U.S. patent application number 14/720723 was filed with the patent office on 2015-11-26 for glycan profiling utilizing capillary electrophoresis.
The applicant listed for this patent is BIOPTIC, INC.. Invention is credited to Varouj D. AMIRKHANIAN, Ming-Jhy HSEU, Shou-Kuan TSAI.
Application Number | 20150338347 14/720723 |
Document ID | / |
Family ID | 54555853 |
Filed Date | 2015-11-26 |
United States Patent
Application |
20150338347 |
Kind Code |
A1 |
TSAI; Shou-Kuan ; et
al. |
November 26, 2015 |
GLYCAN PROFILING UTILIZING CAPILLARY ELECTROPHORESIS
Abstract
A method for glycan profiling by capillary electrophoresis (CE),
and a CE system for glycan analysis (N-Glycan). The CE system uses
integrated dual optical fibers for both radiation excitation and
emission detection. The CE system is configured for performing a
two-color detection for data analysis. A single radiation
excitation source is used to excite two emission fluorophores or
dyes in the sample solution to be analyzed. One emission dye is to
tag the sample and the other dye is used to provide a reference
marker (e.g., a Dextran Ladder) for the sample run. Two detectors
(e.g., photomultipler tubes (PMTs)) are applied to simultaneously
detect the fluorescent emissions from the dyes. The data collected
by both detectors are correlated (e.g., synchronized, and/or
super-positioned for analysis) for accurate data peak
identification.
Inventors: |
TSAI; Shou-Kuan; (New Taipei
City, TW) ; HSEU; Ming-Jhy; (New Taipei City, TW)
; AMIRKHANIAN; Varouj D.; (La Crescenta, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOPTIC, INC. |
New Taipei City |
|
TW |
|
|
Family ID: |
54555853 |
Appl. No.: |
14/720723 |
Filed: |
May 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62002142 |
May 22, 2014 |
|
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|
Current U.S.
Class: |
204/452 ;
204/450; 204/600; 204/603; 250/216; 250/459.1 |
Current CPC
Class: |
G01N 27/44791 20130101;
G01N 27/44726 20130101; G01N 27/44721 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; G01N 27/447 20060101 G01N027/447 |
Claims
1. A method of glycan profiling, comprising: providing a separation
channel having a first longitudinal axis along which a glycan
sample undergoes separation into sample components, and defining a
detection zone defined along the separation channel through which
sample components pass; providing a radiation source; providing a
detector; providing an incident light guide having a second
longitudinal axis, directing incident radiation from the radiation
source to the detection zone, causing radiation to be emitted by
sample components as they pass through the detection zone; and
providing an emission light guide having a third longitudinal axis,
collecting and directing emitted radiation from the detection zone
to the detector, wherein the emission light guide and the incident
light guide are positioned on opposite sides of the separation
channel.
2. The method of claim 1, wherein prior to subjecting the sample to
separation, the sample is provided with a sample marker
corresponding to emission of a first characteristic wavelength,
wherein the sample components are labeled by the sample marker for
identification as the sample undergoes separation.
3. The method of claim 2, wherein prior to subjecting the sample
separation, the sample is further provided with a reference marker
corresponding to a second characteristic wavelength, wherein the
reference marker provides a reference to facilitate identification
of the sample components as the sample undergoes separation.
4. The method of claim 3, further comprising subjecting the sample
to high voltage to effect electrophoresis to separate the sample
into the sample components along the separation channel.
5. The method of claim 4, wherein the separation channel is a
capillary channel defined in a capillary column.
6. The method of claim 5, wherein the sample marker comprises a
sample fluorophore and the reference marker comprises a reference
fluorophore, wherein the sample fluorophore and the reference
fluorophore are simultaneously subject to the incident radiation as
they pass through the detection zone, and wherein the incident
radiation induces emitted radiations in the form of fluorescence
emissions of the sample marker and the reference marker as the
sample components pass through the detection zone.
7. The method of claim 6, wherein the reference fluorophore
provides a Ladder as a reference marker as the sample undergoes
separation.
8. The method of claim 7, wherein the sample is N-Glycan.
9. The method of claim 8, wherein the emission light guide and the
incident light guide are positioned on opposite sides of the
separation channel, and wherein the first, second and third
longitudinal axis are substantially coplanar at least at or near
the detection zone.
10. The method of claim 9, wherein at least one of the second and
third longitudinal axis is not perpendicular to the first
longitudinal axis.
11. The method of claim 10, wherein the emission light guide and
the incident light guide are positioned on opposite sides of the
separation channel, wherein the incident light guide comprises a
first optical fiber having a terminating integral ball-end
structure, and the emission light guide comprises a second optical
fiber having a terminating integral ball-end structure, and wherein
the ball-end structures do not touch exterior of the separation
channel.
12. An electrophoresis system for profiling glycan, comprising: a
separation channel having a first longitudinal axis along which a
glycan sample undergoes separation into sample components, and a
detection zone defined along the separation channel through which
sample components pass; a radiation source; a detector; an incident
light guide having a second longitudinal axis, directing incident
radiation from the radiation source to the detection zone, causing
radiation to be emitted by sample components as they pass through
the detection zone; an emission light guide having a third
longitudinal axis, collecting and directing emitted radiation from
the detection zone to the detector, wherein the emission light
guide and the incident light guide are positioned on opposite sides
of the separation channel, and at least one of the incident light
guide and emission light guide comprises an optical fiber; and a
power supply providing a high voltage across ends of the separation
channel to effect electrophoresis separation, wherein separated
sample components pass through the detection zone.
13. The system of claim 12, wherein prior to subjecting the sample
to separation, the sample is provided with a sample marker
corresponding to emission of a first characteristic wavelength,
wherein the sample components are labeled by the sample marker for
identification as the sample undergoes separation, and the sample
is further provided with a reference marker corresponding to a
second characteristic wavelength, wherein the reference marker
provides a reference to facilitate identification of the sample
components as the sample undergoes separation.
14. The system of claim 13, wherein the sample maker comprises a
sample fluorophore and the reference marker comprises a reference
fluorophore, wherein the sample fluorophore and the reference
fluorophore are simultaneously subject to the incident radiation as
they pass through the detection zone, and wherein the incident
radiation induces emitted radiations in the form of fluorescence
emissions of the sample marker and the reference marker as the
sample components pass through the detection zone.
15. The system of claim 14, wherein the sample is N-Glycan.
16. The system of claim 15, wherein the separation channel is a
capillary channel defined in a capillary column.
17. The system of claim 16, wherein the reference fluorophore
provides a Ladder as a reference marker as the sample undergoes
separation.
18. The system of claim 16, wherein the incident light guide
comprises a first optical fiber having a terminating integral
ball-end structure, and the emission light guide comprises a second
optical fiber having a terminating integral ball-end structure,
wherein the ball-end structures do not touch exterior of the
separation channel.
19. The system of claim 18, wherein the separation channel has a
first longitudinal axis along which the glycan sample undergoes
separation into sample components, the incident light guide has a
second longitudinal axis, and the emission light guide has third
longitudinal axis, and wherein at least one of the second and third
longitudinal axis is not perpendicular to the first longitudinal
axis.
20. The detection system of claim 13, wherein the emission light
guide and the incident light guide are positioned on opposite sides
of the separation channel, and wherein the first, second and third
longitudinal axis are substantially coplanar at least at or near
the detection zone.
Description
PRIORITY CLAIM
[0001] This application claims the priority of U.S. Provisional
Patent Application No. 62/002,142 filed on May 22, 2014. This
provisional patent and all documents discussed below are fully
incorporated by reference as if fully set forth herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to bio-analysis, in particular
a method for glycan profiling by capillary electrophoresis and a
capillary electrophoresis instrument for performing the method.
[0004] 2. Description of Related Art
[0005] Post-translational modification of proteins with
oligosaccharides to form glycoproteins is a common biological
motif. These glycoprotein oligosaccharides are involved in a wide
range of biological and physiological processes including
recognition and regulatory functions, cellular communication, gene
expression, cellular immunity, growth, and development. Aberrant
glycosylation of proteins is connected to cancer progression,
invasion, and metastasis. With many biological functions,
glycosylation is one of the most important post-translational
modifications of eukaryotic cell proteins.
[0006] Oligosaccharides are covalently attached to proteins
primarily through two structural motifs. They can be attached to
the amide group of an asparagine, referred to as "N-linked glycans"
or attached to proteins through the hydroxyl group on serine or
threonine, referred to as "O-linked glycans". The biological
activity and function of N-linked glycans are well studied as
compared to O-linked glycans. However, both types of glycans are
investigated as biomarkers in order to understand changes related
to complex organelle development, and as part of therapeutic
protein drug development, which has increasing evidence that
efficacy is effected by glycosylation.
[0007] Most glycoproteins exist as a heterogeneous population of
glycoforms or glycosylated variants with a single protein backbone
and a heterogeneous population of glycans at each glycosylation
site. It has been reported in the literature that for some
glycoproteins, 100 or more glycoforms exist at each glycosylation
site. In view of this heterogeneity and the presence of branched
structures, the analysis of glycans is much more complicated than
protein chemistry. It requires several different strategies to
separate and study the structure of each individual glycan. Once
the glycans have been released from the glycoprotein, the glycan
pool can be analyzed by MALDI-TOF mass spectrometry (MS) or, after
fluorescent labeling, by either HPLC or MS, or both. This strategy
can provide a "glycan profile" or a "glycosylation pattern" that is
highly characteristic of the glycoprotein. The technology can be
applied to compare glycan profiles of glycoproteins found in normal
and diseased states, or to compare different batches of recombinant
protein products. Both these techniques provide valuable
information in terms of composition, linkage and arm specificity
(using various exoglycosidases) from which structural information
on individual glycans can be elucidated.
[0008] Separation of glycans by electrophoresis in polyacrylamide
gel has been widely used and different methods are described in the
literature for analysis of monosaccharides and oligosaccharides.
The most commonly used system is the electrophoresis of
fluorophore-labeled glycans in highly cross-linked polyacrylamide
gels and is termed as Fluorophore-Assisted Carbohydrate
Electrophoresis (FACE). The glycans are usually labeled with a
fluorescent tag, mainly ANTS or AMAC and separated on 20-40% gels.
The extent of cross-linking means that extra precautions should be
taken to prevent heating and warping of the gel during the run.
After electrophresis, the band patterns are visualized by
illuminating the gel under UV light and photographing the image.
Although this technique is sensitive in the sub-pico molar range,
the resolution between the glycans can be poor due to the
limitation on the size of the gel.
[0009] The slab gel electrophoresis separation method of FACE is
based on the use of high concentration polyacrylamide gel
electrophoresis to separate intact oligosaccharides released from
several glycoproteins. However, slab gel electrophoresis for
bio-analysis is labor intensive and needs to be drastically
improved in terms of resolving power, throughput and cost per
sample.
[0010] Recently, a complete method for analysis of N-glycans has
been derived from glycoproteins. It is based on a combination of
specific chemical and enzymatic conversions coupled with Capillary
Electrophoresis (CE) with Laser-Induced Fluorescence (CE/LIF).
N-Glycans are released enzymatically from glycoproteins and
derivatized with APTS under mild reductive amination conditions to
preserve sialic acid and fucose residues. The method successfully
profiled the heavily sialylated N-glycans. A method for
multistructure sequencing of N-glycans by gel CE and exoglycosidase
digestions has also been devised.
[0011] Without a doubt, CE with laser-induced fluorescence (LIF) is
one of the most powerful analytical tools for rapid, high
sensitivity and high-resolution dsDNA analysis and immunoassay
analysis applications. However, the current selling price for
CE-based LIF systems is much more expensive than traditional
slab-gel based bio-analysis systems due to the complicated optical
detection mechanism. The expensive CE-based systems are thus out of
reach for all but a few well-funded laboratories and seems to be a
high-cost barrier. Further, CE is commonly avoided in routine
analysis because it is reputed to be a troublesome technique with
high failure rates. However this is no longer true because
instrument manufacturers have drastically improved instrument
design and overall CE knowledge has increased. There are three key
factors for reducing failure rate and producing accurate, precise
and robust CE data: operator training, system stability, and
operation ease of the instrument with low maintenance.
[0012] There is a need for a method for glycan profiling by
capillary electrophoresis and a capillary electrophoresis
instrument for performing the method that reduces costs, with
simplicity in operation, and offers rapid analysis with high
efficiency, sensitivity and throughput.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method for glycan profiling
by capillary electrophoresis (CE), and a cost-effective capillary
gel-electrophoresis system for highly efficient, high speed, high
throughput, glycan analysis (N-Glycan). The novel method and system
significantly increase the pace at which glycoprotein research is
performed in the laboratory, saving hours of preparation time and
assuring accurate, consistent and economical results.
[0014] In one aspect of the present invention, a high-performance
capillary gel electrophoresis analyzer system has been optimized
for glycoprotein analysis application. The system uses integrated
dual fiber optic radiation induced fluorescence detection
technology (i.e., fibers for both radiation excitation and emission
detection). Using commercially available labeling agent such as
ANTS as an indicator, the capillary gel electrophoresis-based
glycan analyzer (FIG. 1) provides high resolving power within a
relatively short run time (e.g., a separation period of 2-5 minutes
of separations). The system can hold multiple samples (e.g., a
total of 96 samples), which can be automatically analyzed within,
e.g., 4-5 hours. This affordable fiber optic based fluorescence
detection system can be used in laboratories for high speed glycan
profiling applications.
[0015] In one embodiment, the glycan analyzer system utilizes
relatively short capillary columns (e.g., 15 cm long, 75 .mu.m ID)
filled with linear polymer format for the separation of
ANTS-labeled complex carbohydrates.
[0016] In another embodiment, the analyzer is configured for
performing a two-color detection for data analysis (e.g., for
accurate data peak identifications). A single radiation excitation
source (e.g., LED or Laser) is used to excite two emission
fluorophores or dyes in the sample solution to be analyzed. One
emission dye is a marker that tags the sample and the other dye is
used to provide a reference marker (e.g., a Dextran Ladder) for the
sample run. Two detectors (e.g., photomultipler tubes (PMTs)) are
applied to simultaneously detect the fluorescent emissions from the
dyes. The data collected by both detectors are correlated (e.g.,
synchronized, and/or super-positioned for analysis) for accurate
data peak identification. The two-color detection simplifies and
shortens sample separation and detection into a single run and
assures accurate data analysis for peak identification. The dual
dye detection (i.e., two dye labeling) is a very robust and
accurate way to provide reproducible peak identification and sizing
for glycan profiling.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] For a fuller understanding of the nature and advantages of
the invention, as well as the preferred mode of use, reference
should be made to the following detailed description read in
conjunction with the accompanying drawings. In the following
drawings, like reference numerals designate like or similar parts
throughout the drawings.
[0018] FIG. 1 is a schematic view of a capillary electrophoresis
system that incorporates the optical detection configuration in
accordance with one embodiment of the present invention.
[0019] FIG. 2 illustrates the detection region, showing the
configuration of the excitation fiber, emission fiber and the
capillary column.
[0020] FIG. 3 illustrates the external view of a CE instrument, in
accordance with one embodiment of the present invention.
[0021] FIG. 4 illustrates the internal view of the CE instrument of
FIG. 4, in accordance with one embodiment of the present
invention.
[0022] FIG. 5 is a schematic view illustrating the components of
the CE instrument of FIGS. 3 and 4, in accordance with one
embodiment of the present invention.
[0023] FIG. 6 schematically illustrates a two-color detection
scheme in accordance with a first embodiment of the present
invention.
[0024] FIG. 7 schematically illustrates a two-color detection
scheme in accordance with a second embodiment of the present
invention.
[0025] FIGS. 8-11 illustrate results of fluorescence detection of
glycan profiling by HPLC.
[0026] FIGS. 12-13 illustrate results of fluorescence detection of
glycan profiling by the inventive CE instrument and method in
accordance with the present invention.
[0027] FIGS. 14 and 15 illustrate results of fluorescence detection
of glycan profiling using a two-color detection scheme, by the
inventive CE instrument and method in accordance with the present
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] This invention is described below in reference to various
embodiments with reference to the figures. While this invention is
described in terms of the best mode for achieving this invention's
objectives, it will be appreciated by those skilled in the art that
variations may be accomplished in view of these teachings without
deviating from the spirit or scope of the invention.
[0029] The present invention provides a method for glycan profiling
by capillary electrophoresis (CE), and a cost-effective capillary
gel-electrophoresis system for highly efficient, high speed, high
throughput, glycan analysis (N-Glycan). The novel method and system
significantly increase the pace at which glycoprotein research is
performed in the laboratory, saving hours of preparation time and
assuring accurate, consistent and economical results.
[0030] In one aspect of the present invention, a high-performance
capillary gel electrophoresis analyzer system has been optimized
for glycoprotein analysis application. The system uses integrated
dual fiber optic radiation induced fluorescence detection
technology (i.e., fibers for both radiation excitation and emission
detection). Using commercially available labeling agent such as
ANTS as an indicator, the capillary gel electrophoresis-based
glycan analyzer (FIG. 1) provides high resolving power within a
relatively short run time (e.g., a separation period of 2-5 minutes
of separations). The system can hold multiple samples (e.g., a
total of 96 samples), which can be automatically analyzed within,
e.g., 4-5 hours. This affordable fiber optic based fluorescence
detection system can be used in laboratories for high speed glycan
profiling applications.
[0031] Reference is made to the bioanalytical system including
detection system disclosed in U.S. Pat. Nos. 8,778,155 and
8,784,626, the entirety of which are incorporated by reference as
if fully set forth herein. This patent is commonly assigned to
BiOptic, Inc., the applicant and assignee of the present invention.
In particular, these patents disclosed a simplified, low cost, high
efficiency, highly sensitive, high throughput bio-separation system
(e.g., capillary electrophoresis (CE) system). The bio-separation
system includes an instrument that is configured to work with a
capillary cartridge, and that is provided with a detection
configuration that includes optics for application of incident
radiation at and detection of output radiation from a detection
zone along the separation channel, for the detection of radiation
emitted by sample analytes (e.g., radiation induced fluorescence
emission), without requiring fine alignment of the optics to the
separation column. The instrument is configured to conduct
bio-separation in the separation channel of the bio-separation
cartridge in an automated manner. The CE system has a less complex
optical detection mechanism to reduce costs, which complements
simplicity in operation, rapid analysis with high efficiency,
sensitivity and throughput. The present invention adopts and
modifies this system to perform a novel method of glycan profiling,
including improvements to the system in accordance with the
disclosure hereinbelow.
[0032] For purpose of illustrating the principles of the present
invention and not limitation, the present invention is described by
reference to embodiments directed to capillary electrophoresis
using a capillary separation column. Further, the present invention
will be described, without limitation, in connection with radiation
induced fluorescence detection (e.g., using a laser or LED source).
Fluorescence is a spectrophotometric method of analysis where the
molecules of the analytes are excited by irradiation at a certain
wavelength and emit radiation at a different wavelength. The
emission spectrum provides information for both qualitative and
quantitative analysis. Generally, the advantage of fluorescence
detection over absorbance detection is the superior detectability
(detection sensitivity). For efficient fluorophores, single
molecule detection in small volumes has been demonstrated. This is
in part because fluorescence signal is measured against a
relatively dark background, as a result of the emitted radiation
being detected at a wavelength that is different from the
wavelength of the incident radiation (e.g., the wavelength of the
emitted fluorescence is at longer wavelengths than the excitation
radiation).
[0033] Referring to FIG. 1, a capillary electrophoresis (CE) system
100 incorporates the detection configuration as schematically
illustrated. The CE system 100 generally comprises a capillary
separation column 10 (e.g., 200-500 .mu.m O.D.), which defines an
internal separation channel 12 (e.g., 25-150 .mu.m I.D.). The
capillary column 10 may be made of fused silica, glass, polyimide,
or other ceramic/glassy materials. The inside walls of the
separation column 10 (i.e., the walls defining the separation
channel 12) may be coated with a material that can build up an
electrostatic charge to facilitate electrophoresis and/or
electrokinetic migration of the sample components. The separation
channel 12 may be filled with a separation support medium, which
may be simply a running buffer, or a sieving gel matrix (of a
linear or non-linear polymeric composition) known in the art.
[0034] One end of the capillary column 10 is coupled to a reservoir
14 of running buffer. The other end of the capillary column 10 is
coupled to another reservoir 16, which may alternately contain a
sample (to be injected into the separation channel 12) and running
buffer (after sample injection, to undertake separation). A power
supply 18 supplies a high voltage to the reservoirs 14 and 16 via
electrodes 20 and 22.
[0035] The mechanism of electrophoresis and radiation induced
fluorescence when considered alone are outside the scope of the
present invention. For the sake of completeness, it is sufficient
to briefly mention the operation of the CE system 100. In
operation, a prepared biological sample, tagged with at least one
known fluorophore, is introduced into the far end of the capillary
column away from the detection zone, by any of a number of ways
that is not part of the present invention (e.g., electrokinetic
injection from a sample reservoir or physical pressure injection
using a syringe pump). When a DC potential (e.g., 1-30 KV) is
applied by the power supply 18 to the electrodes 20 and 22, the
sample migrates under the applied electric potential along the
separation channel 12 in the direction 24 (e.g., sample that is
negatively charged travels toward the positive electrode 22 as
shown in FIG. 1) and separates into bands of sample components. The
extent of separation and distance moved along the separation
channel 12 depends on a number of factors, such as migration
mobility of the sample components, the mass and size or length of
the sample components, and the separation support medium. The
driving forces in the separation channel 12 for the separation of
samples could be electrophoretic, pressure, or electro-osmotic flow
(EOF) means.
[0036] When the sample reaches the detection zone 32, excitation
radiation is directed via the excitation fiber 34 in a direction 35
at the detection zone 32. The sample components would fluoresce
with intensities proportional to the concentrations of the
respective sample components (proportional to the amount of
fluorescent tag material). The detector 42 detects the intensities
of the emitted fluorescence via the emission fiber 36 in a
direction 37, at one or more wavelengths different from that of the
incident radiation. The detected emitted radiation may be analyzed
by known methods, as well as further methods discussed below (e.g.,
in connection with the two-color detection scheme discussed in
reference to FIGS. 14 and 15 below). For an automated system, a
controller 26 (e.g., in the form of a notebook computer or a
desktop computer) having a processor, controls the operations of
the various components in the CE system 100 to effect capillary
electrophoresis separation and data collection. Such control is
well within the knowledge of one skilled in the art.
[0037] In the particular illustrated embodiment in FIG. 1, the
detection optics configuration (schematically indicated in the area
30 located about a detection window/zone 32) corresponds to the
embodiment illustrated in FIG. 2. The direction 35 of incident
radiation (e.g., from a laser or LED source), the axis of the
separation channel at the detection zone, and the direction 37 of
collection of the output radiation are all substantially in the
same plane. In the illustrated embodiment, the detection
configuration of the present invention has optical fibers
positioned at opposite sides of the detection zone separation
channel. In one embodiment, the incident radiation is provided to
the detection zone and/or the output radiation is collected from
the detection zone, using light guides in the form of optical
fibers, in particular ball-ended optical fibers (i.e., optical
fibers terminating in a micro ball that is integral to the fiber
end in a unitary structure).
[0038] Referring also to FIG. 2, a ball-ended fiber (the excitation
fiber 34) extends from a radiation source (e.g., LED or laser
source 41, schematically shown in FIG. 1) to direct excitation
radiation in a direction 35 at the detection zone 32. The ball end
of the excitation fiber 34 is positioned at or proximate to the
exterior surface of the separation column 10 about the detection
zone 32. In the illustrated embodiment, the ball end of the
excitation fiber 34 is positioned at a distance spaced from the
exterior surface of the separation column 10 (i.e., non-contact
mode). In this illustrated embodiment, another ball-ended fiber
(the emission fiber 36) extends to a detector (e.g., a fluorescence
detector 42, schematically shown in FIG. 1) to collect emitted
radiation at a direction 37 from the detection zone 32. The ball
end of the emission fiber 36 is positioned at or approximate to the
exterior surface of the separation column 10 about the detection
zone 32. In the illustrated embodiment, the ball end of the
emission fiber 36 is positioned at a distance spaced (in a
non-contact mode) from the exterior surface of the separation
column 10. Both excitation and emission fibers 34 and 36 with ball
tips are positioned at opposite sides of the separation column 10
in a non-contact mode (spaced from the exterior of the capillary
column) to reduce background fluorescence and not cause any
physical damage to either capillary column or the micro-ball.
[0039] In the illustrated embodiment in FIG. 2, the components at
the detection zone 32 as shown in FIG. 2 lie in substantially the
same plane. Specifically, the longitudinal axis of the excitation
fiber 34, the longitudinal axis of the emission fiber 36 and the
longitudinal axis of the capillary channel 12, are substantially
aligned in the same plane (i.e., substantially coplanar), at least
at the region of the detection zone 32. That is, while the lengths
of the excitation fiber 34, the emission fiber 36 and the capillary
column 10 may be bent overall, however at least near the detection
zone region, the axis of the excitation fiber 34, the axis of the
emission fiber 36 and the axis of the capillary channel 12 are
substantially aligned in the same plane, such that the direction 35
of incident radiation from the excitation fiber 34 towards the
detection zone 32, the axis of the separation channel 12 at the
detection zone 32, and the direction 37 of collection of the output
radiation away from the detection zone along the emission fiber 36
are all substantially in the same plane.
[0040] Further, at the detection zone 32, the angle between the
axis of the excitation fiber 34 and the axis of the emission fiber
36 are not aligned in a straight line. At least one of the axis of
the excitation fiber 34 and the axis of the emission fiber 36 is
not perpendicular to the axis of the separation channel 12 at the
detection zone 32. In the illustrated embodiment shown in FIG. 2,
both the axis of the excitation fiber 34 and the axis of the
emission fiber 36 are not perpendicular to the axis of the
separation channel, and are at angles 39 and 40, respectively, to
the axis of the separation channel 12 at the detection zone 32. The
angle 39 and the angle 40 may be substantially the same or
different, and may be less than or greater than 90 degrees measured
with respect to a reference direction of the axis of the separation
channel 12 or a reference section of the capillary column 10 (e.g.,
the section of capillary column 10 between the fibers 34 and 36 as
shown in FIG. 2). For example, the angle 39 may be less than 90
degrees and the angle 40 may be greater than 90 degrees, measured
from the same reference section. In the illustrated embodiment in
FIG. 2, the angles 39 and 40 are same and substantially in the same
plane.
[0041] In the embodiment illustrated in FIG. 2, both the excitation
fiber 34 and the emission fiber 36 each has a 200 micron diameter
core as light guide within an external cladding, and a 350 micron
diameter ball shaped tip (i.e., the ratio of the fiber core
diameter to the ball diameter is 1:1.75), which comprises fused the
core and cladding material. The ball shaped tip has a substantially
spherical profile. The ball-end fibers may be formed by using a
fusion splicer, or are available from a number of available
suppliers. The capillary column 10 has an outside diameter of 200
to 370 micron (e.g., 360 micron) and an internal diameter of 20 to
150 micron (e.g., 75 micron). The tip of the ball end of the
excitation fiber 34 is spaced at approximately 50-500 micron from
the external surface of the capillary column, and the tip of the
ball end of the emission fiber 36 is spaced at approximately 10 to
500 microns (e.g., 50-200 micron) from the external surface of the
capillary column. Alternatively, the emission fiber 36 may have a
300 micron diameter core with a 500 micron diameter ball shaped tip
at its distal end (i.e., the ratio of the fiber core diameter to
the ball diameter is 1:2.5). The angles 39 and 40 each may range
from greater than 0 to less than 90 degrees, preferably between 20
to 70 degrees, and more preferably at 30 to 45 degrees. In the
illustrated embodiment of FIG. 2, both angles 39 and 40 are about
70 degrees. The ball ends of the fibers 34 and 36 are not touching
the capillary column 10.
[0042] In one embodiment, the optical detection system is
structured with a super-bright UV LED (e.g., LG Innotek/IRTronix or
Dowa) as excitation radiation source for the fluorescent labeled
(FITC) antibody fragment detection. The modular design and fiber
optic coupling provides flexibility for exchanging the excitation
radiation to a laser module (for LIF applications) or other type of
inexpensive light sources.
[0043] It has been found that compared with flat-end fibers (bare
fiber, with no micro ball lens), the ball-ended fibers provide good
focusing of incident radiation (light concentration/power density)
for the excitation fiber 34 and high collection efficiency (high
Numerical Aperture; NA) for the emission fiber 36 as a high angle
fluorescence collector for increased fluorescence signal collection
capability and improved detection sensitivity. Using large core
(e.g., 100-1000 micron) and high NA (0.15-0.5) multi-mode fibers,
it allows high power light coupling from LED or laser into the
excitation fiber 34. By producing an integrated micro ball lens at
the distal output end of the excitation fiber 34, it allows good
coupling efficiency inside the separation channel 12 (e.g., 20-200
micron micro-fluidic channel) for high fluorescence detection
sensitivity.
[0044] A smaller diameter excitation fiber 34 having 200 micron
core diameter with a 330-350 micron diameter ball (see FIG. 2)
directed at the capillary separation channel 12 results in a
smaller focal spot with higher power density, thereby optimizing
the fluorescence excitation signal. If an emission fiber 36 having
a 300 micron core diameter and a 500 micron diameter ball lens is
used for emission collection, the emission collection efficiency is
increased. The outside diameter of the capillary column is 360
micron, and the inside diameter is 75 micron.
[0045] The excitation and emission fibers could be pre-positioned
fixed within the body/assembly of a capillary cartridge (see
cartridge 60 shown in FIG. 5; which may include a separation
support medium such as a gel). The 2-fiber detection configuration
with ball-end fibers has been applied to a disposable
single-channel, single capillary cartridge concept with an
integrated buffer reservoir. A higher throughput instrument
utilizing 4 or 8 gel-cartridges (of the same design) could be
designed to speed up the separation time (cycle) by a factor of
4.times.-8.times. (1 hour for full 96-well sample plate run).
[0046] The test samples are introduced to the separation capillary
column 10 by electro kinetic injection. The high voltage power
supply (e.g., EMCO, Sutter Creek, Calif.) is used to deliver, e.g.,
500V to 20 KV of electrical field to the capillary for the electro
kinetic injection and separations of bio-molecules. An excitation
LED having broad band light energy (e.g., FWHM=20-50 nm) and 20-100
degrees of viewing angle is coupled to the large core excitation
fiber (e.g., 100-1000 micron) at the flat end (polished or cleaved
end). A line filter (e.g., FWHM=2-50 nm Band Pass line filter) is
placed in front of the LED before coupling the light into the 200
micron diameter core with 350 diameter micron ball-ended excitation
fiber to reduce background noise. The micro-ball lens end of the
fiber is produced by fusion splicing (high voltage heat melting)
with a well controlled ball diameter to create a well defined exit
NA and spot size for coupling the excitation radiation energy into
the inner diameter (the separation channel) of the capillary
column. The fluorescence emission signal produced by the separated
analytes are then collected at the detection zone of the capillary
channel using a similar ball-ended fiber (larger core fiber with
500 micron diameter ball) and is relayed to an external detector
module (e.g., fluorescence detector 42 schematically shown in FIG.
1), which may include one or more photomultiplier tubes (PMTs) or
SiPMTs or CCDs, and may also include beam-splitters, built-in
emission filters (e.g., Band Pass Filters) for glycan profiling, in
accordance with further disclosure below.
[0047] FIG. 3 illustrates the external view of a CE instrument 200
in accordance with one embodiment of the present invention. The CE
instrument 200 includes essential components including the
detection configuration schematically shown in FIG. 1. FIG. 4
illustrates that internal view of the CE instrument 200 with the
front and side housing 203 removed, in accordance with one
embodiment of the present invention. FIG. 5 is a schematic view
illustrating the components of the CE instrument 200, some of which
reside within the instrument housing, and some outside of the
housing. The CE instrument 200 comprises a system board 201,
operatively coupled to a sample transport mechanism 202, a
cartridge interface mechanism 204, an optical signal detector such
as a photomultiplier tube (PMT) 206, a power supply 208 (which
includes a high voltage power supply 223, and may further include a
system power supply 222; the power supply 222 may reside outside of
the CE instrument 200), detection optics (e.g., as shown in FIG.
2), and a pressurized gas source 212 (which may reside outside the
CE instrument 200, but connected to a port in the instrument
housing).
[0048] A controller 26 is provided for user interface and
programming of experiment/test settings and parameters. The
controller includes the necessary application software routines,
which may also include data reduction applications. The controller
26 may be an integral part of the instrument 200 (e.g., as part of
the system board 201, with application routines coded in ASICs), or
it may be a separate unit coupled/interfaced to the CE instrument
200. In the illustrated embodiment, the controller is external to
the housing of the CE instrument 200, in the form of a desktop
computer or notebook computer, which is coupled to the CE
instrument 200 via the system board 201 via a USB interface. The
external controller 26 may include mass storage devices, display,
keyboard, etc., or some of these user interface components may be
configured integral to the CE instrument (e.g., a display and a
keyboard on the front housing). Alternatively, the system board 201
may be incorporated as part of the external controller 26, without
departing from the scope and spirit of the present invention.
[0049] The system board 201 includes the necessary electronics to
drive the various components in the CE instrument, e.g., the
movements of the transport mechanism 202, the output of the power
supply 208, the PMT 206, the valve release of the pressurized gas
212, the movements of the cartridge interface 204, an RFID
transmitter/reader, etc. It is noted that the system board 201 is
schematically represented in the figures. It may include other
electronic boards for controlling specific components (e.g.,
electronic board for controlling motors in the sample transport
mechanism 202), or these other boards may be separate from and in
communication with the system board 201 to perform the intended
function. The exact electronic board configuration is not critical
to the present invention, and it is well within the knowledge of
one skill in the art to configure the boards to achieve the desired
functions and features disclosed herein.
[0050] The sample transport mechanism 202 includes a table 221
supporting a sample and buffer tray 220 having multiple wells
(e.g., a standard 96-well titer plate, and larger wells for buffer,
cleaning solutions and waste collection) to move with three degrees
of freedom. The multiple wells may include wells containing
cleaning solutions and samples and also for waste collection. It is
noted that in the figures, X, Y and Z are orthogonal axes. Y is the
vertical axis; X is in a horizontal direction across the instrument
(parallel to the rear of the instrument); and Z is in a horizontal
direction into and out of the instrument. The table 221 is
controlled by the transport mechanism 202 to move up and down, and
to move within a plane in a straight line and rotate within the
plane. That is, the table 221 moves in a single horizontal
direction (Z-direction), and in a vertical (Y-direction), and
rotation about the vertical axis (Y-axis). The combination of
rotation and translation motions would be able to place any of the
multiple wells in the tray 220 for access by the tip of the
depending capillary column 60. The front panel 203 of the
instrument housing includes an opening with a door 260 to allow
user access to place and remove the tray 220.
[0051] The pressurized gas source 212 (e.g., pressurized air or N2)
may be a gas cartridge installed within the housing of the CE
instrument, or may be an external source (e.g., air-pump) providing
pressurized gas to the CE instrument via a gas connection port at
the instrument housing (in which case, the pressurized gas source
would be the gas connection port to the external gas source). The
pressured gas is fed to the reservoir 62 in the cartridge 60 via
appropriate gas tubing and valves (which is operatively coupled to
the system board 201).
[0052] The power supply 208 includes a system DC power supply 222
(e.g., 12-24 VDC from external AC power) coupled to the system
board 201, and a variable high voltage power supply 223 providing
the necessary high voltage to electrode contacts/probes 224 and
225, for electrical contact with electrodes 66 and 67 in the
cartridge 60 for carrying out electrophoresis therein.
Alternatively, instead of using an internal 12-24 VDC power supply
with external AC power, the CE instrument 200 may use an external
12-24 VDC power supply, which makes the instrument simpler and
safer to use without the internal AC to DC conversion. This would
also allow for battery operation for field portability and
operations. The contact probes 224 and 225 may be actuated
pneumatically (e.g., by regulating pressurized gas from the gas
source 212, or electromechanically, to contact against the exposed
surfaces of the electrodes 66 and 67, or the contact probes 224 and
225 may be simply spring loaded to bias against the exposed
surfaces of the electrodes 66 and 67.
[0053] The excitation fiber 34 is optically coupled to a light
source in the form of an LED 226, which may be part of the system
board 201. The emission fiber 36 is optically coupled to the PMT
206 via appropriate optical filters 226. The electrical output of
the PMT 206 is coupled to the system board 201.
[0054] The cartridge interface mechanism 204 is supported on the
chassis of the instrument, and is configured to receive the
cartridge 60, and support its location positively and accurately
with respect to the detection optics. A cartridge-door 261 (FIG. 3)
is provided at the top panel of the instrument housing. The
cartridge interface mechanism 204 includes a base 227 supporting a
receiver block 228 having a cylindrical opening sized and
configured to receive the cartridge 60 as shown. In this
illustrated embodiment, the cartridge 60 is support by the receiver
block 228 in a vertical orientation, with its longitudinal axis
substantially vertical with respect to the horizontal plane of the
tray 220. It is within the scope of the present invention to have
the cartridge supported with its longitudinal axis horizontal with
respect to reagent/sample containers. A safety interlocking feature
may be provided to engage to prevent the cartridge 60 from being
accidentally removed from the receiver block 228 during
electrophoresis operations. The safety interlock feature could also
include the front door (sample-door) 260 for tray 220 and top door
(cartridge-door) 261 for insertion of the cartridge 60 (FIG. 3), to
prevent user accidentally opening these doors during
electrophoresis operations. The safety interlock (not shown) will
only be released upon execution of termination sequence for an
electrophoresis run (e.g., shutting down high voltage supply, and
outward movement of the fork assemblies 230 described below). The
receiver block 228 also includes an RFID reader/transmitter 226
(e.g., on the outside of the receiver block 228) for communicating
with an RFID label on the capillary cartridge 60.
[0055] In one embodiment of the present invention, the
aforementioned CE instrument/system is adopted and modified and
improved for glycan profiling. In one embodiment, the glycan
analyzer system utilizes relatively short capillary columns (e.g.,
15 cm long, 75 .mu.m ID) filled with linear polymer format for the
separation of Instant Dye-labeled or ANTS-labeled complex
carbohydrates.
[0056] Miniaturization and automation of the CE has many advantages
over conventional labor intensive techniques (i.e. slab-gel
electrophoresis) for glycan profiling. These advantages include
improved data precision and reproducibility, short analysis times,
minimal sample consumption, improved automation and integration of
complex workflows. In particular, the CE system provides automated
sample analysis of Instant Dye-labeled or ANTS-labeled N-glycans by
the use of disposable gel capillary cartridge (e.g., cartridge 60
discussed above). The fluorescence detector in the CE system
includes UV LED (e.g., 270 nm-380 nm) as the excitation source and
the emission Detection uses a PMT (e.g., Hamamatsu R5984) with a
band-pass filter (e.g., 400 nm-550 nm). The fluorescence detection
is done by two optical fibers (i.e., one fiber IN and one fiber
OUT). After the cartridge is installed inside the instrument (FIG.
4), the Instant Dye-labeled or ANTS-labeled Glycan samples are
injected from the sample tray (FIGS. 4 and 5) into the capillary
column for separation and detection.
[0057] Specifically, an example, but not limitation, of the
protocol for glycan profiling includes a single micro-fluidic glass
capillary (75 .mu.m ID) with an effective separation length of 11
cm supported in a capillary cartridge (e.g., cartridge 60 disclosed
above). The shortened capillary length allows for reduced operating
voltages (4-8 KV) and the elimination of expensive cooling systems
such as Peltier or recirculating chillers. The cartridge includes
top and bottom electrodes (anode & cathode), an exposed
detection zone and an imbedded RFID chip/label to provide ID for
the gel-cartridge type and track the number of runs per cartridge.
Each cartridge contains linear gel-matrix and is capable of
analyzing 100-300 (typically 200) samples in as few as 2 minutes
per sample, consuming as little as 1 pl from the 1 .mu.l-20 .mu.l
sample volume.
[0058] The system operations are as follows. Buffer(s), markers and
samples are placed in the buffer/sample tray on the platform within
the CE instrument (e.g., CE instrument 200 in FIGS. 3-5) and the
capillary gel cartridge is inserted. Using the system software
(including user interface), the user selects their preferred
preprogrammed method or programs new run parameters followed by
indicating the location of the samples to be tested and the
analysis is started. Depending on the selected methods associated
with the gel-cartridge type the results are completed and displayed
within 2-5 minutes. For example, the Q-Analyzer.TM. software
(developed by BiOptic, Inc.) automatically identifies and
calculates the glucose units (gu) of detected glycan peaks using a
reference glycan Ladder table.
[0059] Using the CE system described above to perform glycan
profiling, it has been found that the present invention provides a
cost-effective capillary gel-electrophoresis system for highly
efficient, high speed, high throughput, glycan analysis (N-Glycan).
The novel method and system significantly increase the pace at
which glycoprotein research is performed in the laboratory, saving
hours of preparation time and assuring accurate, consistent and
economical results.
[0060] FIGS. 8-11 are results of detected fluorescence in
connection with glycan profiling using traditional HPLC, and FIGS.
12-13 are results of detected fluorescence in connection with
glycan profiling using the inventive system 200 and associated
method described above.
[0061] Specifically, FIG. 8 illustrates the detected fluorescence
for HPLC profile of the 2-AB labeled N-linked glycan library
obtained from Fetuin, requiring a run time of 130 minutes; FIG. 9
illustrates the detected fluorescence for HPLC Profile of the 2-AB
labeled N-linked glycan library obtained from RNase B, requiring a
run time of 130 minutes; FIG. 10 illustrates the detected
fluorescence for separation of the neutral glycan fraction from by
normal phase chromatography, requiring a run time of 140 minutes;
and FIG. 11 illustrates the detected fluorescence for separation of
partially hydrolyzed 2-AB labeled dextran on normal phase HPLC;
requiring a run time of 160 minutes. The numbers at the peaks of
the profile indicate glucose units (gu).
[0062] FIG. 12 illustrates the detected fluorescence for profile of
ANTS labeled N-linked glycan library obtained from Fetuin, IgG and
Rnase B utilizing the novel CE system 200, requiring run time of
less than 5 minutes. FIG. 13 illustrates the detected fluorescence
for separation of ANTS labeled dextran utilizing the novel CE
system 200, requiring a run time of 5 minutes. The numbers at the
peaks of the profile indicate glucose units (gu). The Dextran
Ladder is used as a reference marker for accurate peak
identification.
[0063] Comparing the results of traditional HPLC profiling (FIGS.
8-11) of glycan samples are compared with the results of similar
profiling using the inventive system 200 and associated method
(FIGS. 12-13), it can be clearly realized that the HPLC method of
profiling glycan took more than 2 hours (FIGS. 8-11), as compared
to the significantly shorter run time of less than 5 minutes for
the inventive system 200 (FIG. 12).
[0064] Instead of running the sample and a reference marker in
separate runs, and detecting using a single wavelength detection
for each run, the CE system is configured with an improved
detection scheme requiring dual-color/wavelength detection. In
another embodiment, the analyzer is configured for performing a
two-color detection for data analysis (e.g., for accurate data peak
identifications). A single radiation excitation source (e.g., LED
or Laser) is used to excite two emission fluorophores or dyes
(e.g., UV-type fluorophores) in the sample solution to be analyzed.
One emission dye is to tag the sample and the other dye is used to
provide a reference marker (e.g., a Dextran Ladder) in the same
sample run. Two detectors (e.g., photomultipler tubes (PMTs)) are
applied to simultaneously detect the fluorescent emissions from the
dyes. The data collected by both detectors are correlated (e.g.,
synchronized, and/or super-positioned for analysis) for accurate
data peak identification. The two-color detection simplifies and
shortens sample separation and detection into a single run and
assures accurate data analysis for peak identification. The dual
dye detection (i.e., two dye labeling) is a very robust and
accurate way to provide reproducible peak identification and sizing
for glycan profiling.
[0065] Referring back to FIGS. 1 and 2, in this improved CE
detection scheme, a single detection/emission fiber 36 captures the
fluorescence emission from the detection zone 32 similar to the
prior embodiment discussed above (i.e., one fiber IN/one fiber
OUT). However, from the single emission fiber 36, the fluorescence
emission is detected in at least two different wavelengths, by
"splitting" the fluorescence emission into at least two signals for
detection at two different wavelengths (e.g., at .lamda.1 and
.lamda.2 in FIG. 6). In this embodiment, the fluorescence detector
42 in FIG. 1 includes at least two corresponding detectors for at
least two wavelengths of fluorescence emissions. For example, the
excitation wavelength may be 270-380 nm and emission detection
wavelength of 400-550 nm for the Instant Dye-labeled or
ANTS-labeled fluorophores as glycan labeling.
[0066] There are at least two approaches to splitting the
fluorescence emission for detection at different wavelengths.
[0067] FIG. 6 illustrates one embodiment of two-color detection,
including a fiber combiner/splitter 61 for splitting an emission
signal 37 into two signals for fluorescence detection at two
different wavelengths. The 1.times.2 fiber combiner/splitter 61
couples the output signal 37 of the emission fiber 36 to the inputs
of a first emission fiber 36a and a second emission fiber 36b. The
emission fiber 36 includes fluorescence at at least two wavelengths
.lamda.1 and .lamda.2. In this embodiment, .lamda.1 corresponds to
the wavelength of the fluorescence of detected Dextran Ladder and
.lamda.2 corresponds to the wavelength of the fluorescence of
detected glycan profile. The first emission fiber 36a routes
emissions from the emission fiber 36 to a first PMT1 that detects
fluorescence at .lamda.1, and the second emission fiber 36b routes
emissions from the emission fiber 36 to a second PMT2 that detects
fluorescence at .lamda.2. The fiber combiner may be of the type
that splits orthogonal polarizations at at least two wavelengths
(e.g., at .lamda.1 and .lamda.2) or two ranges of wavelengths
(e.g., Thorlabs 1X2 Coupler or Gould 1X2 Fiber Splitter). In
addition, while not shown in FIG. 6, one or more band-pass filters
may be provided between the PMT1 and/or PMT2 and the corresponding
outputs of the first and/or second emission fibers 36a and 36b.
[0068] FIG. 7 illustrates another embodiment of two-color
detection, which includes a dichroic filter/beam-splitter 62 for
splitting an emission signal 37 into two emission signals for
fluorescence detection at two different wavelengths. The
beam-splitter 62 splits the output signal 37 of the emission fiber
36 into the input signals 37a and 37b of a first emission fiber 36a
and a second emission fiber 36b, respectively. The emission fiber
36 includes fluorescence at at least two wavelengths .lamda.1 and
.lamda.2. In this embodiment, .lamda.1 corresponds to the
wavelength of the fluorescence of detected Dextran Ladder and
.lamda.2 corresponds to the wavelength of the fluorescence of
detected glycan profile. The first emission fiber 36a routes
emissions from the emission fiber 36 to a first PMT1 that detects
fluorescence at and the second emission fiber 36b routes emissions
from the emission fiber 36 to a second PMT2 that detects
fluorescence at .lamda.2. An example of an appropriate
beam-splitter may be a model no. DMLP P425 longpass type Dichroic
Mirror available from ThorLabs, Inc., which has a 45.degree. angle
of incident, a cutoff wavelength of 425 nm, with a transmission
band of 440-700 nm, and a reflectance band of 380-410 nm. In
addition, while not shown in FIG. 6, one or more band-pass filters
may be provided between the PMT1 and/or PMT2 and the corresponding
outputs of the first and/or second emission fibers 36a and 36b. As
shown in FIG. 7, the ends of the optical fibers 36, 36a and 36b are
inserted/supported in corresponding terminating optical couplers
63, 63a and 63b, which each has a collimating lens (64, 64a,
64b).
[0069] Referring to FIG. 5, the above described two-color detection
configurations can be modified by implementing the above-noted
beam-splitter 62 and/or combiner/splitter 61, emission fibers 36a
and 36b, and another PMT in addition to the PMT 206 for the
additional color detection.
[0070] FIGS. 14-15 are results of detected fluorescence in
connection with glycan profiling using the system 200 modified with
two-color detection scheme and associated method discussed above.
Specifically, FIG. 14 illustrates the detected fluorescence 70 for
a Glycan Ladder (by PMT1 at .lamda.1) transposed on the detected
fluorescence 72 for a glycan sample 72 (by PMT2 at .lamda.2). FIG.
15 illustrates the detected fluorescence 70 for the same Glycan
Ladder (by PMT1 at .lamda.1) and the detected fluorescence 72 for
the same glycan sample (by PMT2 at .lamda.2) displayed separately
and aligned by arrows 75.
[0071] The two-color detection simplifies and shortens sample
separation and detection into a single run and assures accurate
data analysis for peak identification. The dual dye detection
(i.e., two dye labeling) is a very robust and accurate way to
provide reproducible peak identification and sizing for glycan
profiling.
[0072] The simplicity of the micro-optical detection also provides
flexibility in designing higher throughput (i.e. multi-channel,
e.g., 4-12-channel) type gel-cartridge without the use of optics
(excitation or emission optics) inside the cartridge assembly,
hence reducing costs for the cartridge.
[0073] Accordingly, the new fluorescence fiber-based detection for
the CE system in accordance with the present invention provides
simplicity in design, ease of operation and lower cost consumable
for glycan profiling. It provides a good solution particularly for
the research and clinical diagnostic laboratories/industry that
demands sustained and stable recurring revenue streams from both an
installed base of instruments and recurring need for consumables
such as testing reagents and buffer containing capillary
cartridge.
[0074] While the invention has been particularly shown and
described with reference to the preferred embodiments, it will be
understood by those skilled in the art that various changes in form
and detail may be made without departing from the spirit, scope,
and teaching of the invention.
[0075] For example, the excitation radiation source could be, for
example, LEDs, Laser Diodes (semiconductor solid-state lasers),
pulsed lasers (e.g., solid state lasers, gas lasers, dye lasers,
fiber lasers), or other sources of radiation. Alternate relative
inexpensive light source for the present invention could be laser
diodes in the visible, UV and/or infrared range. For example, laser
diodes in the range of 200-900 nm, and more specifically in the
range of 270-380 nm may be used, for example.
[0076] A person skilled in the art will recognize that the
instrument incorporating the essence of this invention can also be
used for bio molecular analysis other than glycan profiling
analysis. For example, by altering the separation gel or buffer,
the system can also be modified to analyze biomolecules like DNA,
immunoassays, proteins, carbohydrates, and lipids.
[0077] By way of example and not limitation, the detection
configuration of the present invention is described in connection
with capillary electrophoresis and radiation induced fluorescence
detection for glycan profiling. It is understood that the present
invention is also applicable to detection of analytes separated
based on bio-separation phenomenon other than electrophoresis, and
detection of radiation emissions other than fluorescence
emissions.
[0078] Instead of positioning the excitation fiber and emission
fiber substantially coplanar with the axis of the separation
channel at the detection zone, the excitation fiber or the emission
fiber may be out of plane, without departing from the scope and
spirit of the present invention.
[0079] Furthermore, while the separation channels in the described
embodiments are defined by cylindrical columns or tubes, it is
understood that the concepts of the present invention is equally
applicable to separation channels defined by open channels, for
example micro-channels defined by etching in a substrate.
[0080] Accordingly, the disclosed invention is to be considered
merely as illustrative and limited in scope only as specified in
the appended claims.
* * * * *