U.S. patent application number 12/634349 was filed with the patent office on 2014-02-20 for capillary electrophoresis fluorescent detection system.
This patent application is currently assigned to Advanced Analytical Technologies, Inc.. The applicant listed for this patent is Ho-ming Pang, Wei Wei. Invention is credited to Ho-ming Pang, Wei Wei.
Application Number | 20140048722 12/634349 |
Document ID | / |
Family ID | 41571488 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140048722 |
Kind Code |
A9 |
Pang; Ho-ming ; et
al. |
February 20, 2014 |
CAPILLARY ELECTROPHORESIS FLUORESCENT DETECTION SYSTEM
Abstract
The invention includes a high sensitivity and high throughput
capillary electrophoresis multiwavelength florescence detection
system. The fluorescent detection system is configured to
illuminate a relatively large volume of a single capillary or a
plurality of capillaries, with a pixelated detection system capable
of imaging an area of each capillary that differentiates the
capillary walls, the space between the capillaries, and the
internal liquid volume within the capillary. Only the desired
pixels or image area are used for processing and generating an
output signal.
Inventors: |
Pang; Ho-ming; (Ames,
IA) ; Wei; Wei; (Ames, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pang; Ho-ming
Wei; Wei |
Ames
Ames |
IA
IA |
US
US |
|
|
Assignee: |
Advanced Analytical Technologies,
Inc.
Ames
IA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20100140505 A1 |
June 10, 2010 |
|
|
Family ID: |
41571488 |
Appl. No.: |
12/634349 |
Filed: |
December 9, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11299643 |
Dec 12, 2005 |
|
|
|
12634349 |
|
|
|
|
61121043 |
Dec 9, 2008 |
|
|
|
Current U.S.
Class: |
250/459.1 ;
250/458.1 |
Current CPC
Class: |
G01N 27/44726 20130101;
G01N 27/44782 20130101; G01N 21/645 20130101; G01N 21/6428
20130101; G01N 27/447 20130101; G01N 27/44721 20130101; G01N
27/44791 20130101 |
Class at
Publication: |
250/459.1 ;
250/458.1 |
International
Class: |
G01J 1/58 20060101
G01J001/58 |
Claims
1. A fluorescence detection system, comprising: a sample vessel; a
light source to emit light to excite a fluorescently labeled
sample, the system configured to direct light on the sample vessel
to illuminate a volume of more than about 100
micrometers.times..pi.r.sup.2, where r is one-half of the inner
diameter of the sample vessel; and a fluorescence detector
positioned to detect the fluorescent emissions of the sample.
2. The fluorescence detection system of claim 1, wherein the light
is directed on the sample vessel to illuminate a volume of more
than about 500 micrometers.times..pi.r.sup.2.
3. The fluorescence detection system of claim 1, wherein the light
is directed on the sample vessel to illuminate a volume of more
than about 1,000 micrometers.times..pi.r.sup.2.
4. The fluorescence detection system of claim 1, wherein the
detection system includes at least two light sources.
5. The fluorescence detection system of claim 1, wherein the light
emitted by the light source is incoherent.
6. The fluorescence detection system of claim 1, wherein the light
source is a LED.
7. The fluorescence detection system of claim 1, wherein the light
source is optically coupled to an optical fiber bundle that
transmits the light emitted by the light source to the sample
vessel.
8. The fluorescence detection system of claim 7, wherein a distal
end of the optical fiber bundle includes a generally rectangular
shape.
9. The fluorescence detection system of claim 7, wherein a distal
end of the optical fiber bundle is oriented such that the light is
angled at about forty-five degrees relative to the sample
vessel.
10. The fluorescence detection system of claim 7, further including
a first filter between the light source and the optical fiber
bundle.
11. The fluorescence detection system of claim 1, further including
a lens to focus the fluorescent emissions onto the fluorescence
detector.
12. The fluorescence detection system of claim 1, further including
a second filter between the sample vessel and the fluorescence
detector.
13. The fluorescence detection system of claim 1, the fluorescent
detector being configured to detect fluorescent emissions at more
than one wavelength.
14. The fluorescence detection system of claim 1, wherein the
sample vessel is a capillary having an inner diameter of about 20
to about 100 micrometers.
15. The fluorescence detection system of claim 1, wherein the
fluorescence detector includes a CCD detector.
16. The fluorescence detection system of claim 1, wherein the
system is configured to direct light on more than one sample
vessel.
17. A method of detecting a fluorescence emission in a sample the
method comprising: introducing a fluorescently labeled sample into
a sample vessel; directing a light from a light source to excite
the fluorescently labeled sample, the light directed on the sample
vessel to illuminate a volume of more than about 100
micrometers.times..pi.r.sup.2, where r is one-half of the inner
diameter of the sample vessel; and detecting a fluorescent emission
from the fluorescently labeled sample with a fluorescence
detector.
18. A fluorescence detection system, comprising: a sample vessel; a
light source to emit light and to excite a fluorescently labeled
sample, the system configured to direct light on the sample vessel
to illuminate a volume of more than about 50
micrometers.times..pi.r.sup.2, where r is one-half of the inner
diameter of the sample vessel; a fluorescence detector positioned
to detect the fluorescent emissions image of the sample, whereby
the fluorescence detector has the resolution to detect three
distinct regions of the image, including a wall of each capillary,
an internal volume of each capillary, and a space between the
capillaries, each region being defined by at least one pixel; and a
processor capable of selecting which pixels to process for an
integration of light intensity, whereby pixels from the walls of
the capillary and pixels from the space between the capillaries are
specifically excluded from the integration of the signal.
19. The fluorescence detection system of claim 18, wherein at least
5 pixels are used to define the internal volume of each
capillary.
20. The fluorescence detection system of claim 18, wherein the
light is directed on the sample vessel to illuminate a volume of
more than about 100 micrometers.times..pi.r.sup.2.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 61/121,043, filed Dec. 9, 2008, titled
Capillary Electrophoresis Fluorescent Detection System, the
contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to capillary electrophoresis (CE)
fluorescence detection systems.
BACKGROUND OF THE INVENTION
[0003] Capillary electrophoresis (CE) instruments use electric
fields to separate molecules within narrow-bore capillaries
(typically 20-100 .mu.m internal diameter). CE techniques are
employed in numerous applications, including DNA sequencing,
nucleotide quantification, and mutation/polymorphism analysis.
Samples analyzed by CE are often detected by fluorescence emission
of the sample which has been tagged with a fluorophore. The
fluorophores are excited with a light source, and the intensities
of the fluorescence emission represent the concentration or amount
of the sample components. Generally, the light source is focused on
a narrow point on the sample to maximize the energy available for
the excitation of fluorophore within the illuminated volume. The
detector, which is usually a photomultiplier, photodiode, diode
array, or CCD, is positioned to capture the maximum amount of light
from the sample, without specific discrimination of the capillary
walls or the background.
SUMMARY OF THE INVENTION
[0004] Embodiments of the invention provide a high sensitivity and
high throughput capillary electrophoresis multiwavelength
florescence detection system. The fluorescent detection system is
configured to illuminate a single capillary or a plurality of
capillaries, with a pixelated detection system capable of imaging
an area of each capillary that differentiates the capillary walls,
the space between the capillaries, and the internal liquid volume
within the capillary. The detector is coupled to a computer
processing system capable of selecting pixels or areas of the image
to process (e.g., integrate). The pixels or image area is selected
such that only fluorescent light from the internal volume of the
capillary, without light from the capillary walls or background
light from between the capillaries, is integrated. This results in
a larger signal to noise ratio relative to methods that integrate
the light from the entire capillary cross-section. The system is
configured so that a width of at least one pixel defines the middle
liquid volume of each capillary.
[0005] Embodiments of the invention also illuminate a relatively
large volume of the capillary to maximize the number of
fluorophores available for excitation within the illuminated area.
This allows for a larger signal to noise ratio relative to methods
that integrate light from only a narrowly focused point on the
capillary. Further, since embodiments of the invention are able to
differentiate between the capillary walls, the space between the
capillaries, and the internal liquid volume within the capillary,
the computer processing system can process and display the data
from the internal liquid volume and exclude the data from the
capillary walls and space between the capillaries to provide an
excellent signal output even though the large beam area may
illuminate capillary walls and space between capillaries.
[0006] Some embodiments of the invention include more than one
detection window to detect fluorophore emissions at different
wavelengths. This allows for detection of multiple compounds within
the same column. For example, a series of unknown DNA strands can
be labeled with Fluorophore X, while a known standard ladder of DNA
strands with known molecular weights can be labeled with
Fluorophore Y. The use of multiple detection windows (coupled with
multiple light sources), allows for the independent measure of the
unknown DNA samples with known standards in a single capillary.
This eliminates the need to run a standard compound and unknown
compound in two separate capillaries.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a partially exploded schematic diagram
illustrating a large volume multi-wavelength fluorescence detection
system for multiplexed capillary electrophoresis in accordance with
an embodiment of the invention.
[0008] FIG. 2 is a schematic diagram illustrating an optical fiber
bundle in accordance with an embodiment of the invention.
[0009] FIG. 3A is a close up view illustrating an optical path in
accordance with an embodiment of the invention.
[0010] FIG. 3B is a close up view illustrating the optical path in
accordance with an embodiment of the invention.
[0011] FIG. 4 is a schematic of a CCD array image showing a
differentiation of internal capillary volume, liquid capillary
walls, and space between the capillaries.
[0012] FIG. 5 is a picture of an imaged capillary array, showing a
clear differentiation of internal capillary liquid, capillary
walls, and space between the capillaries.
[0013] FIG. 6A is an enlarged view of one of the capillaries shown
in FIG. 5.
[0014] FIG. 6B is a capillary electropherogram trace that
represents the summation of all pixels in each column from FIG.
6A.
[0015] FIG. 7 is a series of electropherograms of single stranded
DNA that represents the summation of all pixels in each column from
edge to edge of the capillary shown in FIG. 6.
[0016] FIG. 8 is an electropherogram constructed from the middle 6
electropherograms from FIG. 7 (i.e. excluding light from the walls
of the capillary).
[0017] FIG. 9 is an electropherogram measured with large volume
fluorescence detection as discussed in FIG. 8 (lower trace)
compared with an electropherogram using a small volume fluorescence
detection (top trace).
[0018] FIG. 10 is a double stranded DNA electropherogram with
single wavelength large volume fluorescence detection as discussed
in Example 1, wherein each separate electropherogram is from a
separate capillary.
[0019] FIG. 11 is a carbohydrate (MALTRIN.RTM. M-200) separation
electropherogram with single wavelength large volume fluorescence
detection as discussed in Example 2.
[0020] FIG. 12 is two electropherograms (one for each wavelength)
generated with a two wavelength large volume fluorescence detection
as discussed in Example 3.
DETAILED DESCRIPTION OF THE INVENTION
[0021] For the purpose of promoting an understanding of the
principles of the invention, reference will now be made to the
embodiments illustrated in the drawings and specific language will
be used to describe the same. It will, nevertheless, be understood
that no limitation of the scope of the invention is thereby
intended; any alterations and further modifications of the
described or illustrated embodiments, and any further applications
of the principles of the invention as illustrated therein, are
contemplated as would normally occur to one skilled in the art to
which the invention relates.
[0022] In some embodiments, the invention includes a fluorescence
detection system. The detection system includes a sample vessel
(e.g., a capillary) in which a sample is placed. A light source is
included to emit light to excite a fluorescently labeled sample,
and the system is configured to direct light on the sample vessel
(sometimes referred to herein as a "detection window") to
illuminate a volume of more than about 100
micrometers.times..pi.r.sup.2, where r is one-half of the inner
diameter of the sample vessel or capillary. Although the light is
intended to illuminate the internal capillary volume, it will also
necessarily at least partially illuminate the capillary wall and
the space between capillaries. The problem of inadvertent
illumination degrades the quality of the output signal, and this
problem is exacerbated by the relatively large detection window as
described herein.
[0023] Embodiments of the invention also include a fluorescence
detector capable of imaging the entire cross section of the
capillary (or multiple capillaries), and has the resolution to
allow it to clearly differentiate between the capillary wall, the
internal capillary volume, and the space between capillaries. The
detector is positioned to detect the fluorescent emissions of the
sample. The detector has the resolution to image distinct parts of
the image. For example, the detector can have at least one pixel
defining the internal volume of each capillary, at least one pixel
defining each capillary wall, and at least one pixel defining the
space between the capillaries. Any suitable detector may be used.
However, detectors such as charge coupled devices (CCDs) are
particularly useful with embodiments of the invention. An example
of such a CCD is made by Starlight Xpress Ltd., model #: SXVR-H9,
equipped with an ICX285 CCD chip with 1392.times.1040 pixels in a
two-third inch format interline camera and a pixel size of 6.45
.mu.m.times.6.45 .mu.m.
[0024] The detector is attached to a computer system or processor
capable of selecting the pixels for the final detection of
fluorescent light--whereby only the pixels corresponding to the
internal capillary volume are chosen. Pixels corresponding to the
capillary walls or the space between capillaries are excluded from
the final fluorescent signal. In some embodiments, after the
detector (e.g., CCD) records the images, the processor calculates
the time lapsed signal to noise ratio of the pixels along the
x-axis. The capillary walls always have a lower signal to noise
ratio than the illuminated internal volume of the capillary and the
space between the capillaries has no signal. Accordingly, the
processor (e.g., with software) can use these unique
characteristics of each region to define the regions. For example,
these data discrimination and analysis functions can be written on
Labview version 7.0 form National Instruments run on a personal
computer. Accordingly, embodiments of the invention are useful for
illuminating a relatively large volume of a fluorescently labeled
sample, while excluding stray light from the capillary walls and
light from between the capillaries, thereby increasing the
signal-to-noise ratio of the illuminated volume to provide a higher
quality output.
[0025] The fluorescence excitation light source can be a gas
discharge lamp (mercury or xenon), a laser (gas, solid state, dye,
or semiconductor) or a light-emitting-diode (LED). In some
embodiments the detection system includes non-coherent light
sources as the excitation light source. In some embodiments, the
light source is a high power LED, which operates at a current
rating of least 100 milliAmps, preferably at 500 milliAmps, and
even more preferably 700 to 1000 milliAmps.
[0026] An optical fiber bundle can be provided to direct the
emitted light from the light source to the sample vessel detection
window without focusing the irradiation light. A large volume of
each sample vessel is illuminated due to the non-focused
illumination. In some embodiments, the detection window includes a
bandpass filter for specific wavelength detection. A fluorescence
detector capable of imaging the entire capillary cross-section,
with a differentiation of the walls of the capillary, the internal
capillary volume, and the space between the capillaries, such as a
CCD, is positioned to detect the fluorescent emissions of the
sample. In addition, the use of the optical fiber bundle allows the
illumination of multiple sample vessels simultaneously in
multi-channel systems and the detector can monitor fluorescence
signals of multiple channels.
[0027] FIG. 1 illustrates a fluorescent detection system 10 in
accordance with an embodiment of the invention. The embodiment of
FIG. 1 includes a multiplexed capillary array electrophoresis
system with a capillary array 20 and a high power LED 30 for large
volume illumination fluorescence detection. A "high power" LED is
one which operates at a current rating of least 100 milliAmps,
preferably at 500 milliAmps, and even more preferably 700 to 1000
milliAmps. As shown, the high power LED's light output is coupled
to an optical fiber bundle 40 through optical couplers 50. In the
embodiment shown, the fiber bundle light entrance end 60 (i.e.,
proximal end) has a round shape to match the output of the LED
while the exit end 70 (i.e., distal end) has a rectangular shape
with the long side having similar or larger dimension than the
detection window on the multiple capillaries, as shown in FIG. 2.
In some embodiments, the optical fiber bundles include about 16,600
optical fibers. Each optical fiber can be about 50 .mu.m in
diameter with numeric aperture (N.A.) of about 0.55. The light
entrance end can have a diameter of about 7.11 mm to match the
optical coupler output area. The exit end of the optical fiber can
have dimensions of about 1.5 mm.times.about 25.4 mm. With this
dimension, roughly 30.times.510 optical fibers are packed into a
rectangular shape at the distal end of the optical fiber bundle. In
addition, the exit end of the fiber bundle can be positioned at an
angle relative to longitudinal axis of the capillary (e.g., between
about 30 degrees and 60 degrees, such as about 45 degrees). The
angle helps to eliminate the direct irradiation of the excitation
light onto the camera lenses to eliminate background noise.
[0028] As shown, filters 80 (which can be the same or dissimilar
for each other) can be included to block off unwanted excitation
wavelengths from the LEDs. Filters 90 (which can be the same or
dissimilar for each other) can be used to select the desired
fluorescent wavelength for detection. Also as shown, a camera lens
100 can be used to collect the fluorescent emission from the
detection windows of the multiple capillaries while a
two-dimensional detector such as a CCD 110 can be used to monitor
the fluorescent emission. A processor, which would be connected to
the CCD to process the output from the CCD (e.g., differentiate the
regions and provide an integrated output signal), is not shown.
[0029] Embodiments of the invention include configuring the CCD
array in such a way as to enable differentiation of the light
coming from the capillary walls, the internal capillary volume, and
the space between each capillary. This allows one to select and
detect light only from the internal volume of each capillary. A CCD
with a two-dimensional array area of 1392 by 1040 pixels is
preferred for imaging from about 1 up to about 96 capillaries,
while enabling differentiation of the walls of the capillary, the
internal capillary volume, and the space between the
capillaries.
[0030] The capillary array electrophoresis system shown in FIG. 1
has capillary windows arranged on the same plane at the detection
region to simultaneously illuminate the detection window for
on-column detection. During use, the capillary array has both ends
immersed into buffer solution in which a high voltage is applied
for electrophoresis separation. The ends of the capillary array may
be separated for individual sample loading.
[0031] FIGS. 3A and B illustrate close up sections of detection
windows in accordance with embodiments of the invention. As shown,
the exit end of the optical fiber bundle is positioned less than 1
mm away from the capillaries and at about 45.degree. against the
capillaries. The light exits each fiber of the optical fiber bundle
with a divergence angle based on the numeric aperture (N.A.) of the
optical fiber. For example, an optical fiber has a N.A. of 0.25
will have a divergence angle of about 29.degree. while a N.A. of
0.66 will have divergence angle of about 83.degree.. When the
optical fiber bundle is positioned 1 mm or less from the capillary
tubing window, approximate 2 mm of the tubing length will be
illuminated by the exit light from the optical fiber bundle. In
addition, each capillary's detection window will be illuminated by
more than one optical fiber from the optical fiber bundle.
[0032] Further, in some embodiments, the optical fiber position
from the light entrance and exit are randomized. In such
embodiments, the uneven light distribution from the LED output is
homogenized at the exit end of the optical fiber bundle, which
provides more consistent illumination to the sample volume.
[0033] Typical fluorescent detection systems focus the light source
onto the sample with as small an area as possible. Fluorescent
signal intensity is proportional to the incident light power and
the amount of fluorophore molecules present in the irradiation
volume. Capillaries generally have an internal diameter of about
100 um or less, and fluorescence detection systems for HPLC or
capillary electrophoresis system generally focus the light source
to a point much less than 100 um. Focusing the light source
increases the power density of incident light at the small
detection volume. Therefore, more photons are available to excite
the sample molecules within the small detection zone (<100
.mu.m.times..pi.r.sup.2, where r is the 1/2 of the radius inner
tubing of the capillary). Further, focusing the light source into a
small area maintains high resolution of separation. If the CE
separation resolution is smaller than the illumination area, the
detection resolution lost. However, in most of multiplexed
capillary electrophoresis applications, the separation resolution
does not require the tight focusing (<<100 um).
[0034] Therefore, instead of focusing the light to a small volume
to obtain high power density for illumination, embodiments of the
invention use a high power LED to provide high photon flux to
illuminate a relatively large volume in which more molecules are
excited to fluorescence because more sample molecules are available
for excitation. In some embodiments, the system is configured to
direct light on the sample vessel to illuminate a volume of more
than about 50 micrometers.times..pi.r.sup.2, where r is one-half of
the inner diameter of the sample vessel. In other embodiments, the
system is configured to direct light on the sample vessel to
illuminate a volume of more than about 500
micrometers.times..pi.r.sup.2. In yet other embodiments, the system
is configured to direct light on the sample vessel to illuminate a
volume of more than about 1,000 micrometers.times..pi.r.sup.2. In
certain embodiments, the system is configured to direct light on
the sample vessel to illuminate a volume of more than about 1,500
micrometers.times..pi.r.sup.2. In some embodiments, the system is
configured to direct light on the sample vessel to illuminate a
volume of less than about 2,000 micrometers.times..pi.r.sup.2. In
certain embodiments, the system is configured to direct light on
the sample vessel to illuminate a volume of about 2,000
micrometers.times..pi.r.sup.2.
[0035] As shown in FIG. 4, the CCD detector should be configured to
detect three regions of the capillary (for a single capillary) or
capillary array (for multiple capillaries). In FIG. 4, region A
includes the wall of each capillary, region B includes the internal
volume of each capillary, and region C includes the space between
capillaries. Each region should correspond to at least one distinct
pixel in the detector. In some embodiments, region B (of each
capillary) has at least 3 pixels. In other embodiments, region B
(of each capillary) has at least 5 pixels.
[0036] The height of the entire capillary array image may range
from 1 pixel up to the y-axis length (in pixels) of the CCD. For
example, A CCD array with width of 1392 pixels and length (y-axis)
of 1040 pixels may be used to image a 12-capillary system wherein
the internal liquid volume width (x-axis) of each capillary is at
least 6 pixels, the walls of each capillary (x-axis) is at least
1-pixel, and the space between each capillary is at least 20
pixels. The height of each image is at least 60 pixels, but may be
up to 1040 pixels, depending on how the capillary image is focused
onto the CCD window. FIG. 5 shows an imaged capillary array, in
which the internal volume of each capillary is about 6 pixels, each
capillary wall is 2 pixels, and the space between capillaries is
about 80 pixels. The length of the capillary image is about 60
pixels. A CCD detector with 1392 by 1040 pixels was used to capture
this image. FIG. 6 shows an enlarged view of one of the capillaries
from FIG. 5. The two vertical lines represent the wall of the
capillary. The uneven light distribution from the capillary is due
to scattering from imperfect surface, dust, and incomplete removal
of polyimide coating. FIG. 6B shows the one dimensional display
when summing all vertical pixels intensities together for each
column.
[0037] FIG. 7 shows the electropherograms of single stranded DNA
separation. A sample similar to that illustrated in example 3 was
used here for demonstration. Only one LED and fiber optical bundle
was used for the excitation. A LED with 470 nm emission was used
for the excitation while monitoring the fluorescence signal through
a band pass filter with bandwidth from 500 nm to 550 nm. The
samples were injected into one end of the capillaries by applying 5
kV for 20 seconds. After the sample injection, the injection ends
of the capillaries were immersed into buffers for separation under
150 V/cm separation field strength. The fluorescent signal was
recorded by a CCD capable of imaging the entire cross-section of
the capillary, including the capillary walls, the volume within
each capillary, and the space between each capillary. Each
electropherogram represents the time lapse signal from the
summation of all vertical pixels signal of each column (vertical
line to vertical line) shown in FIG. 6A. The top trace to the
bottom trace in FIG. 7 shows 10 electropherograms that represent
each column's (i.e. a column with a width of 1 pixel) signal during
the time of electrophoresis separation. The top trace is the left
edge of the capillary, and the bottom trace is the right edge of
the capillary. FIG. 7 indicates that the capillary image had two
pixels for each side of wall with significantly lower S/N and 6
pixels for the internal volume with high S/N. An average of all 10
electropherograms, including the wall, results in a lower signal to
noise ratio than an average of the middle 8 electropherograms. To
obtain a higher signal to noise ratio the wall's signal should be
excluded and only the internal volume signal used to construct the
final electropherogram. FIG. 8 shows the final electropherogram
excluding the signal from the capillary's wall.
[0038] With the same irradiance, large volume fluorescence
detection provides better S/N compared to small volume fluorescence
detection since more sample molecules are available for detection.
In FIG. 9, the bottom electropherogram was constructed using large
volume fluorescence detection, in which a 1.5 mm section of
capillary was illuminated; while the top electropherogram
represented the small volume fluorescence detection, in which only
a 50 .mu.m section of capillary was illuminated. The
electropherogram has much better S/N for large volume fluorescence
detection (lower trace) than the small volume fluorescence
detection (top trace).
[0039] In addition, as shown in FIG. 1, embodiments of the
invention provide for multi-wavelength excitation and fluorescent
detection. If the sample has been labeled with two different
fluorophores, it may require two different wavelengths for
excitation because of the absorption coefficiency is different for
different fluorophores. Embodiments of the invention provide for
multiple wavelength excitation and detection with the use of LEDs
at different wavelengths to irradiate at different locations for
different wavelength excitation and detection.
[0040] In such embodiments, as shown in FIG. 1, multiple light
sources and optical fiber bundles are used for excitation at
multiple detection windows to excite the fluorophore at multiple
wavelengths. In some embodiments, each detection window comprises a
bandpass filter for specific wavelength detection. All detection
windows can be monitored simultaneously with a two-dimensional
detector such as a charged coupled device (CCD) capable of imaging
the entire cross-section of the capillaries, with a clear pixelated
differentiation of the capillary walls, the internal capillary
volume, and the space between the capillaries, as described in
detail above. When coupled with a computer system capable of
selecting individual pixels for integration, such that pixels
corresponding to the walls of the capillary are excluded from
integration, this allows for an increase in the signal-to-noise
ratio. When multiple detection windows are used, multiwavelength
fluorescence signals from the same separation sample vessel are
obtained. In addition, the use of optical fiber bundle allows the
illumination of multiple sample vessels simultaneously in
multi-channel systems and the two-dimensional detector can monitor
multiwavelength fluorescence signals of multiple channels.
Examples
[0041] The examples below are merely illustrative and are not
intended to limit the scope of the invention.
Example 1
Double Stranded DNA Electropherogram and a Large Volume Detection
System
[0042] FIG. 10 shows the electrophoretic pattern of double-stranded
DNA 100 b.p. ladders obtained by an embodiment of the invention.
This was measured in a multicapillary system, and each trace
represents a different capillary. In this example only one LED
light source and fiber optical bundle was used for the fluorophore
excitation. The LED emitted at 470 nm for excitation while a band
pass filter transmitted from 500 nm to 600 nm was used to select
the desire fluorescent wavelength for detection. The separation
matrix contained SYBR Green dye. When double-stranded DNA binds to
SYBR Green, the resulting DNA dye complex absorbs the LED light and
fluoresces at 522 nm. The samples were injected into one end of the
capillaries by applying 5 kV for 5 seconds. After the sample
injection, the injection ends of the capillaries were immersed into
buffers for separation under 150 V/cm of a constant electric field.
The volume of illuminated liquid was 1500
micrometers.times..pi.r.sup.2, where r is one-half of the inner
diameter of the sample vessel, which was 75 um.
Example 2
Carbohydrate Separation Electropherogram With Large Volume
Fluorescence Detection
[0043] FIG. 11 shows the high resolution oligosaccharide profiling
by electrophoretic separation of carbohydrate MALTRIN.RTM. M-200
labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) using an
embodiment of the invention. In this example only one LED and fiber
optical bundle was used for the excitation. A LED with 470 nm
emission was used for the excitation while monitoring the
fluorescence signal through a band pass filter with bandwidth from
500 nm to 600 nm. The volume of liquid illuminated was 1500
micrometers.times..pi.r.sup.2, where r is one-half of the inner
diameter of the sample vessel, which was 75 um. The samples were
injected into one end of the capillaries by applying 5 kV for 10
seconds. After the sample injection, the injection ends of the
capillaries were immersed into buffers for separation under 300
V/cm of a constant electric field. The fluorescent signal was
recorded by the CCD. MALTRIN.RTM. M-200 is a maltooligosaccharide
ladder which contains at least 16 individual oligomers as shown in
FIG. 11.
Example 3
Multiple Wavelength Detection
[0044] The Staphylococcus aureus tuf gene has the following DNA
sequence:
TABLE-US-00001 5'-TATTCTCAATCACTGGTCGTGGTACTGTTGCTACAGGCCGTGTTGAA
3'-ATAAGAGTTAGTGACCAGCACCATGACAACGATGTCCGGCACAACTT
CGTGGTCAAATCAAAGTTGGTGAAGAAGTTGAAATCATCGGTTTACATGA
GCACCAGTTTAGTTTCAACCACTTCTTCAACTTTAGTAGCCAAATGTACT
CACATCTAAAACAACTGTTACAGGTGTTGAAATGTTCCGTAAATTATTAG
GTGTAGATTTTGTTGACAATGTCCACAACTTTACAAGGCATTTAATAATC
ACTACGCTGAAGCT-3' TGATGCGACTTCGA-5'
[0045] The following DNA sequences were selected as primers for PCR
amplification:
TABLE-US-00002 5'-TATTCTCAATCACTGGTCGT-3'
5'-AGCTTCAGCGTAGTCTA-3'.
5'-TATTCTCAATCACTGGTCGT-3' was labeled with a fluorescence dye
(FAM) in the 5' position, and 5'-AGCTTCAGCGTAGTCTA-3' was labeled
with a different fluorescence dye (Cy-5) at the 5' position. After
the PCR amplification for Staphylococcus aureus DNA, one strand of
PCR product contained a green fluorescence dye while other strand
of DNA contained a red fluorescence dye. After purification of the
PCR product, 80% of N-methylformamide was used to cleave the DNA at
110.degree. C. for 30 minutes. Samples were then separated with
electrophoresis without further purification.
[0046] A capillary fluorescent detection system in accordance with
the invention was used to simultaneously separate and detect the
fragments that labeled with the different dyes. The embodiment of
the invention shown in FIG. 1 was used for the separation and
detection. FIG. 12 shows the electropherograms obtained with the
present two wavelength (i.e., colors) detection system. The bottom
trace represents the emission wavelength from 500 nm to 550 nm with
excitation at 470 nm while the top trace represents the emission
wavelength from 620 nm and up with the excitation at 560 nm. The
volume of illuminated liquid was 1500
micrometers.times..pi.r.sup.2, where r is one-half of the inner
diameter of the sample vessel, which was 75 um. The 480 nm to 550
nm represents the emission from FAM labeled DNA fragments while the
other wavelength represents the emission from Cy-5 labeled DNA
fragments.
[0047] While the invention has been described in conjunction with
specific embodiments thereof, it is evident that many alternatives,
modifications, and variations will be apparent to those skilled in
the art in light of the foregoing description. Accordingly, it is
intended to embrace all such alternatives, modifications, and
variations, which fall within the spirit and broad scope of the
invention.
* * * * *