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Claims  |
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What is claimed is:
1. A multiplexed fluorescence detection system for use with capillary
electrophoresis to detect target species in a sample, comprising:
(a) an array of at least two capillaries each having an intake end, an
outflow end, and an annular wall wherein at least a portion of the wall is
translucent to light having a wavelength of about 200-1,500 nm, the intake
end being in fluid communication with the sample so that the sample is
drawn into the capillary;
(b) an array of at least two optical fibers, each optical fiber having a
first and second end, the second end of each optical fiber being coupled
to a capillary in the array of capillaries;
(c) means for generating coherent light positioned to direct the coherent
light onto the first end of the optical fibers, wherein the coherent light
is of a wavelength suitable to cause fluorescence in target species
present in the capillaries; and
(d) means for substantially simultaneously detecting changes in fluorescent
emission by the target species through the annular wall of each capillary.
2. The detection system of claim 1 wherein the capillaries have an inside
diameter of about 20-500 microns and at least one optical fiber is coupled
to each capillary in a manner to transmit the coherent light axially into
the capillary.
3. The detection system of claim 2 wherein the optical fiber is coupled to
the capillary by insertion of the second end of the optical fiber into the
outflow end of the capillary, the optical fiber being of a suitable
diameter for affixing the fiber inside the capillary without substantially
hindering the sample flow.
4. The detection system of claim 1 wherein at least one optical fiber is
coupled to each capillary and positioned adjacent to and orthogonally in
relation to the capillary.
5. The detection system of claim 1 further comprising a means for focusing
light.
6. The detection system of claim 5 wherein the focusing means is a lens for
focusing fluorescent light emitted from the target species in the
capillaries to the means for detecting fluorescent light.
7. The detection system of claim 1 wherein the means for generating
coherent light includes collimating means for distributing the coherent
light into the first end of the optical fibers in a substantially uniform
manner.
8. The detection system of claim 1 wherein the means for detecting the
fluorescent emission comprises a charged-coupled device based imaging
system.
9. The detection system of claim 1 wherein the means for detecting the
fluorescent emission comprises a charge injection device based imaging
system.
10. The detection system of claim 1 further comprising a spacer positioned
between at least two capillaries to reduce optical crosstalk between the
capillaries.
11. The detection system of claim 1 wherein the means for generating
coherent light delivers about 0.05-10 mW per fiber.
12. The detection system of claim 1 wherein the coherent light has a
wavelength of about 250-700 nm.
13. The detection system of claim 1 wherein the array of capillaries
comprises at least 100 capillaries.
14. The detection system of claim 1 wherein the array of capillaries
comprises at least 1000 capillaries.
15. The detection system of claim 1 wherein at least one of the capillaries
contains a gel that is suitable for capillary gel electrophoresis systems.
16. The detection system of claim 1 wherein at least one of the capillaries
contains no gel.
17. A multiplexed method for detecting the presence of fluorescent target
species in a sample in a plurality of capillaries using capillary
electrophoresis, comprising the steps of:
(a) introducing samples containing a fluorescent species into intake ends
of a plurality of capillaries, each capillary having an annular wall, and
an outflow end, wherein the samples migrate through the capillaries
towards the outflow ends;
(b) irradiating the samples in the capillaries with coherent light
transmitted through optical fibers, the optical fibers having first ends
coupled to a coherent light source and second ends individually coupled to
the capillaries;
(c) substantially simultaneously detecting change in fluorescent emission
by the fluorescent species through the wall of each of the capillaries.
18. The method of claim 17 wherein the step of irradiating comprises
transmitting coherent light axially into a capillaries by inserting the
second ends of the optical fibers into the outflow ends of the
capillaries, the optical fiber having a diameter suitable for affixing the
fiber in position in the capillary without substantially hindering sample
flow.
19. The method of claim 17 wherein the step of irradiating comprises
irradiating coherent light orthogonally into the capillaries through an
optical fiber that is coupled adjacent to, and perpendicular in relation
to the capillary.
20. The method of claim 17 wherein the step of detecting fluorescent
emission comprises using a charged-coupled device based imaging system for
detecting the fluorescent emissions.
21. The method of claim 17 wherein the step of detecting fluorescent
emission comprises using a charge injection device based imaging system
for detecting the fluorescent emission.
22. The method of claim 17 further comprising a step of using a lens for
evenly distributing the coherent light generated by a laser axially into a
plurality of optical fibers at an end distal to the capillaries.
23. The method of claim 17 wherein the step of irradiating comprises
irradiating coherent light at a power of about 0.05-10 mW per fiber.
24. The method of claim 17 wherein the step of irradiating comprises
irradiating coherent light with a wavelength of about 250-700 nm.
25. The method of claim 17 wherein the step of introducing samples into
capillaries comprises introducing samples into an array of at least 100
capillaries.
26. The method of claim 17 wherein the step of introducing samples into
capillaries comprises introducing samples into an array of at least 1000
capillaries.
27. The method of claim 17 wherein the step of introducing samples into
capillaries comprises introducing a sample into at least one capillary
that contains a gel that is suitable for capillary gel electrophoresis.
28. The method of claim 17 wherein the step of introducing samples into
capillaries comprises introducing a sample into at least one capillary
that contains no gel.
29. A multiplexed method of detecting the presence of macromolecule in a
sample present in a plurality of capillaries in capillary electrophoresis,
comprising the steps of:
(a) linking a fluorophore to the macromolecule in a sample to transform the
macromolecule into a fluorescent species with fluorescence that can be
excited by light of suitable wavelength;
(b) introducing samples containing fluorescent species into each of a
plurality of capillaries, each capillary having an annular wall, an intake
end and an outflow end;
(c) irradiating each sample in each capillary with coherent light to cause
fluorescence of the macromolecule, the coherent light being transmitted
though optical fibers individually coupled to the capillaries; and
(d) substantially simultaneously detecting changes in fluorescent emission
through the wall of each of the capillaries by the fluorescent species
using a charge transfer device based imaging system.
30. The method of claim 29 wherein the step of detecting changes in
fluorescence comprises using a charged-coupled device based imaging
system.
31. The method of claim 29 wherein the step of detecting change in
fluorescence comprises using a charged injection device based imaging
system.
32. The method of claim 29 further comprising a step of introducing a light
absorber into the capillaries for reducing photochemical bleaching of
fluorescent species.
33. The method of claim 29 wherein the step of introducing samples into
capillaries comprises introducing samples into an array of at least 100
capillaries.
34. The method of claim 29 wherein the step of linking a fluorophore to the
macromolecule comprises linking a fluorophore to the macromolecule in a
material selected from the group consisting of proteins, amino acids,
polypeptides, carbohydrate, polysaccharides, oligonucleotides, nucleic
acids, RNA, DNA, bacteria, viruses, chromosomes, genes, organelles, as
well as fragments and combinations thereof.
35. The method of claim 29 wherein the step of linking a fluorophore to the
macromolecule comprises linking the macromolecule to a fluorophore
selected from the group consisting salicylate,
3,3'-diethylthiadicarbocyanine iodide (DTDCI), dansyl chloride,
fluorescein, fluorescein isothiocyanate, ethidium bromide, rhodamine, and
combinations thereof.
36. A multiplexed method of detecting the presence of macromolecules in
samples present in a plurality of capillaries in capillary
electrophoresis, comprising the steps of:
(a) providing a buffer solution containing a fluorophore;
(b) introducing samples into a plurality of capillaries in a capillary
electrophoresis system, each capillary containing the buffer;
(c) irradiating the samples in the capillaries with coherent light to cause
fluorescence, the coherent light being transmitted though optical fibers
individually coupled to the capillaries; and
(d) substantially simultaneously detecting changes in fluorescent emission
through the wall of each the capillaries by the fluorescent species by
using a charge transfer device based imaging system.
37. A multiplexed method of detecting the presence of DNA fragments in
samples present in a plurality of capillaries in capillary
electrophoresis, comprising the steps of:
(a) linking a fluorophore to a DNA fragment in a sample to transform it
into a fluorescent species;
(b) introducing samples containing fluorescent species into a plurality of
capillaries in a capillary electrophoresis system, the system comprises an
array of at least about 100 capillaries each having an inside diameter of
about 20-500 microns, and an annular wall;
(c) irradiating the samples in the capillaries with coherent light having a
wavelength of about 250-700 nm to cause native fluorescence of the DNA
fragment, the coherent light being transmitted though optical fibers
individually coupled to the capillaries; and
(d) substantially simultaneously detecting change in fluorescence emitted
by the fluorescent species through the wall of each of the capillaries by
focusing the fluorescence onto a charge transfer device based imaging
system. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The present invention relates to fluorescence detector systems for
capillary electrophoresis. Specifically, the present invention relates to
fluorescence detector systems with the ability to substantially
simultaneously excite fluorescence and to substantially simultaneously
monitor separations in multiple capillaries.
BACKGROUND OF THE INVENTION
Electrophoresis is an electrochemical process in which molecules with a net
charge migrate in a solution under the influence of an electric current.
Traditionally, slab gel electrophoresis has been a widely used tool in the
analysis of genetic materials. See, for example, G. L. Trainor, Anal.
Chem., 62, 418-426 (1990). Recently, capillary electrophoresis (CE) has
emerged as a powerful separations technique, with applicability toward a
wide range of molecules from simple atomic ions to large DNA fragments. In
particular, capillary gel electrophoresis (CGE) has become an attractive
alternative to slab gel electrophoresis (SGE) for biomolecule analysis,
including DNA sequencing. See, for example, Y. Baba et al., Trends in
Anal. Chem., 11, 280-287 (1992). This is generally because the small size
of the capillary greatly reduces Joule heating associated with the applied
electrical potential. Furthermore, CGE produces faster and better
resolution than slab gels. Because of the sub-nanoliter size of the
samples involved, however, a challenging problem in applying this
technology is the detecting and monitoring of the analytes after the
separation.
Currently, sophisticated experiments in chemistry and biology, particularly
molecular biology, involve evaluating large numbers of samples. For
example, DNA sequencing of genes are time consuming and labor intensive.
In the mapping of the human genome, a researcher must be able to process a
large number of samples on a daily basis. If many capillaries of CE can be
conducted and monitored simultaneously, i.e., multiplexed, cost and labor
for such projects can be significantly reduced. Attempts have been made to
sequence DNA in slab gels with multiple lanes to achieve multiplexing.
However, slab gels are not readily amenable to a high degree of
multiplexing and automation. Difficulties exist in preparing uniform gels
over a large area, maintaining reproducibility over different gels, and
loading sample wells. Furthermore, difficulties arise as a result of the
large physical size of the medium, the requirement of uniform cooling,
large amounts of media, buffer, and samples, and long run times for
extended reading of nucleotides. Unless gel electrophoresis can be highly
multiplexed and run in parallel, the advantages of capillary
electrophoresis cannot produce substantial gain in shortening the time
needed for sequencing the human genome.
Significantly, the capillary format is in fact well suited for
multiplexing. The substantial reduction of Joule heating per lane makes
the overall cooling and electrical requirement more manageable. The cost
of materials per lane is reduced because of the smaller sample sizes. The
reduced band dimensions are ideal for excitation by laser beams and for
imaging onto solid-state array detectors. The use of electromigration
injection, i.e., applying the sample to the capillary by an electrical
field, provides reproducible sample introduction with little band
spreading and with little labor.
Among the techniques used for detecting target species in capillary
electrophoresis, laser-excited fluorescence detection so far has provided
the lowest detection limits. Therefore, fluorescence detection has been
used for the analysis of chemicals, especially macromolecules in capillary
electrophoresis. For example, Zare et al. (U.S. Pat. No. 4,675,300)
discussed a fluoroassay method for the detection of macromolecules such as
genetic materials and proteins in capillary electrophoresis. Yeung et al.
(U.S. Pat. No. 5,006,210) presented a system for capillary zone
electrophoresis with laser-induced indirect fluorescence detection of
macromolecules, including proteins, amino acids, and genetic materials.
Systems such as these generally involve only one capillary. There have been
attempts to implement the analysis of more than one capillary
simultaneously in the electrophoresis system, but the number of
capillaries has been quite small. For example, S. Takahashi et al.,
Proceedings of Capillary Electrophoresis Symposium, December, 1992,
referred to a multi-capillary electrophoresis system in which DNA fragment
samples were analyzed by laser irradiation causing fluorescence. This
method, however, relies on a relatively poor focus (large focal spot) to
allow coupling to only a few capillaries. Thus, this method could not be
applied to a large number of capillaries, such as 1000 capillaries, for
example. This method also results in relatively low intensity and thus
poor sensitivity because of the large beam. Furthermore, detection in one
capillary can be influenced by light absorption in the adjacent
capillaries, thus affecting accuracy.
Attempts have been made to perform parallel sequencing runs in a set of up
to 24 capillaries by providing laser-excited fluorometric detection and
coupling a confocal illumination geometry to a single laser beam and a
single photomultiplier tube. See, for example, X. C. Huang et al., Anal.
Chem., 64, 967-972 (1992), and Anal. Chem., 64, 2149-2154 (1992). However,
observation is done one capillary at a time and the capillary bundle is
translated across the excitation/detection region at 20 mm/sec by a
mechanical stage.
There are features inherent in the confocal excitation scheme that limit
its use for very large numbers (thousands) of capillaries. Because data
acquisition is sequential and not truly parallel, the ultimate sequencing
speed is generally determined by the observation time needed per DNA band
for an adequate signal-to-noise ratio. Having more capillaries in the
array or being able to translate the array across the detection region
faster will not generally increase the overall sequencing speed. To
achieve the same signal-to-noise ratio, if the state-of-the-art sequencing
speed of 100 nucleotides/hour/lane is used, the number of parallel
capillaries will have to be reduced proportionately regardless of the scan
speed. Moreover, the use of a translational stage can become problematic
for a large capillary array. Because of the need for translational
movement, the amount of cycling and therefore bending of the capillaries
naturally increases with the number in the array. It has been shown that
bending of the capillaries can result in loss in the separation
efficiency. This is attributed to distortions in the gel and multipath
effects. Sensitive laser-excited fluorescence detection also requires
careful alignment both in excitation and in light collection to provide
for efficient coupling with the small inside diameter (i.d.) tubing and
discrimination of stray light. The translational movement of the
capillaries thus has to maintain stability to the order of the confocal
parameter (around 25 .mu.m) or else the cylindrical capillary walls will
distort the spatially selected image due to misalignment of the
capillaries in relation to the light source and photodetector.
Further, the standard geometry for excitation perpendicular to the axis of
the capillary requires the creation of an optically clear region in the
capillary. This makes the capillary fragile and complicates the
preparation of capillaries for use. Moreover, the excitation path length,
and hence tee fluorescence signal, is restricted to the small diameter of
the capillary. Therefore, there is a need for a fluorescence detection
system that can be utilized to analyze a large number of samples
substantially simultaneously without bulky equipment and unduly
complicated procedures such as careful alignment.
SUMMARY OF THE INVENTION
The present invention provides a fluorescence detection system for
capillary electrophoresis wherein a laser can be used to substantially
simultaneously excite fluorescence in multiple capillaries and a detector
can substantially simultaneously monitor analyte separations by detecting
the fluorescence in a plurality of separation capillaries. This
multiplexing approach involves laser illumination of a bundle of optical
fibers that are coupled individually with the capillaries in a capillary
array. The coupling can be done by inserting at least one optical fiber
into each capillary that contains sample. It can also be accomplished by
placing the optical fiber adjacent to and perpendicular in relation to the
capillary. The fluorescence of the array of capillaries is focused
orthogonally, i.e., perpendicularly in relation to the length of the
capillaries, through a lens such as a microscope or camera lens and imaged
onto a charge-coupled-device camera for signal analysis. The technique can
be used for monitoring as many capillaries as desired, from at least 2 to
more than 1000 capillaries.
The multiplexed fluorescence detection system contains an array of at least
two (but possibly thousands) of capillaries, each preferably having an
inside diameter of about 20-500 microns, and an array of at least two (but
possibly thousands) of optical fibers of a suitable outside diameter. Each
capillary has an annular wall, an intake end, and an exit end. A more
preferable inside diameter of each capillary is about 40-100 microns. The
multiplexed fluorescence detection system contains a laser for generating
coherent light of a wavelength suitable for exciting fluorescence in a
fluorescent species and a means for detecting the fluorescent light
emitted by the fluorescent species through the wall of each capillary.
An argon laser is a preferred means for generating coherent light in the
present invention, although any laser with the proper power and wavelength
for excitation of the fluorescent species can be used. A beam from the
laser is focused through a microscope objective onto the ends of the
optical fibers distal to the capillaries. Light is therefore transmitted
axially, i.e., parallel to the length of the capillaries, into the cores
of the capillaries by means of the optical fibers. Alternatively, light is
transmitted through the capillary wall of each of the capillaries with an
optical fiber placed perpendicular in relation to each capillary. Each
capillary has a detection zone where the fluorescence is transmitted
through the capillary wall. The detection zones of the capillaries are
imaged onto a charged-coupled device imaging system, or (CCD) camera,
through a microscope. Data is extracted from the memory of the CCD camera
and analyzed by a computer. Instead of a CCD system, a charge injection
device based (CID) imaging system can also be used.
The present invention can be applied to a direct fluorescence system or an
indirect fluorescence system. In a direct fluorescence system, the
presence of a target fluorescent species is detected by a change,
typically an increase in the fluorescence recorded by the detector as the
target fluorescent species passes the detection zone. In an indirect
fluorescence system, the buffer solution contains a background
fluorophore. As the target analyte species traverses the detection zone,
laser induced fluorescence results in either displacement or ion pairing
of an ionic analyte with the fluorophore.
The present invention can be implemented utilizing an array of capillaries
containing preferably at least about 100 capillaries, and more preferably
at least about 500 capillaries, and most preferably at least about 1000
capillaries. It is necessary to select a material of construction for the
capillaries such that at least part of each capillary is translucent to
light of a wavelength of about 200-1,500 nm, so that fluorescent light can
pass through the capillary wall. Inorganic materials such as quartz,
glass, fused silica, and organic materials such as teflon and its related
materials, polyfluoroethylene, aramide, nylon, polyvinyl chloride,
polyvinyl fluoride, polystyrene, polyethylene and the like can be used.
Any conventional fluorescence labels such as a salicylate, dansyl chloride,
ethidium bromide, rhodamine, fluorescein, fluorescein isothiocyanate, and
the like can be used. To eliminate or reduce photochemical bleaching
(i.e., a reduction in the fluorescent properties of fluorophores) of the
fluorescent labels, an organic absorber, such as Orange G, which is a dye,
can be used to reduce bleaching of the fluorophores before they reach the
detection zone of the capillaries. In the case of a partial internal
reflection system, a bend can be made in the capillary after the detection
zone to prohibit any further light travel through the liquid core.
Spacers are preferably placed between each of the separation capillaries to
reduce crosstalk, which is the interference between the separation
capillaries due to the emission of fluorescence light in the adjacent
capillaries. Fibers, capillaries, thin pieces of paper of dark color and
the like can be used as spacers.
This multiplexed detection system can be used for analyzing macromolecules
such as proteins, amino acids, polypeptides, carbohydrates,
polysaccharides, oligonucleotides, nucleic acids, RNAs, DNAs, bacteria,
viruses, chromosomes, genes, organelles, fragments, and combinations
thereof. This invention is also equally applicable whether a gel is used
in the capillary electrophoresis system or not.
A method for detecting macromolecules, such as biological molecules, using
a multiplexing approach is also provided. According to this method,
samples are introduced, optionally with fluorophores, into capillaries of
a capillary array in a capillary electrophoresis system. Coherent light
emitted by a laser is transmitted through optical fibers coupled to the
capillaries. The change in fluorescence emitted by the fluorescent species
is detected orthogonally by a CCD or CID camera through a microscope.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is schematic representation of a preferred multiplexed detection
system in capillary electrophoresis utilizing axial irradiation.
FIG. 2 is a schematic representation of a portion of an interface between
capillaries and optical fibers.
FIG. 3 is a schematic representation of an alternative multiplexed
detection system in capillary electrophoresis wherein orthogonal
irradiation is used.
FIG. 4 shows graphs of fluorescence intensity versus time in three
different capillaries. These graphs show crosstalk between separation
channels in the absence of spacers. The eluted fluorophore is
3,3'-diethylthiadicarbocyanine iodide (DTDCI). The elution times are
different in the three graphs due to variations among the capillaries. The
scale is deliberately magnified.
FIG. 5 shows graphs of fluorescence signals from 10 different capillaries.
These graph show the results of simultaneous electrophoretic separations
of riboflavin and fluorescein. The concentrations of riboflavin and
fluorescein were 5.times.10.sup.-5 M and 6.times.10.sup.-7 M,
respectively. The injection time at 7500 volts was 5 seconds. The time
axis is in minutes.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a fluorescence detector system, i.e.,
fluorescence detection system, for capillary electrophoresis wherein a
laser can substantially simultaneously excite fluorescence in analyte in
multiple capillaries and a detector can substantially simultaneously
monitor separations of analytes, i.e., target specie, in a plurality of
capillaries. Herein, "a plurality" means at least two. In this system, an
excitation laser is coupled to a plurality of optical fibers that are in
turn individually coupled with the capillaries. The technique of the
present invention can be used for monitoring as many capillaries as
desired, even up to thousands of capillaries and more. Furthermore, data
collection rates are much faster in this system than in conventional CGE.
Significantly, the present invention allows for future increases in
sequencing rates as permitted by advances in optics and capillary
technology.
In capillary electrophoresis (CE), typically analytes, i.e., target
species, are detected by measuring laser-induced fluorescence emitted by
the target species. Fluorescence is a phenomenon in which an atom or
molecule emits light when passing from a higher to a lower electronic
state. A fluorescent species is excited by absorption of electromagnetic
radiation of a proper wavelength. It emits light of a longer wavelength
when it passes from the high energy state to a low energy state. The time
interval between absorption and emission of energy is extremely short,
typically within a range of about 10.sup.-8 -10.sup.-3 seconds. Some
analyte molecules such as DNAs can be fluorescent naturally and gives off
native fluroescences when irridated with a light of suitable wave length.
Other molecules, such as proteins, may need to be tagged with fluorophores
to become fluorescent. The present invention is a system for detection of
such laser-excited fluorescence. This invention can also be used for the
detection of phosphorescence in which the time interval between absorption
and admission of energy is much longer. As used herein, the term
"fluorescence" includes fluorescent phenomenon as well as phosphorescent
phenomenon.
Fluorescence is measured by a detector at a detection zone of a capillary.
During the process of electrophoresis, as a fluorescent species traverses
the detection zone, it is excited by an incident laser beam. In a direct
fluorescence detection system, either the target species is fluorescent,
or it is transformed into a fluorescent species by linking with a
fluorophore. The passing of the fluorescent species across the detection
zone results in a change, typically an increase in the fluorescence that
is detectable.
It is also possible to employ other schemes of fluorescence. For example,
instead of producing an increase in fluorescence it is possible to have a
change in the spectral characteristics of the fluorescent species. The
change in the emitted fluorescence spectrum can be detected as a change in
the intensity of light of particular wavelengths. Another applicable
system is the use of indirect fluorometry. Such a system is described by
Yeung et al. in U.S. Pat. No. 5,006,210. In this method, a fluorescent ion
or fluorophore is added to a buffer solution as a principal component of
the buffer. As these fluorophore components in the buffer pass the
detection zone, laser induced fluorescence results in chemical reaction of
the ionic analyte with the fluorophore. This produces a change in
fluorescence, a signal which can be detected by a camera through a
microscope.
The Detection System
FIG. 1 is a schematic representation of the application of the multiplexed
detection system 100 in capillary electrophoresis. Container 12 contains a
buffer solution 25 that is in fluid communication with each individual
capillary 20 of an array 23 of capillaries 20 of the electrophoresis
system 100. Each capillary 20 has a high voltage end 13 and a low voltage
end 33 (see FIG. 2). The high voltage end 13 of each capillary 20 is
immersed in buffer 25 and the low voltage end 33 of each capillary 20 is
in contact with buffer 30 in container 32. Buffer 30 is at ground
potential in this embodiment. A sample to be analyzed is injected into
each capillary 20 at the high voltage end 13. The samples migrate through
the array 23 of individual capillaries 20 and flow out of the low voltage
end 33 of each capillary 20 into buffer 30.
For the sake of simplicity, the voltage source equipment for applying the
high potential to the capillaries is not shown. In this embodiment, the
high potential end of the capillary electrophoresis system is at the
entrance end of the capillary. Herein, the high voltage end of each
capillary is also referred to as the intake end, and the low voltage end
as the outflow end. For specifics regarding a description of the
components and operation of capillary electrophoresis systems, see, for
example, H. Swerdlow et al., Anal. Chem., 63, 2835-2841 (1991).
The system also includes a bundle 10 of optical fibers 15 wherein each
optical fiber 15 is coupled to an individual capillary 20 by insertion
into the outflow end 33 of the capillary 20. Each optical fiber 15 has a
first end 42 and a second end 70. Excitation laser 40 is coupled into the
individual optical fibers 15 of bundle 10 at first ends 42 by means of a
microscope objective 45. First ends 42 of the fibers are distal to the
capillaries 20. The laser 40 and the microscope objective 45 are
positioned so that the laser beam, represented by arrow 47, is focused
onto the ends 42 of the optical fibers 15. The capillaries 20 with optical
fibers 15 inserted at the outflow ends 33 of the capillaries 20 are
arranged in an array 102, which is held in a fixed position on a stage 52
under an objective 65 of a microscope 60. The capillaries 20 as well as
the optical fibers 15 are held firmly in place in an array 102 by guides
(not shown). The fluorescent light emitted from the fluorescent species in
the array 102 of capillaries 20 through capillary walls 104 is imaged
orthogonally through the microscope 60 by means of an adapter 55 onto a
charge-coupled device camera 50 for signal analysis.
FIG. 2 (not to scale) shows the array 102 of capillaries 20 with optical
fibers 15 inserted at the outflow end 33 of the capillaries 20 in
capillary electrophoresis. The outflow end 33 of the capillaries 20 are
aligned substantially evenly in the field of the microscope 60. Optical
fibers 15 are positioned into the capillaries 20 so that the second ends
70 of the optical fibers 15 are also aligned substantially evenly.
Capillaries 80 with black coating are placed between each of capillaries
20 that are used for separation, to act as spacers for reducing the
crosstalk between the separation capillaries 20. Detection zone 90 is the
area of a capillary 20 before the end 70 of the optical fiber. The
detection zones 90 of the capillaries 20 are arranged such that the
capillary images are detected by the pixel columns of the camera 50 (not
shown) coupled to the microscope 60.
Referring again to FIG. 1, the microscope objective 45 is a means for
collimating the laser beam 47 onto the ends of a plurality of optical
fibers 15. Depending on the power of the laser 40, the diameter of the
laser beam 47, and the number and size of the optical fibers 15, the
collimating means 45 can either be focusing or diverging the laser beam 47
to irradiate the ends 42 of the optical fibers 15. If the laser has a
narrow beam of high power and the number of optical fibers is large, the
microscope objective 45 can be used to diverge and spread the laser beam
47 evenly over the ends 42 of the optical fibers 15. If the laser 40 has a
wide beam 47 of low power, it may be necessary to use the microscope
objective 45, to focus the beam 47 onto the ends 42 of the optical fibers
15.
The light source used for excitation of the fluorophores in the samples of
interest is a means for generating a coherent light, or a laser. The
wavelength of the laser radiation is selected to match the excitation
wavelength of the particular fluorophore. Suitable fluorescent species
typically absorb light at a wavelength of about 250-700 nm, preferably
about 350-500 nm, and emit light at a wavelength of about 400-800 nm.
Preferably a laser that can deliver about 0.05-10 mW per fiber, and more
preferably about 0.1-0.5 mW per fiber is used, although lasers with power
outside these ranges can be employed. Any appropriate laser of the proper
wavelength and energy can be used. A commonly used laser is an Argon laser
that produces coherent light of 488 nm. Such lasers are commercially
available from, for example, Cyonics of San Jose, Calif.
Any appropriate optical fibers can be used for illuminating the samples in
the capillaries. Optical fibers are selected based on cost, size, and the
attenuation of light intensity related to length. Most typical commercial
optical fibers of the appropriate diameter are suitable. The selection of
the size of the optical fiber is dependent on the capillary inside
diameter (i.d.). The fibers should be small enough to facilitate smooth
insertion into the capillaries. The fibers should not be so large,
however, that they hinder substantially the flow of analytes and buffer in
the capillaries. Preferably, the reduction of flow rate due to the
presence of the optical fiber is less than about 50%. Generally the size
of the optical fiber is not critical as long as the detection zone is well
irradiated. The fibers and the capillaries are held in place by guides.
Generally, however, the friction between the fiber and the capillary is
adequate to affix the fibers in place so that fluid motion or minor
vibration does not cause a fiber to move in relation to the capillary.
Although in this embodiment each capillary is coupled to one optical
fiber, more than one fiber can be used to irradiate each capillary. There
can be a slight pressure differential because of the restricted cross
section of the capillary bore. Therefore, the overlap region should be
short compared to the length of the capillary. Typically, the overlap is
about 0.1- 2 cm. Optical fibers are commercially available from, for
example, Edmond Scientific Co., Barrington, N.J. The insertion of an
optical fiber to the end of the capillary reduces contributions from the
surfaces of the optical fiber to the separation.
Capillaries are often coated with a polymer, typically polyimide. In
fluorescence detection, the capillary wall and the polyimide coating
fluoresce if they are irradiated by the laser light. In certain cases,
this may interfere with the small fluorescence signal from the analytes.
Thus, the preferred embodiment of the present invention focuses the
excitation laser beam directly into the capillary core axially by focusing
the laser beam into an optical fiber, which has been inserted into the
separation capillary. Because of the unique light transmitting property of
the optical fiber, light is efficiently transmitted to the liquid core.
Although the preferred embodiment is to irradiate the sample axially, a
viable application of the present invention is to irradiate orthogonally,
i.e., perpendicular to the capillary 20 length. The illuminating ends of
the optical fibers 15 are located on the opposite side of the array 102 of
capillaries 20 in relation to the microscope 60 and the camera 50. Optical
fibers 15 are again coupled individually to the capillaries 20. FIG. 3
(not to scale) illustrates this scheme. A camera 50 (not shown) is coupled
to a microscope objective 65 in substantially the same manner as in FIG. 1
for detecting fluorescence. Optical fibers 15 are oriented at an angle
that is perpendicular to capillaries 20 but is 45.degree. in relation to
the light path between the capillaries 20 and the microscope objective 65.
(For the purpose of simplicity, the electrophoretic equipment, camera,
laser and microscope body are not shown. They can be the same as in FIG.
1.) Again, as in the previously described embodiment, the capillaries 20
as well as the optical fibers 15 are held by guides (not shown in FIG. 3)
and affixed in place to prevent movement. Although 45.degree. is
preferred, the angle of incident light on the surface of the capillary 20
can be varied as long as care is taken to reduce stray laser light from
interfering with the fluorescent light as detected by the camera. Although
not shown in FIG. 3, spacers similar to those in FIG. 2 can be used for
reducing stray light. The size of the optical fiber 15 can be selected so
that the fibers 15 and spacers 80 may be conveniently held in place.
The laser/capillary interface of individually coupling optical fibers to
capillaries 20 represented by FIG. 3 is also advantageous because
alignment of the optical fiber 15 with the laser beam 47 can be made
permanent. Changing the separation capillary is then simplified. With
fibers or capillaries of larger than 50 .mu.m, alignment does not even
require the use of a microscope. Because there is no moving part, once the
alignment is made, no further alignment is needed. Single-mode optical
fibers with diameters down to 5 .mu.m are presently available. Therefore
capillaries with inside diameters of slightly larger diameters may be
utilized in electrophoresis.
The emitted fluorescent light that passes through the capillary wall 104 is
detected by a charge transfer device imaging system 50 through coupling
with a microscope 60. Any appropriate microscope or camera lens system may
be used so long as it adequately transmits the image of the separation
zone of the capillary array to the imaging camera. A Bausch and Lomb
Stereo Zoom 7 binocular microscope with camera extension is suitable.
The present invention is capable of multiplexing more than 1000, e.g.,
1024, capillaries in, for example, a DNA sequencing run. This number is
based on the number of column elements already available in modern
solid-state imaging devices. The principle is the same as an embodiment of
an optical system device based on 10 capillaries. Fiber bundles with up to
1000 fibers are readily available at a low cost.
For an embodiment of 1024 capillaries, an argon ion laser of 1-5 Watt power
or any appropriate laser of suitable power and wavelength can be used to
illuminate one end 42 of the fiber bundle, conveniently distributing about
0.4-2 mW of light to each fiber. The other end 70 of the fiber bundle 10
can be fanned out into a flat sheet on a guide (not shown), which is a set
of parallel grooves to fix the location of each fiber 15 to maintain a
constant spacing. Similarly, 1024 separation capillaries can also form a
flat sheet on a suitable guide. Insertion of the fibers 15 (smaller than
50 .mu.m) into the capillaries 20 (about 75 .mu.m i.d.) as in the
preferred embodiment, or positioning the optical fibers 15 at a 42.degree.
angle to the capillaries 20 as in another embodiment, can be readily
accomplished. The imaging optics 50 can be | | |
