noise here. After 25-point boxcar smoothing, the average rms noise
was lowered to 1.33.times.10.sup.-5 AU at 85% saturation level with
the tungsten lamp as the light source. One can consider smoothing
as increasing the dynamic range (electron well depth) of the diodes
after the fact. The observed enhancement factor is close to the
factor of 5 predicted by Equation (1).
[0138] To probe the actual detection limits achievable using this
capillary array absorption detection system, electrophoresis of
rhodamine 6G, at the concentration of 4.times.10.sup.-7 M in
1.times.TBE buffer solution was performed using 1.times.TBE as the
running buffer. The sample was hydrodynamically injected for 6 sec
at a height difference of 8 cm. No sample stacking was expected
under these conditions. Capillary electrophoresis was run at 250
V/cm. Detection was performed at a wavelength of 552 nm, defined by
an interference filter with 10-nm bandwidth. The electropherogram
from one capillary in the array is shown in FIG. 3, which is a
graph of absorbance vs. frame number, in which the top trace
represents the raw data and the bottom trace represents the data
with a 25-point boxcar smoothing. The S/N for the rhodamine 6G peak
was about 8 (based on a peak height of 0.0002 and an rms noise
between frame 1950 and frame 2250 of 2.6.times.10.sup.-5), which
was near the detection limit predicted by Eq. (1). After 25-point
boxcar smoothing, the S/N ratio was improved to about 45, as shown
in FIG. 3. The resulting 1.8.times.10.sup.-8 M LOD (S/N=2) for
rhodamine 6G injected is comparable to what most commercial
single-capillary machines could achieve.
[0139] The hand-held mercury lamp used in this experiment had much
more fluctuation than the tungsten lamp did, but less than the
pen-light mercury lamp used in previous work (Culbertson et al.
(1998), supra). This is inherent to the discharge nature of the
mercury source as compared to Joule heating in the tungsten source.
While the battery-operated tungsten lamp provided negligible
flicker noise in the system, a double-beam scheme was employed to
cancel the flicker noise due to the mercury lamp. Certain
capillaries in this 96-capillary array were injected with blank
samples (buffer solution), and the signals from them were used as
reference signals. Signals from other capillaries were normalized
to the level of the reference signal from the nearest reference
capillary, and then the reference signal was subtracted from the
normalized signals. FIG. 4, which is a graph of light intensity
(counts) vs. frame number vs. value after subtraction (in counts),
in which (A) is the electropherogram before noise cancellation, (B)
is the reference signal from a blank capillary, and (C) is the
electropherogram after noise cancellation, shows the effect of the
noise cancellation scheme for a signal at about 85% saturation
level. The baseline drift and the intermediate-term noise (i.e.,
those on the time scale of the signal peaks) were reduced. After
normalization to the reference signal, the rms noise of the signal
was lowered to 6.0.times.10.sup.-5 AU from 9.1.times.10.sup.-5 AU.
The short-term (high frequency) noise was actually a bit higher.
However, these were adequately smoothed out by the boxcar algorithm
described above. It was found that, in this 96-capillary array, the
blank signal from one capillary could act well enough as the
reference for signals from ten capillaries on each side. So only
five reference capillaries were needed in the entire 96-capillary
array. We note that since the data in each diode was acquired
consecutively by the digitizer, true temporal correlation of the
flicker noise still does not exist between the reference and the
measurement channels. This contributes to the short-term noise.
This aspect of the system could potentially be improved in the
future with more sophisticated diode arrays with flexible clock
functions.
[0140] FIG. 5, which is a graph of intensity vs. frame number,
shows the result of the MEKC separation of five neutral
(polyaromatic hydrocarbons) compounds, which are, in order,
9,10-diphenyl-anthracene (9.times.10.sup.-5M), benzo[ghi]perylene
(1.times.10.sup.-4 M), benzo[a]pyrene (6.times.10.sup.-5M),
benz[a]anthracene (4.times.10.sup.-5M), fluoranthene
(1.times.10.sup.-4M) and anthracene (5.times.10.sup.-5M). The LOD
(SIN=2) was 1.9.times.10-6 M before smoothing and 9.2.times.10-7 M
after smoothing. The final noise level was higher by about 2-fold
compared to the CZE separation experiment due to the higher
intensity fluctuation of the hand-held mercury lamp, as discussed
above.
[0141] The MEKC separation also generated very high current, which
is 30 .mu.A per capillary and about 3 mA for the whole array.
Therefore, a large amount of heat was produced during the
separation. Some cooling approaches needed to be employed to help
the heat dissipation. It was found that the hottest part in this
setup during the separation was the detection window. This was
because all of the capillaries were densely packed together in this
region. To avoid mechanical vibrations in the capillary array,
which would bring about excess amounts of noise, a laminar flow of
nitrogen gas was created in parallel to the detection window of the
capillary array to carry away the heat generated in this region.
After the nitrogen cooling approach was employed, heat dissipation
was not a problem in this setup.
[0142] To minimize the cross-talk between adjacent capillaries, the
image of a capillary on the face of the PDA needs to be big enough
to ensure that the diode corresponding to the center of the
capillary receives minimal stray light from adjacent capillaries.
It was found if the image of one capillary covers more than 8
diodes in the PDA, cross-talk was less than 0.2%, which is
negligible for the multiplexed analysis. Cross-talk was also found
to be related to the spatial alignment of the capillary array. We
found that the image of each capillary in the array needs to be
parallel to the diodes in the PDA. Otherwise the signal from more
than one capillary may cross over each diode (which is narrow but
long) and cause more cross-talk. The array also needs to be
confined to a plane, or else imaging will not be uniform for each
capillary. Finally, vibrations in the capillaries, especially when
high voltage is applied or when cooling fans are improperly
situated, will cause additional flicker noise in the system. Rigid
mounting of the array and of the optics is, therefore,
critical.
[0143] FIG. 6, which is the result of CZE separation of four
visible dyes in the 96 capillary array, in which the order of dyes
is 5CF (4.times.10.sup.-5 M), 6CF (4.times.10.sup.-5 M), F
(8.times.10.sup.-5 M) and DADCF (1.2.times.10 .sup.-4M), the
horizontal direction represents the location of the capillaries,
the vertical direction represents migration time from 5.3 to 7.0
min, the top plot represents intensity across the array, and the
left plot represents intensity along one of the capillaries.
Relatively uniform separation resolution and S/N distribution can
be observed from the reconstructed image file. Cross-talk between
adjacent capillaries was not observable, as expected for this
analyte concentration range.
[0144] The results for multiplexed CE indicate that the migration
times are highly nonuniform among the capillaries. This is to be
expected from the absence of temperature regulation and variations
in the column surfaces. We have demonstrated that an internal
standardization scheme can be employed to normalize the results
among the capillaries so that the migration times and the peak
areas are reliable enough for high-throughput applications. FIG. 7,
which is graph of migration time vs. capillary number in which the
open symbols represent raw data and the closed symbols represent
data normalized by two internal standards for two fluoresceins,
confirms that, by using the first and the last peaks as the two
internal standards, both the migration times and the peak areas
become uniform across the array. For the second and third peaks the
relative standard deviations (RSDs) of migration times were reduced
from 17% and 22% to 1.9% and 2.0%, respectively, and the RSDs of
peak areas were reduced from 65% and 87% to 3.8% and 8.8%,
respectively. In the case of peak areas, two capillaries (#23 and
#45, see FIG. 8, which is a graph of peak area vs. capillary number
in which the open symbols represent raw data and the closed symbols
represent data normalized by two internal standards for two
fluoresceins) dominated the contributions to the RSDs. If these two
capillaries were omitted from the statistical analysis, the RSDs
were lowered to 2.9% and 5.3%, respectively. The fact that this
normalization scheme (Xue et al., Anal. Chem. 71:2642-2649 (1999))
works equally well with the absorption detector here shows that the
data in FIG. 7B are all within the linear range of the detector.
This is not surprising, since at no time did the intensity decrease
by more than 15% (FIG. 6). For larger absorptions, a logarithmic
correction will need to be applied to maintain a linear
response.
[0145] This example demonstrates for the first time a
high-throughput system for analyzing multiple samples
simultaneously using absorption detection. Uniform separation
efficiency and good S/N were obtained. The LOD achievable for such
a system, such as the 96-capillary array electrophoresis system
described above, is comparable to those for commercial
single-capillary electrophoresis machines using absorption
detection. The separation of ionic and of neutral analytes were
demonstrated by zone electrophoresis and by MEKC, respectively.
Consequently, the capillary array electrophoresis system can do
everything single-capillary electrophoresis absorption instruments
can do, only with much higher throughput. Potentially, the present
inventive system also can serve as an alternative to HPLC in many
applications. No moving parts were used in this system. Once the
positions of all components are fixed, the only thing that needs to
be adjusted is the focal point of the camera lens, just like taking
a picture, to get the best focused image of the capillary array.
One focused, no noticeable changes in the system were observed over
many days. Also, no lasers are used, thus the system should be
smaller, more cost effective and easier to use and maintain than
the multiplexed laser-induced fluorescence CE systems. Besides, the
analytes do not have to be fluorescent to be detected. The
absorption wavelength can be selected by simply changing a filter.
Since the sample injection process involves only moving different
microtiter plates under the injection block and can be fully
automated, it should be possible to obtain a true throughput that
is 100 times higher than what conventional single CE absorption
determinations can achieve.
Example 2
[0146] This example demonstrates the application of the present
invention to genetic typing and diagnosis.
[0147] Based on the 96-capillary array electrophoresis system of
Example 1, DNA analysis protocols were designed to take advantage
of capillary array gel electrophoresis and absorption detection
based on the inherent spectral properties of the DNA bases and the
fact that a 100-bp DNA contains 100 absorbing units that can
provide excellent net absorptivity for sensitive detection. The
method was tested on two broadly used PCR protocols using typical
concentrations of starting materials.
[0148] Samples were prepared as follows:
[0149] Polymerase Chain Reaction
[0150] 1. Multiplexed PCR for Variable Number of Tandem Repeats
(VNTR) Loci
[0151] AmpliFLP D1S80 PCR amplification kit was purchased from
Perkin-Elmer (Foster City, Calif.). The kit included D1 S80 PCR
Reaction Mix (containing two D1S80 primers, AmpliTaq DNA polymerase
and dNTPs in buffer), MgCl.sub.2 solution and Control DNA 3 (human
genomic DNA of D1 S80 type 18, 31 in buffer). The PCR mixtures used
were as follows:
[0152] Positive Control: 20 .mu.l of D1 S80 PCR Reaction Mix, 10
.mu.l of MgCl.sub.2 solution and 20 .mu.l of Control DNA3.
[0153] Negative Control: 20 .mu.l of D1 S80 PCR Reaction Mix, 10
.mu.l of MgCl.sub.2 solution and 20 .mu.l of autoclaved DI
H.sub.2O.
[0154] The polymerase chain reactions were performed with the
following parameters: 30 cycles of denaturation at 95.degree. C.
for 15 sec, annealing at 66.degree. C. for 15 sec, and extension at
72.degree. C. for 40 sec. The thermal cycler used was a
Perkin-Elmer GeneAmp PCR system 2400.
[0155] 2. PCR for Human Immunodeficiency Virus (HIV)
[0156] The HIV testing kit (Perkin-Elmer) included positive control
DNA that includes all parts of the HIV-1 genome, negative control
DNA, HIV primers, AmpliTaq DNA polymerase, dNTPs, PCR reaction
buffer and MgCl.sub.2 solution. The PCR mixtures used are listed in
Table 1. The protocol for the Perkin-Elmer GeneAmp thermal cycler
is 40 cycles of denaturation at 95.degree. C. for 30 sec, annealing
and extension at 62.degree. C. for 1 min. The annealing and
extension temperatures were the same for this amplification.
1TABLE 1 PCR mixtures for HIV amplification Addition Component
Order Volume Final Concentration Autoclaved, deionized water 1 32.8
.mu.l 10.times. PCR buffer II 2 5 .mu.l 1.times. DNTPs 3 1 .mu.l
200 .mu.M each dNTP each HIV-1 primer 1 (SK38) 4 1 .mu.l 0.5 .mu.M
HIV-1 primer 2 (SK39) 5 1 .mu.l 0.5 .mu.M AmpliTaq DNA polymerase 6
0.2 .mu.l 1 unit 25 Mm MgCl.sub.2 solution 7 5 .mu.l 2.5 mM
Positive Control DNA or 8 1 .mu.l 0.5 .mu.g human Negative Control
DNA placental DNA
[0157] DNA Purification
[0158] All PCR products were purified with Microcon YM-30
centrifugal filter devices (Millipore, Bedford, Mass.). After the
purification, salts, dNTPs and most HIV-1 primers were eliminated
from the DNA samples.
[0159] The 96 capillary array electrophoresis system with
photodiode array absorption detection as described in Example 1 was
used. A DC-powered mercury lamp (UVP Inc., Upland, Calif.) was used
as the light source, which gave lower noise levels than the
AC-powered mercury lamp used in previous work. The absorption
wavelength was set at 254 nm by an interference filter (Oriel). The
total length of the capillaries was 55 cm, with 35-cm effective
length. The capillary array was first flushed with deionized water
and then with 1 of 2% PVP at a pressure of 100 psi. While the
injection ends were immersed in the buffer reservoir, 0.5 ml of 2%
PEO (600,000 MW) sieving matrix was pushed into the capillary
bundle at 100 psi. The procedure roughly took 20 min. After the gel
filling, ethidium bromide was added to the buffer reservoirs at the
concentration of 11 .mu.g/ml. The system was then pre-run for 10
min with the electric field strength at 150 V/cm. After the pre-run
ethidium bromide should have spread out evenly in the sieving
matrix through electrical migration. Ethidium bromide is known to
make the DNA fragments more rigid, thereby leading to sharper bands
in CGE. It was not expected to alter significantly the absorption
strength of the DNA fragments in this study. The samples were
injected electrokinetically at 150 V/cm for 15 sec. A field
strength of 150 V/cm was employed for the separation. The total
current was about 620 .mu.A during the separation process. Twelve
different samples were used in the 96-capillary array
electrophoresis experiment, which are detailed in Table 2. Each
type of sample was injected into and run in eight different
capillaries in the 96-capillary array.
2TABLE 2 Sample description in the capillary array electrophoresis
Sample No. Description 1 4 .mu.l of HIV-1 primer-1 (SK38) 2 4 .mu.l
of purified HIV-1 negative PCR product 3 4 .mu.l of purified HIV-1
positive PCR product 4 4 .mu.l of 100 bp ladder 5 3 .mu.l of
purified HIV-1 negative PCR product with 1 .mu.l of 100-bp ladder 6
3 .mu.l of purified HIV-1 positive PCR product with 1 .mu.l of
100-bp ladder 7 4 .mu.l of purified D1S80 negative PCR product 8 4
.mu.l of purified D1S80 positive PCR product 9 4 .mu.l of 50-bp
ladder 10 3 .mu.l of purified D1S80 negative PCR product with 1
.mu.l of 50-bp ladder 11 3 .mu.l of purified D1S80 positive PCR
product with 1 .mu.l of 50-bp ladder 12 4 .mu.l of DI H.sub.2O
[0160] FIG. 9, which is a reconstructed two-dimensional
electropherogram for capillary array electrophoresis, in which the
12 capillaries (corresponding to the 12 samples described in Table
2) are aligned vertically and the migration direction is from left
to right, shows the result of the capillary array gel
electrophoresis for DNA analysis as a reconstructed "gel" image.
The vertical direction represents the capillary array arrangement,
while the horizontal direction represents the migration time. All
separations were finished within 25 min. Capillary #84 (Sample type
11, see Table 2) showed bad separation resolution after 350 base
pairs. The other 95 capillaries gave reasonable separation and good
signal-to-noise ratios. The migration times and peak intensities
were highly non-uniform among the capillaries. This was to be
expected from the absence of temperature regulation and variations
in the column surfaces. An internal standardization scheme can be
employed to normalize the results among the capillaries so the
migration times and the peak areas are reliable enough for
high-throughput applications.
[0161] Actual electropherograms were extracted from capillaries #3,
9, 17, 25, 33, 41, 49, 57, 65, 73, 85 and 89 to represent each type
of sample described in Table 2, and are shown in FIG. 10, in which
the 12 capillaries (corresponding to the 12 samples described in
Table 2) are stacked vertically accordingly to the order in FIG. 9
and the migration times are plotted from left to right. In
capillaries #9-16 and #17-24, negative and positive HIV-1 PCR
products were injected respectively. The positive and negative
results can be easily differentiated through the HIV-1 gag fragment
peak (triangle), which appeared only in the electropherograms from
capillaries #17-24. Both the positive and negative HIV-1 PCR
samples also contained the excess primers (circle) and the primer
dimers (cross). To provide an even higher level of confidence for
identification, despite the variation of migration times among
capillaries, the HIV-1 gag fragments, primers and primer dimers can
be sized by mixing the PCR products with 100-bp DNA ladders
(capillaries #25-32), and injecting them into capillaries #33-40
and #41-48. The electropherograms from the latter two groups of
capillaries showed that the HIV-1 gag fragment is about 115 bp and
the primer dimer is about 60 bp.
[0162] In the electropherograms from capillaries #1-8, the HIV-1
primers gave broad peaks, which we believe are due to sample
overloading. Deionized H.sub.2O was injected into capillaries
#89-96, which gave blank electropherograms. These electropherograms
served as blank references and were subtracted from the signals in
the other capillaries to cancel out the flicker noise from the
mercury lamp, as reported before. The electropherograms from
capillaries #49-56 showed negative D1 S80 PCR results, where only
the primer peaks can be observed. The electropherograms from
capillaries #57-64 showed positive D1 S80 genotyping PCR results.
Two component peaks (D1 S 80 type 18 and 31) can be observed as
expected from the heterozygous samples in addition to the very
broad primer peaks. Again, to increase the confidence level for
identification, the two D1 S80 components as well as the primer
were roughly sized by mixing each PCR product with a 50-bp ladder
(capillaries #65-72) and injecting them into capillaries #73-80
(negative) and #81-88 (positive). The results showed the two D1 S80
components to be about 400-bp and 600-bp.
[0163] Current high-throughput approaches to the analysis of PCR
products are based primarily on electrophoretic separation and
laser-excited fluorescence detection. This example demonstrates
that the present invention can be applied to genetic typing and
diagnosis based simply on UV absorption detection. The additive
contribution of each base pair to the total absorption signal
provides adequate detection sensitivity for analyzing most PCR
products. Not only is the use of specialized and potentially toxic
fluorescent labels eliminated, but also the complexity and cost of
the instrumentation are greatly reduced. For example, no lasers are
used. UV absorption detection of DNA products reduces the cost of
analysis since it does not require labeling. The capillary array
was flushed with water in between runs and did not show any
degradation over tens of runs in a one-month period. Since the
sample injection can be fully automated, this example demonstrates
that it should be possible to obtain a true DNA analysis throughput
that is 100 times (scalable to 1,000 times) higher than what
commercial single capillary gel electrophoresis systems can
achieve, at relatively low cost.
Example 3
[0164] This example demonstrates the application of the present
invention to high-throughput comprehensive peptide mapping of
proteins.
[0165] An experimental CE setup for multi-dimensional 96-capillary
array electrophoresis similar to that of Example 1 was used.
Briefly, a total of 96 fused-silica capillaries (Polymicro
Technologies, Inc., Phoenix, Ariz.), 50-.mu.m i.d. and 360-.mu.m
o.d., with 50-cm effective length and 70-cm total length were
packed side by side at the detection window and clamped between two
flat surfaces of a plastic mount. The window was created after
packing by using an excimer laser beam to burn off the polyimide
coating. At the ground end (outlet), every 12 capillaries were
bundled together to allow simultaneous filling of six-different
buffers for six-dimensional peptide mapping. At the injection end,
the capillary array was spread out and mounted on a copper plate to
form an 8.times.12 format with dimensions to fit into a 96-well
microtiter plate for sample introduction. Gold-coated pins (96)
(MillMax Mfg. Corp.) were mounted on the copper plate near the
capillary tips to serve as individual electrodes, with the
capillary tips slightly extended (.about.0.5 mm) beyond the
electrodes to guarantee contact with small-volume samples. A
high-voltage power supply (Glassman High Voltage, Inc., Whitehorse
Station, N.J.) was used to drive the electrophoresis.
[0166] The light source, filter, capillary array holder, and PDA
detector were all contained in a light-tight metal box attached to
an optical table as described above. As the light source, a
213.9-nm zinc lamp (model ZN-2138, Cole-Parmer) was used for UV
absorption detection. The transmitted light from the capillary
array passed through an interference filter (Oriel) and a quartz
lens (Nikon; f.l.=105 mm; F=4.5). An inverted-image of the
capillary array at a nominal magnification factor of 1.2 was
created by the quartz lens on the face of the PDA. The PDA
(Hamamatsu model S5964, Hamamatsu, Japan) incorporated a linear
image sensor chip (1024 diodes, 25-.mu.m in width, 2500-.mu.m in
height), a driver/amplifier circuit, and a temperature controller.
The built-in driver/amplifier circuit was interfaced to an
IBM-compatible computer (233-MHz Pentium, Packard Bell) via a
National Instrument PCI E Series multifunction 16-bit I/O board.
All codes used to operate the PDA and to acquire the data were
written in house using Labview 5.0 software (National Instruments,
Austin, Tex.).
[0167] The raw data sets were converted into single-diode
electropherograms by another in-house Labview program. Data
treatment and analysis were performed using Microsoft Excel 97 and
GRAMS/32 5.05 (Galactic Industries).
[0168] In the multi-dimensional CE experiments, the capillary array
was first flushed with methanol and then water for cleanup. The six
running buffers used for four-dimensional CZE separations and
two-dimensional MEKC separations were as follows: (1) 50 mM
Trizam.RTM..multidot.Phosphat- e buffer (pH 2.5 with
H.sub.3PO.sub.4), (2) 50 mM sodium acetate buffer (pH 5.0 with
acetic acid), (3) 0.1 M Trizma.RTM..multidot.Base/0.1 M Tricine
buffer (pH 8.1), (4) 0.1 M CHES/0.1 M NaOH (pH 9.3), (5) 0.1% Tween
20 in 50 mM sodium acetate buffer (pH 5.0 with acetic acid), and
(6) 7% Tween 20 and 10 mM SDS in 0.1 M
Trizma.RTM..multidot.Base/0.1 M Tricine buffer (pH 8.1). The
samples were put into a 96-well microtiter sample plate (1
.mu.l/well) and gravity injected at the anode for 60 sec at 8-cm
height. The applied electric field was +157 V/cm and
electrophoresis was performed at ambient temperature. After each
run, the capillaries were rinsed with 0.1 M NaOH, water, and
running buffer for 5 min each.
[0169] All CE separations for CZE and MEKC analyses were optimized
on an ISCO (Lincoln, Nebr.) Model 3140 Electropherograph System
before the multi-dimensional multiplexed CE runs. Bare fused-silica
capillaries (Polymicro Technologies, Inc., Phoenix, Ariz.) with
50-cm effective length and 75-cm total length (50-.mu.m i.d. and
361-.mu.m o.d.) were used. Four different buffer systems were
investigated for CZE separations and two different buffer systems
were also investigated for MEKC separations. The optimal
compositions are described above. The samples were introduced with
hydrodynamic flow by placing the inlet of the capillary into the
sample vial and raising the sample vial 30 cm above the exit vial
and allowing the sample to siphon into the capillary for 10 sec.
The applied electric field was +227 V/cm and electrophoresis was
performed at ambient temperature. The detection wavelength was set
at 214 nm for monitoring peptide fragments. After each run, the
capillary was rinsed with 0.1 M NaOH, water, and running buffer in
order for 5 min each.
[0170] Tryptic digestion of BLGA and BLGB was carried out according
to the procedure of Cobb et al. ((1989), supra) without the
dialysis and lyophilization steps. A mixture of 2 mg/ml BLG and
trypsin was prepared with a 10 mM Trizma.RTM..multidot.Base and 50
mM ammonium acetate buffer (pH 8.2) containing 0.1 mM calcium
chloride. Trypsin was added at a trypsin-protein ratio of 1:50
(w/w), and the digestion mixture was incubated at 37.degree. C. for
5 hr. The digest was directly injected into the separation CE
system without filtration.
[0171] Bovine .beta.-lactoglobulin (BLG) is the major whey protein
of cow's milk. Mature bovine BLG has 162 residues as shown in FIG.
11 [SEQ ID NO: 1], which represents the peptide maps of three
variants of BLG. Three variants of BLG, labeled as A, B, and C,
commonly occur in cow's milk. Variants A and B differ at two sites:
aspartic acid (D) 64 in BLGA is changed to glycine (G) in BLGB, and
valine (V) 118 in BLGA is changed to alanine (A) in BLGB. Variants
B and C differ at one site: glutamine (O) 59 in BLGB is changed to
histidine (H) in BLGC (Bin et al., Protein Science 8: 75-83
(1999)). Tryptic digestion is quantitative and very specific
because trypsin cleaves only at the C-terminal side of lysine and
arginine residues. The theoretical fragments are listed in Table 3.
In the case of BLG, seventeen different peptides exist after
tryptic digestion.
3TABLE 3 Theoretical products of tryptic digest of BLGA and BLGB
Expected fragment Sequence Residues 1 L-I-V-T-Q-T-M-K 1-8 2
G-L-D-I-Q-K 9-14 3
V-A-G-T-W-Y-S-L-A-M-A-A-S-D-I-S-L-L-D-A-Q-S-P-L-R 15-40 4
V-Y-V-E-E-L-K-P-T-P-E-G-D-L-E-T-L-L-Q-K 41-60 5 W-E-N-D-E-C-A-Q-K
(W-E-N-G-E-C-A-Q-K).sup..alpha. 61-69 6 K 70 7 I-A-A-E-K 71-75 8
T-K 76-77 9 I-P-A-V-F-K 78-83 10 I-D-A-L-N-E-N-K 84-91 11
V-L-V-L-D-T-D-Y-K 92-100 12 K 101 13
Y-L-L-F-V-M-E-N-S-A-E-P-E-Q-S-L-V-C-Q-C-L-V-R 102-124
(Y-L-L-F-V-M-E-N-S-A-E-P-E-Q-S-L-A-C-Q-C-L-V-R).sup..alpha. 14
T-P-E-V-D-D-E-A-L-E-K 125-135 15 F-D-K 136-138 16 A-L-K 139-141 17
A-L-P-M-H-I-R 142-148 18 L-S-F-N-P-T-Q-L-E-E-Q-C-M-I 149-162
.sup..alpha.The peptides in parenthesis are fragments of BLGB.
[0172] A multiplexed capillary array system allowed high-throughput
characterization and generation of peptide maps of proteins, after
enzymatic digestion, using six-dimensional capillary
electrophoresis at a constant applied electric field. In a
96-capillary array image obtained using a zinc lamp and the PDA,
the center of each capillary corresponds to a "peak" in the image.
Between every two center "peaks," there is a "valley" which
corresponds to the wall of the capillary. When the capillary array
image was well-focused onto the PDA, the intensities of these
valleys became minimized. This feature was used to produce the best
focusing. "Spacing peaks" (see above) were eliminated in this
study. This results from a special treatment on the window of the
capillary array, whereby epoxy glue was applied between the
capillaries on the detection window. The epoxy glue greatly
strengthened the window area of the capillary array and minimized
movement of the capillaries in the electric field. Because the
epoxy glue is not UV transparent, it absorbed all of the light that
would have passed through the spacing of the capillaries and
eliminated the "spacing peaks." This further reduced stray light
for absorption detection. The zinc lamp provided 213.9-nm light
that is well-suited for the absorption detection of peptides. The
emitting length of the zinc lamp is about 2 cm, which is long
enough for uniform illumination of the entire capillary array (1.5
cm). There was less than 2.times. variation in optical throughput
from the center to the edge of the array. This means that the
detection limit varied by less than {square root}{square root over
(2)}.times. across the array. The zinc lamp was very stable and
produced negligible flicker noise, so no double-beam subtraction
was necessary in this experiment.
[0173] Up to the present, peptide mapping of proteins is primarily
based on the cleavage of proteins with enzyme or chemical agents,
followed by a one- or two-dimensional separation of the resulting
peptide fragments. While the resolution of peptide fragments
achieved by one-dimensional separation is often insufficient to
resolve the complex mixture of peptides, the conventional
two-dimensional techniques suffer from the difficulty of
efficiently recovering uncontaminated peptides from the first
dimension to transfer to the second dimension. Also,
two-dimensional separation conditions have to be changed according
to the sample protein. To overcome these problems a six-dimensional
system was used. By combining four different CZE conditions at
different pHs and two different MEKC conditions, comprehensive and
complementary information about the peptides of arbitrary proteins
was obtained. As shown in the results of single-capillary runs
(FIGS. 15 and 16), the peptide fragments at different separation
conditions showed different but related peptide maps. FIG. 12 shows
the results of the six-dimensional separations of tryptic digests
of BLGA and BLGB in the 96-capillary array, in which (A) is 50 mM
Trizam.RTM..cndot.phosphate buffer (pH 2.5 with H.sub.3PO.sub.4),
(B) is 50 mM sodium acetate buffer (pH 5.0 with acetic acid), (C)
is 0.1% Tween 20 in 50 mM sodium acetate buffer (pH 5.0 with acetic
acid), (D) is 0.1 M Trizma.RTM..cndot.Base/0.1 M tricine buffer (pH
8.1), (E) is 7% Tween 20 and 10 mM SDS in 0.1 M
Trizma.RTM..cndot.Base/0.1 M tricine buffer (pH 8.1), and (F) is
0.1 M CHES/0.1 M NaOH (pH 9.3). The vertical direction represents
the capillary array arrangement, while the horizontal direction
represents the migration time. The applied electric field was +157
V/cm. A column, bare fused-silica capillary with effective/total
length of 50/70 cm and 50 .mu.m i.d. was used. Hydrodynamic
injection was conducted for 60 sec at 8 cm height.
[0174] In the single-capillary CE system, when the electric field
of +227 V/cm was applied to the 50-.mu.m i.d. capillary, the
current was below 20 .mu.A at ambient temperature. However, the
capillaries were packed side by side in the 96-capillary array
system and the dense packing generated a much higher temperature at
the same separation condition. Consequently, some of the
capillaries can lose current during the separation because of the
formation of bubbles. Thus, a lower electric field of +157 V/cm was
applied in spite of an increased analysis time. All separations
were still completed within 45 min. Although capillaries 87 and 88
showed unusually long separation times and much lower signal
levels, all of the other 94 capillaries gave comparable separation
times and good signal-to-noise ratios. As shown in the
reconstructed image, even for the same separation condition, the
migration times and peak intensities were not uniform among the
capillaries. This phenomenon is to be expected from the absence of
temperature control in the experiment (Xue et al. (1999), supra;
and Gong et al. (1999), supra). However, we have demonstrated that
an internal standardization scheme can be applied to normalize the
results among the capillaries (Xue et al. (1999), supra; and Gong
et al. (1999), supra) so that the corrected migration times and
peak areas are of sufficient reliability for high-throughput
applications.
[0175] FIGS. 13 and 14 show the extracted electropherograms of BLGA
and BLGB, respectively, as derived from the six-dimensional data in
FIG. 12. The individual maps are virtually identical to the
single-capillary results. The peptide patterns of BLGA and BLGB can
be easily differentiated in each of the six-dimensional separation
conditions. Compared with the single-capillary run, 0.1% Tween 20
in 50 mM sodium acetate buffer (pH 5.0) gave slower migration times
in the 96-capillary array. In part, this was due to the lower
applied electric field (+157 vs. +227 V/cm). Tween 20 (0.1%),
instead of Tween 20 (0.2%), at the MEKC condition was used in the
array because the latter would have resulted in even longer
analysis times.
[0176] Since peptides are polymers of amino acids, they typically
have a limited number of charged states in their structure
depending on the presence of amino acid moieties with ionizable
side chains (Landers et al., Handbook of Capillary Electrophoresis,
CRC Press (1997), pp. 219-221). This determines the pH ranges for
CE separations beyond which no theoretical optimization can be
performed. At pH<2, all ionizable groups of peptides will be
protonated. The number of basic residues in the peptide chain will
determine the overall charge-state of the molecule. At pH>10,
all ionizable groups will be de-protonated, resulting in a
negatively charged peptide. At these extreme pH conditions, the
separation of peptides cannot be adjusted. At intermediate pHs,
partly ionized termini and side chain residues allow optimization
of the peptide separation. Therefore, four different pH conditions,
i.e., pH 2.5, 5.0, 8.1, and 9.3, were selected for the separation
of peptide fragments. Since ionization of peptides generally occurs
over a pH range of 2.5-3.0, these pHs are sufficiently far apart to
give independent electropherograms but are close enough to form a
continuous (combinatorial) set of conditions.
[0177] FIG. 15 shows typical peptide maps of BLGA and BLGB at four
pH conditions for CZE using a single capillary after tryptic
digestion, in which (A) is 0.1 M CHES/0.1 M NaOH (pH 9.3), (B) is
0.1 M Trizma.RTM..cndot.Base/0.1 M tricine buffer (pH 8.1), (C) is
50 mM sodium acetate buffer (pH 5.0 with acetic acid), and (D) is
50 mM Trizma.RTM..cndot.phosphae buffer (pH 2.5 with
H.sub.3PO.sub.4). The applied electric field was +227 V/cm.
Separation was at ambient temperature. A column, bare fused silica
capillary with effective/total length of 50/75 cm and 50 .mu.m i.d.
was used. Hydrodynamic injection was conducted for 10 sec at 30 cm
height. All peptide peaks at the four different separation
conditions were well-resolved within about 30 min. Each condition
revealed different peptide maps. The separation of peptides above
pH 5.0 were completed quickly and efficiently within 15 min (FIG.
15A, B and C). The electropherograms at pH 2.5 showed good
resolution in spite of a relatively long analysis time (FIG. 15D).
Adsorption of the proteins on the capillary wall in CE can be a
serious problem. This can lead to variable migration times, band
broadening, and peak tailing. At pH 2.5, much of the negative
charge had been titrated off the silica walls of the capillary such
that there was little coulombic interaction between the peptide and
the wall. This is not true of other pH conditions. Therefore, high
ionic strengths were used in the running buffers to provide
efficient separations because of reduced interaction between the
peptide fragments and the capillary wall.
[0178] FIG. 15 (FIG. 15A at pH 9.3; FIG. 15B at pH 8.1; FIG. 15C at
pH 5.0; and FIG. 15D at pH 2.5) shows the differences in the
peptide maps of BLGA and BLGB at four different CZE conditions.
Although the amino acid sequences of bovine BLGA and BLGB differ
only at two sites (64 and 118, FIG. 11, SEQ ID NO: 11) among the
162 amino acids, the differences in the peptide fragments of BLGA
and BLGB could be clearly identified. Peptide mapping by CE is
increasingly being utilized as a complement, if not a viable
substitute, for the already established technique of HPLC. CE has
several advantages over HPLC, including much higher efficiency and
a smaller sample requirement. Moreover, CE also offers a
straightforward correlation of migration time with physiochemical
properties. According to the dependence on pH, the net charges of
each peptide fragment can be confirmed. Interpretation of the
combined peptide maps at the four different conditions (FIGS.
15A-D) showed comprehensive data with overlapping redundancy for
the peptides that cannot be obtained using only one separation
condition.
[0179] The addition of surfactants to the running buffer can add
several new aspects to the separation mechanism. Above their
critical micelle concentration, the surfactants form micelles,
introducing a pseudo-stationary phase into the running buffer. FIG.
16 shows MEKC peptide maps of BLGA and BLGB obtained at two
different MEKC conditions using a nonionic surfactant, Tween 20
(i.e., 0.2% Tween 20 in 50 mM sodium acetate buffer (pH 5.0 with
acetic acid) (A and B), and/or the combination of nonionic and
anionic surfactant, Tween 20+SDS (i.e., 7% Tween and 10 mM SDS in
0.1 M Trizma.RTM..cndot.Base/0.1 M tricine buffer (pH 8.1) (C and
D). The use of the surfactants in the intermediate-pH range was
appropriate because more neutral peptides should exist there than
at the extreme pHs such as pH 2 or 10.
[0180] In the MEKC conditions using a nonionic surfactant (MEKC I
conditions) (FIG. 16, A and B), the electropherograms showed higher
resolution compared to the electropherograms obtained using CZE
conditions (FIG. 15C). There are some minor shifts in relative peak
positions, mostly for the early eluting components. At pH 5.0, as
the concentration of the nonionic surfactant Tween 20 is increased,
the resolution of peptide fragments increased without an increase
in the current. Although all of the peptide fragments showed
baseline separation above 0.3% Tween 20, the analysis time was
approximately 1 hr. Under the applied electric field of +227 V/cm,
0.2% Tween 20 was sufficient to improve the resolution compared to
CZE. This buffer was selected so that the analysis time can be kept
under 20 min.
[0181] In the MEKC conditions using the combination of nonionic and
anionic surfactants (MEKC II conditions) (FIG. 16, C and D), the
higher concentration of Tween 20 and the anionic surfactant SDS
were needed to increase the resolution. Because higher pH caused
larger mobilities for the peptide fragments in solution, a lower
frequency of dynamic partition into the micelles resulted. However,
as the concentration of surfactants in the running buffer is
increased to favor partition, the analysis time also increased and
the sensitivity decreased. So, 7% Tween 20 and 10 mM SDS were
chosen as optimal surfactant concentrations for MEKC II. The unique
advantage of combining Tween 20 and SDS led to the full resolution
of all peptide fragments, enabling higher resolution for both
neutral peptides and same-charged peptides. FIG. 17 shows the
effect of Tween 20 concentration at pH 8.1 on the MEKC peptide maps
of BLGB in terms of migration time (min). It is clear that both
resolution and selectivity are affected by the surfactant. The
patterns of the peptide maps obtained at the six different CE
conditions above (FIGS. 15 and 16) were reproducible as judged by
comparing the results of five repeated injections in each case.
[0182] This example demonstrates high-throughput, multi-dimensional
peptide mapping of proteins in accordance with the present
invention, such as by using multiplexed capillary electrophoresis.
Unique fingerprints of closely related sample proteins, BLGA and
BLGB, on a 96-capillary array with six different separation
conditions were obtained within 45 min. The 214-nm monitoring of
peptide bonds is universal but also very complex because a typical
map generally contains 20-150 peaks (Dong et al. (1992), supra) and
all of the fragments should ideally be totally resolved. Maps of
unknown proteins are not unambiguous by using only one- or
two-dimensional separation. With six complementary separation
conditions, there is no need to reoptimize the protocol for each
new sample. The instrumental set-up is simplified and automation of
the method becomes possible. Most importantly, this
multi-dimensional and multiplexed peptide mapping technique is
inherently a small-volume and high-throughput approach. For
example, although only two proteins are studied here, sixteen
different proteins can be mapped at the same time starting with
.mu.l samples. Alternatively, different enzymes or chemical agents
can be employed to provide complementary sets of peptide maps for
further confirmation. For complex unknown samples, internal
standards can be used to normalize the migration times and peak
areas (Xue et al. (1999), supra; and Gong et al. (1999), supra).
The six separation conditions then can be used to rank order the
peptide fragments with respect to their isoelectric points (pIs),
very much like a high-resolution gradient elution. The use of
different sets of buffer systems in each capillary in the array is
in effect a combinatorial approach to developing the best
separation conditions for a given group of analytes. This last
feature should be generally useful in all applications of CE.
Finally, capillary arrays are compatible with on-column digestion
of proteins (Chang et al., Anal. Chem. 65: 294-2951 (1993)) so that
full automation in multiple channels (Chang et al. (1992), supra;
Zhang et al. (1999), supra; and Zhang et al., Anal. Chem. 71:
1138-1145 (1999)) is possible with sub-microliter volumes of
samples and reagents.
Example 4
[0183] This example demonstrates the use of the present invention
in combinatorial screening of enzyme activity.
[0184] The multiplexed capillary system described above was used. A
total of 96 fused-silica capillaries (50-.mu.m i.d., 150-.mu.m
o.d.; Polymicro Technologies, Phoenix, Ariz.) packed side by side
with 50-cm effective length and 70-cm total length were used for
separating reactants, products and enzymes. Preloaded (0.2 ml)
96-well plates were used as reactors for carrying out the enzyme
reactions. A 254-nm mercury lamp was used for UV absorption
detection. A voltage of +11 kV (.about.157 V/cm) was applied across
the capillaries for separation.
[0185] During the period of incubation, the plates were covered by
plastic film to minimize evaporation of the reaction solution. This
allowed the concentrations of enzyme and substrate to remain as
stable as possible.
[0186] Because of the inhibition of pyruvate on the catalysis of
LDH, an optimal concentration of pyruvate is important. At pH 7.2
and 25.degree. C., 2 mM pyruvate is normally suitable for the
M.sub.4 isoenzyme. So, in the reaction buffers (20 mM phosphate, pH
ranges from 5.8.about.8.0), 2.0 mM NADH and .about.2.0 mM pyruvate
were added as the substrates for the enzymatic reaction. The
direction of reaction was chosen as follows: 1 NADH + pyruvate LDH
lactate + NAD + .
[0187] The reaction was allowed to proceed for a fixed period prior
to hydrodynamic injection of the reactants, products and enzymes
into the capillaries. A solution of 10 mM phosphate with pH of 8.0
was used as the separation buffer. After applying voltage, all
components were readily separated due to different mobilities.
Since low concentrations of enzyme
(5.times.10.sup.-10.about.1.times.10.sup.-8 M) (pseudo-first-order
reaction) were used, the amount of NAD.sup.+ formed during a given
period of time at a fixed temperature is linearly proportional to
the LDH activity. Therefore, the LDH activity can be quantified by
measuring the peak area of NAD.sup.+ formed during the fixed
incubation period. The areas of the NAD.sup.+ peak and the NADH
peak were integrated. Because NAD.sup.+ and NADH both absorb at 254
nm, while the enzyme does not contribute much to the background at
this wavelength, a 254-nm mercury lamp was used. By measuring the
absorption coefficients of NAD.sup.+ and NADH at 254 nm in a
conventional spectrophotometer, the peak areas were converted to
amounts. The ratio between the NAD.sup.+ amounts formed and the
original NADH amounts was calculated. Since a small background
reaction exists, blank reactions were monitored and subtracted.
[0188] Separate 20 mM phosphate buffers with pH values of 5.8, 6.3,
6.5, 6.7, 7.0, 7.3, 7.6 and 8.0 were prepared. Buffer solution (175
.mu.l), 10 .mu.l enzyme solution, 10 .mu.l 40 mM NADH and 5 .mu.l
90 mM pyruvate were added into 96 wells for reaction. Reactions
progressed at room temperature. The various final concentrations of
enzyme in those solutions were 5.times.10.sup.-10 M,
2.times.10.sup.-9 M, 3.times.10.sup.-9 M, 4.times.10.sup.-9 M,
5.times.10.sup.-9 M, 6.times.10.sup.-9 M, 7.times.10.sup.-9 M,
8.times.10.sup.9 M and 1.times.10-8 M. Thus, for every
concentration of enzyme, there were 8 different buffer solutions.
At the same time, for every buffer solution with a different pH
value, there were 9 different concentrations of the enzyme. The
reaction mixtures were incubated for 30 min, 78 min, 128 min, 180
min, 308 min, 420 min, 480 min and 1,477 min. After each incubation
period, hydrodynamic injection was used to initiate CE analysis.
The volumes withdrawn each time from the reaction vials were
negligible compared to the starting volumes. Therefore, essentially
non-intrusive monitoring was achieved.
[0189] The reason for using hydrodynamic injection is that
electrokinetic injection would have caused serious errors. This is
because of different surface conditions from capillary to
capillary, different compositions of the buffer solutions,
different capillary temperatures, and different migration
velocities of NADH and NAD.sup.+ (Lee et al., Anal. Chem. 64:
1226-1231 (1992)). The effect is obvious from a comparision of FIG.
18A, which is a graph of the ratio of the amount of NADH (injected)
to the amount of NAD (injected) vs. the results from nine
electrokinetic injections, and FIG. 18B, which is a graph of the
ratio of the amount of NADH (injected) to the amount of NAD
(injected) vs. the results from nine hydrodynamic injections.
There, a solution with 1 mM NADH and 1 mM NAD.sup.+ was prepared
and injected separately by hydrodynamic injection and
electrokinetic injection into 9 different capillaries. A 5 cm
difference in height was maintained for 60 sec for the former and
+11 kV was applied for 30 sec for the latter. By applying +11 kV to
the capillaries, the NADH and NAD.sup.+ sample zones were separated
and driven across the detection windows. Since all of the solutions
had the same NADH and NAD.sup.+ concentrations, identical peak
areas were expected for the NADH peaks and NAD.sup.+peaks.
According to FIG. 18, hydrodynamic injection only caused a very
small standard deviation (SD), which was about 0.027 with a mean of
0.817, while electrokinetic injection caused serious errors, with
an SD of 0.483 and a mean of 3.66. Such an injection problem is
more serious in capillary arrays compared to repeated injections in
a single capillary because of surface heterogeneity.
[0190] The migration times varied from capillary to capillary,
because the conditions of the capillaries were different. Despite
the different migration times, the NADH and NAD.sup.+ peaks were
easily identified in this simple mixture. These peak areas were
used for quantitation. After each incubation period, the fraction
of NADH converted to NAD.sup.+ was calculated using the following
equations: 2 amount of NADH ( reacted ) = NAD + area .times. NADH
NAD + fraction of NADH ( reacted ) = amount of NADH ( reacted )
amount of NADH ( reacted ) + NADH area .
[0191] As stated before, the fraction of NADH (reacted) then could
be used to represent the activity of the enzyme whenever this
reaction is pseudo-first-order. The use of a ratio avoids problems
with variations in detection sensitivity and injected amounts among
capillaries. Additional corrections for the different speeds of the
analytes passing the detector were made since hydrodynamic
injection was employed (Lee et al. (1992), supra).
[0192] Since 8 pH conditions and 9 enzyme concentrations were
screened, there were a total of 72 channels (capillaries) where
products were detected. In addition 16 channels of background
reaction (8 pH conditions with substrates but no enzymes) were
monitored. The other 8 channels in the array were filled with the
buffer (no substrate and no enzyme) as the absorption reference.
The entire data set for the 96-capillary array is shown as a
reconstructed image in FIG. 19, which is a reconstructed absorption
image of combinatorial screening of enzyme activity in a 96
capillary array in which the capillaries (1-96) are arranged from
top to bottom and migration time (0-33 min) is plotted from left to
right. The change in electroosmotic flow from one capillary to the
next and temperature variations are the main reasons for
substantial variations in migration times among the channels. Even
larger variations are expected because the sample pH and sample
ionic strengths are all different. The capillary walls will become
dynamically altered as a result of injection. Internal standards
can be employed to normalize the migration times, but were not
needed for the simple electropherograms in this study. The
intensity variations in FIG. 19 are partly due to uneven
illumination but mostly due to the expected variations in the
extents of reaction in each channel.
[0193] Extracted electropherograms for activity screening are shown
in FIGS. 20 and 21. At a constant pH (FIG. 20), it is easy to see
that the extent of reaction increases with LDH concentration (left
peak is NAD.sup.+ (product); right peak is NADH (reactant); from
top to bottom, the concentrations are 0, 0.5 nM, 2 nM, 3 nM, 4 nM,
5 nM, 6 nM, 7 nM, 8 nM and 10 nM; the enzyme is not detected at
this concentration). At a fixed LDH concentration, i.e.,
5.times.10.sup.-9 M (FIG. 21), it can be seen that there is an
optimum pH where the LDH activity is the highest (left peak is
NAD.sup.+ (product); right peak is NADH (reactant); from top to
bottom, the pH are 5.8, 6.3, 6.5, 6.7, 7.0, 7.3, 7.6 and 8.0; the
enzyme is not detected at this concentration).
[0194] The quantitative optimization results shown in FIG. 22,
which is a graph of NADH conversion percentage vs. pH for series
1-9 at 180 min incubation, need to be examined more carefully by
repeated sampling of the reaction mixture at well-defined time
intervals. For short reaction times (FIG. 23, which is a graph of
reaction percentage vs. pH for series 1-9 for 30 min of LDH
catalysis), there is a linear increase in the extent of reaction as
a function of LDH concentration for all pH conditions. This is
ideal behavior. For long reaction times (FIG. 24, which is a graph
of reaction percentage vs. pH value for series 1-9 for 24 hr of LDH
catalysis), nonlinearity is observed, especially when the fraction
reacted exceeds 0.6. The reason for this is saturation of the
reaction. When a significant fraction of the reagent (NADH) is
consumed, the pseudo-first-order reaction description fails. The
remedy is to use higher reagent concentrations or to stay with
short reaction times. This feature shows the importance of
monitoring the full kinetics of reactions as opposed to
single-point monitoring. FIG. 24 by itself would have led to the
incorrect conclusion that there is not much difference in enzymatic
activity over a broad pH range.
Example 5
[0195] This example demonstrates the use of the present invention
in combinatorial screening of homogeneous catalysis and the
optimization of a homogeneously catalyzed synthetic organic
reaction.
[0196] The present inventive method was used to analyze a new
palladium-catalyzed annulation reaction (Zhang et al., J.
Organometal. Chem. 576: 111-124 (1999)), which readily affords
.gamma.-carbolines, noteworthy for their biological activity. The
optimal reaction conditions and the regiochemistry for this type of
annulation are generally highly dependent on the nature of the
palladium catalyst and the base employed. Previous efforts to
optimize this process employed 5% Pd(OAc).sub.2, 10% PPh.sub.3 and
Na.sub.2CO.sub.3 as base and afforded a 1:1 ratio of isomers A/B in
essentially a quantitative yield. 1
[0197] The nature of this and other catalytic reactions is that a
lot of parameters can affect the yield and "optimum" conditions are
often found by trial and error. The above reaction was run using
0.25 mmole in 5 ml of dimethyl formamide (DMF). The volume was
reduced to 120 .mu.l by using 6 mm O.D. glass tubes sealed at one
end and arranged in a 96-well format. The individual components
were added as a DMF solution or as a slurry by pipetting. Septums
were used to cap the reaction tubes to prevent evaporation. All
reactions were thus run on a 5 .mu.mole scale. Heating was provided
by a dry heat bath kept at 110.degree. C. As an internal standard,
1 .mu.mole of norharman was added to the reaction mixture. No
catalytic effect on the system from the addition of the norharman
was observed in control experiments. FIG. 25 shows the separation
of the two isomeric forms (A and B) of the product from the
reagents and the internal standard using two different buffers (40
mM NH.sub.4OAc and 0.75% formic acid in methanol for 1a; 40 mM
NH40ac and 0.75% formic acid in 80% DMf/20% H.sub.2O for 1b), with
an applied electric field of 140 V/cm, using bare, fused-silica
capillaries with an effective/total length of 50/75 cm and 50 .mu.m
I.D. hydrodynamic injection for 15 sec at 8 cm height. Ethanol and
pure DMF were also tested, but the separation was not acceptable.
No bubbles were found in CAE, even when a low boiling point
solvent, such as methanol, was used.
[0198] One important feature of the experimental protocol is that
the reaction mixture was injected into CAE without diluting or
quenching before analysis. At predetermined times during the
reaction, the reaction block was removed from the heating platform,
quickly cooled and put under the injection ends of the capillary
array. No deleterious effect on the catalytic system was observed
by this operation. By avoiding sample manipulation (e.g. by
pipetting out of the reaction vials), errors associated with
transfer and contamination can be reduced. The CAE running buffer
should be compatible with the reaction buffer for hydrodynamic
injection. When using methanol as the buffer, injection was not
uniform. Only about half of the 96 capillaries had adequate signal.
It was not possible to increase the injection time, because some
capillaries then became overloaded. When DMF-based buffer was used,
all 96 channels had uniform signal over three consecutive runs.
This buffer compatibility issue for CAE may be attributed to the
differences in solution properties, such as viscosity and surface
tension, and was not observed in single-capillary experiments. The
total analysis time is typically 60 min, plus 30 min for capillary
cleaning. Judging from the resolution in FIG. 25, the capillaries
could have been shortened to 25% of the effective length to provide
analysis times of 15 min.
[0199] By choosing 8 different Pd catalysts and 11 different bases,
88 different combinations were tested. FIG. 26 shows such a
96-capillary separation for the reaction conditions for 1b in FIG.
25 and a hydrodynamic injection of 1 min, in which the horizontal
direction spans 88 capillaries (the remaining 8 capillaries
contained solvent only and were not plotted) and the vertical
direction represents time. Information on the total yield (FIG. 27,
which is a 3-dimensional bar graph of yield vs. catalyst vs. base,
for reaction after 17 hr at 110.degree. C., in which dppe is
bis(diphenylphosphino)ethane, TABC is tetra-n-butylammonium
chloride, DABCO is 1,4-diazabicyclo[2.2.2]octane, and dba is trans,
transdibenzylidene-acetone), selectivity (FIG. 28, which is the
selectivity plot of two isomers produced by the reactions, wherein
P1/P2 is the ratio of the two isomers A and B, respectively) and
reaction kinetics (FIG. 29, which is a line graph of fractional
conversion vs. time (hr) vs. base, for the reaction using
Pd(PPh.sub.3).sub.4 as the catalyst and various bases) can be
obtained from the electropherograms. By using Pd(OAc).sub.2 with
the ligand PPh.sub.3 as catalyst and Na.sub.2CO.sub.3 as the base,
a total yield of 84% was achieved with virtually no
regioselectivity in the microreactor, compared with a quantitative
conversion (90% after 17 hours) with no selectivity under the
protection of N.sub.2 in a 5 ml reaction. Among all of the bases,
inorganic bases proved to be more effective in promoting the
reaction. When pyridine or other organic bases were used, the yield
was low and some side products appeared. The ability to detect side
products is clearly an advantage of CAE. Preliminary results also
reveal several new conditions which are quite effective in this
annulation reaction. They are Pd(PPh.sub.3).sub.4 with
Na.sub.2CO.sub.3 (C9, 74%), Pd(dba).sub.2 with K.sub.2CO.sub.3
(E10, 72%), PdBr.sub.2 plus 2PPh.sub.3 with Na.sub.2CO.sub.3 (G9,
88%) and PdBr.sub.2 plus 2PPh.sub.3 with K.sub.2CO.sub.3 (G10,
96%). The latter two are in fact superior to the previous best
catalytic condition (Zhang et al. (1999), supra). Complete
regioselectivity is not observed in any of the test conditions
(FIG. 28), even though some prove to be better than other systems.
The conditions G2, H2, and B1 have some selectivity, but
unfortunately their yields are low. The differences in the rates
and the shapes of the plots in FIG. 29 illustrates the need to
monitor the reactions at several points in time. No attempt was
made to correlate the reaction mechanism with the kinetics in this
work.
[0200] In summary, a new methodology, nonaqueous capillary array
electrophoresis coupled with microreaction, is developed to address
the throughput needs of combinatorial approaches to homogeneous
catalysis and reaction optimization. Catalytic activity,
selectivity and kinetics of the various combinations are determined
quickly. This method is potentially useful in the screening for
asymmetric catalysts and drugs and combinatorial library
synthesis.
INCORPORATION BY REFERENCE
[0201] All sources (e.g., inventor's certificates, patent
applications, patents, printed publications, repository accessions
or records, utility models, World-Wide Web pages, and the like)
referred to or cited anywhere in this document or in any drawing,
Sequence Listing, or Statement filed concurrently herewith are
hereby incorporated into and made part of this specification by
such reference thereto.
GUIDE TO INTERPRETATION
[0202] The foregoing is an integrated description of the invention
as a whole, not merely of any particular element or facet thereof.
The description describes "preferred embodiments" of this
invention, including the best mode known to the inventors for
carrying it out. Of course, upon reading the foregoing description,
variations of those preferred embodiments will become obvious to
those of ordinary skill in the art. The inventors expect ordinarily
skilled artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
as specifically described herein. Accordingly, this invention
includes all modifications and equivalents of the subject matter
recited in the claims appended hereto as permitted by applicable
law.
[0203] As used in the foregoing description and in the following
claims, singular indicators (e.g., "a" or "one") include the
plural, unless otherwise indicated. Recitation of a range of
discontinuous values is intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, and each separate value is incorporated into the
specification as if it were individually listed. As regards the
claims in particular, the term "consisting essentially of"
indicates that unlisted ingredients or steps that do not materially
affect the basic and novel properties of the invention can be
employed in addition to the specifically recited ingredients or
steps. In contrast, the terms "comprising," "having," or
"incorporating" indicate that any ingredients or steps can be
present in addition to those recited. The term "consisting of"
indicates that only the recited ingredients or steps are present,
but does not foreclose the possibility that equivalents of the
ingredients or steps can substitute for those specifically
recited.
* * * * *