Title: Separation device substrate including non-fluorescent quencher dye
Abstract: A device is provided for the analysis of samples. The device includes a plurality of sample-containment features and a non-fluorescent quenching material for minimizing cross-talk between optical signals emitted from or directed toward the sample-containment features.
Patent Number: 7,019,831 Issued on 03/28/2006 to Grossman, et al.
| Inventors:
|
Grossman; Paul D. (Hillsborough, CA);
Frazier; Jeffery D. (Portola Valley, CA);
Harding; Ian (San Mateo, CA)
|
| Assignee:
|
Applera Corporation (Foster City, CA)
|
| Appl. No.:
|
623913 |
| Filed:
|
July 21, 2003 |
| Current U.S. Class: |
356/318; 356/344; 356/246 |
| Current Intern'l Class: |
G01J 3/30 (20060101); G01N 21/01 (20060101); G01N 1/10 (20060101) |
| Field of Search: |
356/318,344
204/450,601,603
435/288.2,288.4,288.7
|
References Cited [Referenced By]
U.S. Patent Documents
| 4832815 | May., 1989 | Kambara et al.
| |
| 5114551 | May., 1992 | Hjerten et al.
| |
| 5192412 | Mar., 1993 | Kambara et al.
| |
| 5268080 | Dec., 1993 | Kambara et al.
| |
| 5277780 | Jan., 1994 | Kambara.
| |
| 5314602 | May., 1994 | Kambara et al.
| |
| 5439578 | Aug., 1995 | Dovichi et al.
| |
| 5498324 | Mar., 1996 | Yeung et al.
| |
| 5529679 | Jun., 1996 | Takahashi et al.
| |
| 5552028 | Sep., 1996 | Madabhushi et al.
| |
| 5582705 | Dec., 1996 | Yeung et al.
| |
| 5695626 | Dec., 1997 | Yeung et al.
| |
| 5741411 | Apr., 1998 | Yeung et al.
| |
| 5790727 | Aug., 1998 | Dhadwal et al.
| |
| 5833827 | Nov., 1998 | Anazawa et al.
| |
| 5867266 | Feb., 1999 | Craighead.
| |
| 5910287 | Jun., 1999 | Cassin et al.
| |
| 6017434 | Jan., 2000 | Simpson et al.
| |
| 6027695 | Feb., 2000 | Oldenburg et al.
| |
| 6159353 | Dec., 2000 | West et al.
| |
| 6171780 | Jan., 2001 | Pham et al.
| |
| 6236945 | May., 2001 | Simpson et al.
| |
| 6246046 | Jun., 2001 | Landers et al.
| |
| 6413782 | Jul., 2002 | Parce et al.
| |
| 6906797 | Jun., 2005 | Kao et al.
| |
| 2002/0003091 | Jan., 2002 | Kojima et al.
| |
| 2002/0176804 | Nov., 2002 | Strand et al.
| |
| 2003/0190608 | Oct., 2003 | Blackburn.
| |
| 2004/0026252 | Feb., 2004 | Li.
| |
| Foreign Patent Documents |
| 0 840 115 | May., 1998 | EP.
| |
Other References
Huang et al., Acousto-Optical Deflection-Based Laser Beam Scanning for Fluorescence
Detection on Mulitchannel Electrophoretic Microships, Anal. Chem., vol. 71,
No. 23, pp. 5309-5314 (1999).
Wang et al., Microfabricated Electrophoresis Chips for Simultaneous Bioassays
of Glucose, Uric Acid, Ascorbic Acid, and Acetaminophen, Anal. Chem., vol.
72, No. 11, pp. 2514-2518 (2000).
Cheng et al., Development of a Multichannel Microfluidic Analysis System Employing
Affinity Capillary Electrohoresis for Immunoassay, Anal. Chem., vol. 73, No.
7, pp. 1472-1479 (2001).
Tian et al. Capillary and Microchip Electrophoresis for Rapid Detection of
Known Mutations by Combining Alle-Specific DNA Amplification with Heteroduplex
Analysis, Clinical Chem., vol. 47, No. 2, pp. 173-185 (2001).
|
Primary Examiner: Toatley, Jr.; Gregory J.
Assistant Examiner: Chisdes; Sarah J.
Attorney, Agent or Firm: Kilyk & Bowersox, P.L.L.C.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part application of U.S. patent application
Ser. No. 10/455,986, filed Jun. 7, 2003, which is a divisional of U.S. patent application
Ser. No. 09/938,767, filed Aug. 24, 2001 now U.S. Pat. No. 6,627,433, both of which
are incorporated herein in their entireties by reference.
Claims
What is claimed is:
1. A device for the analysis of one or more samples, comprising:
a substrate;
a plurality of adjacently arranged channels formed in the substrate, with each
channel having an inlet end and an outlet end, the channels being disposed spaced
apart from one another, with each adjacent pair of channels being separated by
a respective portion of the substrate that includes at least a region that is transparent
and at least a region that comprises a non-fluorescent quencher; and
an excitation-beam source adapted to direct an excitation beam of light along
a beam path that intersects each of the channels at a region between the inlet
and outlet ends and further intersects the transparent region of the substrate
separating adjacent pairs of channels.
2. The device of claim 1, further comprising a cover member positioned adjacent
the substrate, over the channels.
3. The device of claim 2, further comprising an emission detection system optically
coupled to a region within each channel along the beam path.
4. The device of claim 1, wherein the substrate is a plate, slide, wafer, or
chip comprised at least in part of an optically clear material.
5. The device of claim 1, wherein the substrate is a monolithic structure.
6. The device of claim 1, wherein the substrate is a multi-laminate structure.
7. The device of claim 1, wherein each channel includes opposed sidewall regions
including portions that are substantially parallel to one another.
8. The device of claim 7, wherein the transparent region comprises, at least
in-part, the parallel portions, and wherein the beam path extends through the parallel portions.
9. The device of claim 1, further comprising a coating on one or more portion
of the plurality of separation channels, wherein the coating includes the non-fluorescent quencher.
10. The device of claim 1, wherein the non-fluorescent quencher is incorporated
into the substrate.
11. The device of claim 1, wherein at least a portion of one or more of the plurality
of separation units comprises a reporter dye, wherein the reporter dye includes
FAM, and wherein the non-fluorescent quencher includes at least one of Methyl Orange,
Disperse Red 13, Basic Violet 14, Basic Red 9, and non-fluorescent dyes having
an absorbance with a λ-max at about 520 nm.
12. The device of claim 1, wherein at least a portion of one or more of the plurality
of separation units comprises a reporter dye, wherein the reporter dye includes
ROX and wherein the non-fluorescent quencher includes at least one of Malachie
Green, Ethyl Violet, Fast Green FCF, Brilliant Green, Crystal Violet, and non-fluorescent
dyes having an absorbance with a λ-max at about 605 nm.
13. The device of claim 1, wherein at least a portion of one or more of the plurality
of separation units comprises a plurality of reporter dyes and a plurality of non-fluorescent
quenchers, wherein the plurality of non-fluorescent quenchers includes at least
two non-fluorescent quenchers with different λ-max absorbance values.
14. A device for the analysis of one or more samples, comprising:
a substrate including one or more regions, the one or more regions including
a material that comprises a non-fluorescent quencher;
a plurality of adjacently arranged channels formed in the substrate, wherein
each channel includes an inlet end and an outlet end and the channels are disposed
in spaced relation relative to one another, with each adjacent pair of channels
being separated by a respective portion of the substrate;
a transverse channel in the substrate transverse to and passing through each
of the plurality of adjacently arranged channels in the substrate; and
an excitation-beam source adapted to direct an excitation beam of light along
a beam path that intersects each of the channels at a region between the inlet
and outlet ends, wherein the beam path is along the transverse channel.
15. The device of claim 14, wherein at least a portion of the substrate in each
of the separation channels includes a non-fluorescent quencher dye.
16. The device of claim 14, wherein the separation channels are non-intersecting.
17. The device of claim 14, wherein the substrate is a plate, slide, wafer, or
chip; and wherein the separation channels are microfabricated therein.
18. A device for the analysis of one or more samples, comprising:
a plurality of sample-containment units, each sample-containment unit including
an open end and a closed end and an interior portion between the ends;
an excitation source adapted to direct an excitation beam of light along a beam
path that intersects the interior portion of each of the sample-containment units
at a region between the open and closed ends; and
an emission detection system optically coupled to the interior portion of the
separation units, in the vicinity of the beam path,
wherein at least a portion of one or more of the plurality of sample-containment
units comprises a non-fluorescent quencher.
19. The device of claim 18, further comprising an optical coating or element
on one or more regions of the sample-containment units.
20. The device of claim 18, further comprising a cover member positioned over
the sample-containment units.
21. The device of claim 20, further comprising an optical coating or element
on the cover.
22. The device of claim 18, wherein each sample-containment unit is continuous
from its open end to its closed end.
23. The device of claim 18, wherein the units comprise separate respective sample vials.
24. The device of claim 18, wherein the excitation-beam source comprises at least
one laser.
25. The device of claim 24, wherein the device further comprises a substrate,
the substrate comprises first and second lateral sides, the beam path extends between
the first and second lateral sides, and at least one laser is configured to emit
a beam that enters the device along the beam path from each of the first and second
lateral sides.
26. The device of claim 25, wherein the substrate includes a transparent region
between the first and second lateral sides, and the beam path extends along the
transparent region.
27. The device of claim 18, wherein each sample-containment unit includes opposed
sidewall regions including portions that are substantially parallel to one another.
28. The device of claim 27, wherein the beam path extends through the parallel portions.
29. The device of claim 28, wherein the parallel portions, through which the
beam path extends, are transparent to at least a selected wavelength range of light.
30. The device of claim 18, wherein the device further comprises a substrate,
and the substrate includes at least one transverse channel transverse to and passing
through at least some of the plurality of units, and wherein the beam path extends
through the transverse channel.
31. The device of claim 18, wherein the non-fluorescent quencher is coated on
one or more portion of one or more of the plurality of separation units.
32. The device of claim 18, wherein the non-fluorescent quencher is incorporated
into the sample-containment units.
33. The device of claim 18, further comprising a substrate, wherein the sample-containment
units are an array of sample-containment units that are adapted to be placed into
the substrate.
34. A method of forming a device, comprising:
providing a substrate material and a non-fluorescent quencher;
forming a substrate from the substrate material and the non-fluorescent quencher,
wherein the substrate includes a plurality of adjacently arranged channels, each
channel having an inlet and an outlet end, the channels being disposed in spaced
relation to each other.
35. The method of claim 34, further comprising coating at least a portion of
the substrate with a coating material, wherein the coating material comprises the
at least one non-fluorescent quencher.
36. The method of claim 34, further comprising:
mixing the substrate material with the at least one non-fluorescent quencher.
37. The method of claim 34, further comprising:
retaining one or more non-fluorescent quenchers in alternating channels of the
plurality of adjacently ranged channels.
38. A method of forming a device, comprising:
providing a substrate material and a non-fluorescent quencher;
forming a substrate from the substrate material and the non-fluorescent quencher,
wherein the substrate includes a plurality of adjacently arranged separation units,
each separation unit having an inlet and an outlet end, the separation units being
disposed in spaced relation to each other.
39. The meted of claim 38, further comprising coating at least a portion of the
substrate with a coating material, wherein the coating material comprises the at
least one non-fluorescent quencher.
40. The method of claim 38, further comprising:
mixing the substrate material with the at least one non-fluorescent quencher.
41. The method of claim 38, further comprising:
retaining one or more non-fluorescent quenchers in alternating separation units.
Description
FIELD
The present invention relates to a multi-channel analyte-separation device.
REFERENCES
Backhouse et al., "DNA sequencing in a monolithic microchannel device,"
Electrophoresis 2000, 21, 150-156.
Dolnik et al., "Capillary electrophoresis on microchip," Electrophoresis 2000,
21, 41-54.
Grossman and Colburn, "Capillary Electrophoresis Theory and Practice," Chapter
1, Academic Press (1992).
Kambara et al., U.S. Pat. No. 5,192,142 (1993).
Madabhushi et al., U.S. Pat. No. 5,552,028 (1996).
Sambrook et al., eds., "Molecular Cloning: A Laboratory Manual," Second
Edition, Chapter 5, Cold Spring Harbor Laboratory Press (1989).
Woolley et al., "Ultra-high-speed DNA fragment separations using microfabricated
capillary array electrophoresis chips," Proc. Natl. Acad. Sci., vol. 91, pp. 11348-11352,
November 1994, Biophysics.
Yeung et al., U.S. Pat. Nos. 5,741,411 and 5,582,705.
BACKGROUND
Devices for carrying out separations of analytes, such as biomolecules, for
example, proteins, DNA, RNA, and the like, have gained widespread use in recent years.
In electrophoretic separations, it is often desirable to illuminate a plurality
of migrating analytes, tagged with excitable reporters (for example, fluorescent
dyes), to stimulate detectable emission indicative of the nature (for example,
identity or composition) of the tagged analytes.
SUMMARY
According to various embodiments, an analyte detection device is provided
that includes a substrate defining an array of channels, wells, vials, or the like,
wherein the substrate includes a light-absorbing or light-quenching material. The
light-absorbing and/or light-quenching material can be in the form of a quencher
dye or pigment composited or mixed with the substrate material or provided as a
separate layer coated on top of a base substrate material. The light-absorbing
and/or light-quenching material is referred to herein as a non-fluorescent quencher.
According to various embodiments, adjacent channels or wells of the device are
separated by respective wall structures. Each wall structure can include at least
a portion that is substantially transparent. The transparent portions can be disposed
along a beam path or line that intersects or crosses (for example, is substantially
co-planar and substantially normal to) the longitudinal axes of the channels, for
example, adjacent or near one end of each channel.
According to various embodiments, a system can be provided that includes
an excitation-beam source, for example, a laser, that can be adapted to direct
an excitation beam along the beam path, such that the beam can simultaneously pass
through each of the transparent portions and each of the channels, wells, vials,
or the like. Plural samples in various channels, wells, vials, or the like can
thus be simultaneously irradiated and detected.
According to various embodiments, methods are provided for sequencing biomolecules
(for example, DNA, RNA, PNA, or the like) or other analysis methods in which each
of a plurality of different nucleic acid sequence fragment types is labeled with
a spectrally distinctive emitting or fluorescing dye. According to various embodiments,
a side-entry laser arrangement is provided at a detection zone of a multi-channel
electrophoresis device, and the arrangement excites dyes, while in respective channels,
causing them to excite and emit detectable emission beams.
According to various embodiments, light emitted from samples in the respective
channels can pass through a laser light filter, through a collection lens, through
a transmission dispersion element that spectrally separates the light, and/or through
a focusing lens. Focused light can be directed to be incident on a detector array
(for example, a CCD) capable of detecting the simultaneously spatially focused
and spectrally divergent light from the detection regions of all the channels.
Electronic signals from the detector array can provide information about the character
or sequence of the DNA sample. The laser light can impinge on a sample in each
channel by passing through a substantially transparent region of each channel,
or by passing through a groove transverse to and passing through each channel.
Stray light, and extraneous light can be absorbed by the light-quenching or light-absorbing
material in or on the substrate so as not to interfere with excitation or detection.
BRIEF DESCRIPTION OF THE DRAWINGS
The structure and manner of operation according to the present teachings can
further be understood by reference to the following description taken in conjunction
with the accompanying drawings, in which identical reference numerals identify
identical or similar elements, and in which:
FIG. 1 is a perspective view from above of an electrophoresis system, showing
a multi-channel analyte-separation device including a plurality of separation channels,
a detection zone, an excitation beam source, an optical detection system, and a
programmed computer control/analysis system, according to various embodiments;
FIGS. 2A, 2B, and 2C are partial, cross-sectional views of multi-channel
analyte separation devices, according to various embodiments;
FIGS. 3A, 3B, and 3C are cross-sectional views of substrates
with channels formed therein having various geometries, according to various embodiments; and
FIG. 4 is a perspective view from above of an electrophoresis system, including
a multi-channel analyte-separation device including a plurality of separation channels,
an excitation beam source, and optics directing an excitation beam for entry into
the channel device from each lateral side thereof, according to various embodiments.
Other various embodiments of the present teachings will be apparent to those
skilled in the art from consideration of the specification and practice of the
devices, systems, and methods described herein, and the detailed description that
follows. It is intended that the specification and examples be considered as exemplary only.
DETAILED DESCRIPTION
Unless stated otherwise, the following terms and phrases as used herein are
intended to have the following meanings:
The term "channel" as used herein refers to an elongate, narrow passage or other
structure, for example, a groove, formed in a substrate and capable of supporting
a volume of separation medium and/or buffer solution, for example, such as is used
in carrying out electrophoresis. The geometry of a channel can vary widely. For
example, a channel can have a circular, oval, semi-circular, semi-oval, triangular,
rectangular, square, or other cross-section, or a combination thereof. Channels
can be fabricated by a wide range of technologies, including microfabrication techniques.
As used herein, the term "channel' is not intended to encompass a capillary tube.
The terms "capillary" and "capillary tube" as used herein refer to an elongated
tubular or cylindrical structure defining an inner lumen. For example, a capillary
can be an elongated capillary or micro-capillary tube made, for example, from fused
silica, quartz, silicate-based glass, such as borosilicate glass, phosphate glass,
alumina-containing glass, and the like, or other silica-like material(s). As used
herein, "capillary" does not encompass a channel in a substrate such as a plate,
slide, chip, wafer, or the like.
The term "separation unit" as used herein can refer to any device adapted to
separate a sample including nucleic acid or amino acid polymers. The separate unit
can be, for example, a channel or capillary.
The term "channel device" refers to a substrate, such as a plate, slide, chip,
wafer, or similar structure, including one or more channels, for example, grooves.
A channel device can be adapted, at least in part, for carrying out electrophoresis.
Channel devices can take the form, for example, of microfabricated devices for
example, a plate, a slide, a chip, a wafer, or other substrate that is grooved,
etched, or fluted.
The term "well" as used herein refers to a tubular, cylindrical, or rounded structure
having an open end and a closed end. One or both ends can be tapered, for example.
The well can be sealed by, for example, placing a sealing film over the open end
of the well. A plurality of wells can be sealed using a single film. For example,
a plurality of wells can be fixed to a plate. The plate can be adapted to be placed
in the detector device or can be fixed to the detector device. The plate can include,
for example, 96 wells, 284 wells, or the like.
The term "vial" as used herein refers to a tubular or cylindrical structure having
an open end and a closed end. The vial can be sealed with, for example, a cap secured
to the open end of the vial. A plurality of vials can be placed in the detection
device. A plurality of vials can be placed in a holder or template that is adapted
to be placed in the detection device.
As used herein, the terms "sample zone" and "analyte zone" refer to a collection
of molecules comprising a subset of sample or analyte components having similar
electrophoretic migration velocities such that the molecules of a sample zone or
analyte zone migrate as a defined zone. A zone can be made up of molecules having
identical electrophoretic migration velocities. Sample zones and analyte zones
are often referred to as "bands."
As used herein, the terms "separation medium" and "separation matrix" refer to
a medium in which an electrophoretic separation of sample components can take place.
Separation media typically comprise several components, at least one of which is
a charge-carrying component, or electrolyte. The charge-carrying component can
be part of a buffer system for maintaining the separation medium at a defined pH.
Media for separating polynucleotides, proteins, or other biomolecules, having different
sizes but identical charge-frictional drag ratios in free solution, can further
include a sieving component. The sieving component can be composed of a cross-linked
polymer gel, for example, a cross-linked polyacrylamide, a cross-linked agarose
(Sambrook), or a polymer solution, for example, a solution of polyacrylamide, hydroxyethyl
cellulose, and the like (Grossman; Madabhushi).
By way of example, a capillary array will be described. However, the present
teachings
should not be so limiting. The teachings contained herein can be used with, for
example, devices found in FIGS. 2-24 of U.S. Pat. No. 6,159,368, which is incorporated
herein by reference in its entirety.
According to various embodiments, channel devices are provided that are
useful, for example, in electrophoretic separations of bio-molecules. According
to various embodiments, the channel devices can employ side-entry excitation geometry.
Channel devices herein are to be contrasted with capillary arrangements that employ
multiple capillaries, that is, elongated tubular structures. The channel devices
herein can be comprised of a substrate, such as a plate, slide, chip, wafer, or
similar structure, including one or more channels (for example, grooves) and a
light-absorbing or light-quenching material disposed in or on the substrate. According
to various embodiments, channel devices can take the form, for example, of microfabricated
devices, for example, a plate, a slide, a chip, a wafer, or other substrate, that
is grooved, etched, or fluted. It has previously been suggested by others that
channel-device technology was not well developed enough to employ side-entry illumination
(See Yeung et al., U.S. Pat. No. 5,741,411, Col. 8, lines 14-24, and U.S. Pat.
No. 5,582,705, Col. 8, lines 9-19).
According to various embodiments, a channel device, not a capillary tube
device, is provided. The channel device can include a substrate with a plurality
of channels formed in the substrate, and a light-absorbing or light-quenching material
dispersed in or on the substrate. The light-absorbing or light-quenching material
can be dispersed, mixed, or dissolved in the substrate material, for example, to
be homogeneously or uniformly disposed throughout the substrate material. According
to various embodiments, the light-absorbing or light-quenching material can be
in the form of a coating or layer disposed on one or more surfaces of the substrate.
The coating or layer can be formed on the substrate or pre-formed and then applied
to the substrate. Each channel can include an inlet end and an outlet end. The
channels can be disposed in spaced relation relative to one another, with each
adjacent pair of channels being separated by a respective portion of the substrate
that includes at least one region that is substantially transparent. An excitation-beam
source can be adapted to direct a beam of coherent light along a beam path that
intersects each of the channels at a region that is substantially transparent between
the inlet and outlet ends.
According to various embodiments, each channel can include opposed sidewalls
with portions that are substantially parallel to one another. The parallel portions
can include at least a portion that is at least partially transparent, for example,
transparent. The beam path can extend through the partially transparent, parallel
portions of the sidewalls. Such construction can avoid or reduce loss of light
intensity as the beam travels through the device from channel to channel. The side
walls of the channels can be coated or layered with a coating or layer of light-absorbing
or light-quenching material, except at the transparent portion.
According to various embodiments, a transverse channel can be formed in
the substrate such that the transverse channel passes through each of the plurality
of channels in the substrate. The beam path can extend through the transverse channel,
thereby passing through at least a portion of each of the plurality of channels.
Such construction can avoid or reduce loss of light intensity as the beam passes
through the device from channel to channel.
According to various embodiments, separation channels can be formed on
a glass or plastic substrate, such as a plate, slide, wafer, chip, or the like,
by microfabrication techniques known in the art, for example, photolitho and/or
wet-chemical etching procedures, laser ablation, electroforming, microcontact printing,
microstamping, micromolding, microcasting, micromachining, engraving, embossing
techniques, and/or casting in a polymer, and the like. For example, Backhouse et
al., Dolnik et al., and Woolley et al., each of which is incorporated herein in
its entirety by reference, each discusses fabrication techniques that the skilled
artisan can employ in making the devices described herein.
According to various embodiments, the separation channels can be formed
in a generally planar substrate comprised at least in part, for example, of an
electrically insulating material, for example, fused silica, quartz, silicate-based
glass, such as borosilicate glass, phosphate glass, alumina-containing glass, and
the like, or other silica-like material(s) The substrate can be a plastic or polymeric
material, for example, a polyolefin, a polycarbonate, a polyethylene terephthalate,
an acrylic, a polyacrylate, a siloxane, or a comonomer thereof. The substrate can
be formed with or without channels by any standard technique, including molding,
casting, masking, chemical vapor deposition, etching, lithography, soft lithography,
or other forming or depositing techniques. Methods of forming a substrate as taught
in the art can be used, such as, for example, the methods taught in U.S. Pat. No.
6,017,434, issued Jan. 25, 2000, which is incorporated herein in its entirety by reference.
The channel devices described herein can be well suited, for example, to fluorescence
detection of a fluorescent target species in a sample. According to various embodiments,
channels of a channel device can be arranged in a coplanar channel array. The channel
array can include at least about 4 (for example, 8, 12, 16, 24, 48, 96, or more)
coplanar, adjacently-arranged channels. Each channel can have one or more sidewall
that, in combination with a base, forms the channel. Additionally, each channel
can have a ceiling, for example, a glass plate, a polymeric plate, or a polymer
film. The ceiling can include a light-absorbing or light-quenching material disposed
therein or thereon, and the ceiling can be a part of the substrate or a separate
component. The ceiling can include one or more transparent regions or areas. One
or more regions of each sidewall of each channel can include one or more transparent
portions. A transparent portion is transparent to light having a wavelength about
equal to a wavelength of a beam of coherent light used to irradiate a target species
in a channel. A "transparent portion" or "transparent medium" is one that transmits
light with little or no attendant light scattering or absorption. For example,
a transparent portion can be comprised of an optically clear glass or plastic.
According to various embodiments, the transparent portion can be transparent to
light having a wavelength of about 200-1500 nm; for example, about 250-800 nm.
Together, the transparent portions of the sidewalls can define a transparent
substantially linear optical path extending through the channel array, for example,
from one channel to the next all the way through the channel array. The transparent
path can comprise a plane extending through all the channels, for example, where
the channels are fabricated entirely out of transparent material.
The transparent portions of the sidewalls can exhibit little or no fluorescence
when exposed to a beam of coherent light, so as to reduce or eliminate background
fluorescence from the detected fluorescence. For example, the transparent portions
can be selected and designed to exhibit substantially no fluorescence when exposed
to light having a wavelength of from about 200 to about 1500 nm, for example, from
about 250 to about 800 nm. The phrase "substantially no fluorescence" means that
the level of fluorescence emitted, if any, by a transparent portion is less than
an observed background fluorescence.
According to various embodiments, a target species can be detected in a
respective channel through a transparent portion provided in an upper portion of
the channel sidewall or in a portion of a ceiling of the channel. Such an additional
transparent portion can be selected and designed to exhibit substantially no fluorescence
when exposed to light having a wavelength about equal to the wavelength of light
emitted by a fluorescing target species. The entire channel device can be constructed
from a transparent, non-fluorescing material, for example, fused silica. Transparent
windows can alternatively be formed at or along one or more selected regions of
one or more sidewall, ceiling, bottom, or combination thereof, of one or more channels.
Instead of, or in addition to, utilizing such transparent portions, one or
more sidewalls can include a translucent portion defining a translucent linear
path extending through the array perpendicular to the channels. A translucent medium
can produce some light scattering when transmitting light. Transparency can be
preferred over translucency because of greater light throughput and reduced detection
signal-to-noise ratio (S/N).
As indicated above, side-entry irradiation of target species in multiple channels
can be effected through a transparent portion of a sidewall of each channel in
a multichannel array. According to various embodiments, light can pass through
the transparent portions in the array in a sequential manner. A coherent light
source can be positioned to direct a beam of coherent light along the transparent
path. A coherent light source can produce light waves traveling together in phase.
The light can have, for example, a wavelength of from about 200 nm to about 1,500
nm. The coherent light source can be a laser. For example, an argon ion laser operating
simultaneously at one or more visible lines can be used for excitation, although
other light sources and wavelengths can be used. Exemplary excitation wavelengths
include 488 nm and 514 nm. A pure output laser, for example, a laser emitting light
of a single wavelength, can be a useful light source. Alternatively, the wavelength
of the laser can be chosen or manipulated by use of an interference filter, a glass
prism, or another filtering device as known to those skilled in the art of optics.
According to various embodiments, other devices can be used in addition
to or instead of a substrate including separation channels. Devices can include,
for example, a capillary array and sample retaining units. The sample retaining
units can be a plurality of wells or vials. The sample retaining units can include
a plurality of wells or vials. The sample retaining units can be affixed to a substrate
or can be molded such that the substrate and the sample retaining units are a monolithic
structure. For example, the substrate and the sample retaining units can be similar
in form and shape to a standard 96-well reaction plate, available from Applied
Biosystems, Foster City, Calif. The sample retaining units can be a plurality of
individual vials that are placed in a substrate. The sample retaining units can
be an array of wells, such as a 96-well reaction plate, for example, that is placed
in a substrate.
According to various embodiments, a solid state laser can be used as a
light source. Lasers produce monochromatic, coherent, directional light, providing
a narrow wavelength of excitation energy. Solid state lasers use a lasing material
which is distributed in a solid matrix, as opposed to using a gas, dye, or semiconductor,
lasing source material. Examples of solid state lasing material include Ruby (694
nm), Nd:Yag (1064 nm), Nd:YVO
4 (1064 nm or 1340 nm, or doubled to emit
at 532 nm or 670 nm), Alexandrite (655-815 nm), and Ti:Sapphire (840-1100 nm).
Other solid state lasers known to those skilled in the art, including laser diodes,
can also be used. The appropriate lasing material can be selected based on the
desired wavelength. The laser can be selected to closely match the excitation wavelength
of a fluorescent material in a sample in one or more channels. The operating temperature
of the system also can be considered in selecting a laser because the emitted wavelength
of a laser can be affected by the temperature, as known to those of ordinary skill
in the art. The light source for the laser can be any source as known to those
skilled in the art, for example, a flash lamp. Useful information about various
solid state lasers can be obtained, for example, from www.repairfaq.org/sam/lasersl.htm.
Examples of solid state lasers used in various systems for identification of biological
materials include U.S. Pat. No. 5,863,502 to Southgate et al., and U.S. Pat. No.
6,529,275 B2 to Amirkhanian et al., both of which are incorporated herein in their
entireties by reference.
The beam of coherent light can be focused and collimated through a collimating
focusing lens interposed between the coherent light source and the channel array.
For example, the excitation beam can be collimated to have a diameter of less than
about 300 micrometers, for example, less than about 75 micrometers or less than
about 50 micrometers, while traversing the channels in the array. According to
various embodiments including an array comprising about 96 channels, the array
width can be less than about 1.5 cm, and a lens with a focal length of from about
5 cm to about 30 cm, for example, about 10 cm, can focus and collimate the beam
of coherent light such that the beam diameter remains less than about 75 micrometers
while in the channels.
According to various embodiments, the focused line of the laser can be
altered with a beam expander in order to irradiate a large number of channels.
For example, the laser beam can be expanded perpendicular to the channel array.
Such lengthening or "fanning out" of the laser line can facilitate positioning
of the beam so that all channels are adequately irradiated. The beam can optionally
be altered or redirected by use of a mirror, a filter, a lens, another optical
element, or a combination thereof, prior to contacting the array. For example,
mirrors can be used to provide a convenient means for adjusting the direction of
the laser beam to be coplanar with the channel array and perpendicular to the channels.
The use of mirrors, filters, lenses, or any combination thereof, is optional.
The transparent path can be optically coupled to a location external to the channel
array. The location is to be broadly understood as any point, line, or plane, external
to the array, including a single pixel, linear array of pixels, or planar array
(two-dimensional array) of pixels. For example, the location external to the channel
array can be a planar surface parallel to or angled with respect to the channel
array. The location external to the channel array can include an optical detector
capable of detecting fluorescence emissions from a target species in a sample in
a channel of the channel array. The optical detector can be a two-dimensional image
array detector. For example, the optical detector can be a charge-coupled device
(CCD) or a charge-injection device (CID).
Referring now to the drawings, FIG. 1 is a perspective view of an embodiment
of an electrophoresis device 12. Device 12 can include a plurality
of separation channels, such as elongate channels 14, with each channel
having an inlet end 16 and an outlet end 18. A first lead wire 22
connects a power source 20 with a first electrode (not visible in FIG. 1)
disposed in electrical communication with the inlet ends 16 of the separation
channels 14. A second lead wire 24 connects source 20 with
a second electrode (not visible in FIG. 1) disposed in electrical communication
with the outlet ends 18 of the separation channels 14. In operation,
a voltage is applied between the first and second electrodes, and thereby along
the channels 14, such that a sample zone is transported from the inlet ends
16, to the outlet ends 18 of the channels 14, and through
an on-channel detection zone 30 located between the inlet ends 16
and the outlet ends 18.
Device 12 can be comprised of upper plate 26 and lower plate
28, with abutted confronting faces. As shown, lower plate 28 includes
end portions 28
a and 28
c, and lateral side portions
28
b and 28
d. Lower plate 28 is provided with
a plurality of non-intersecting elongate grooves, each of approximately semi-circular
or semi-oval cross-section, positioned at regular intervals, for example, at a
pitch of about 250 μm, and extending along a first face, for example, for
a length of about 5 cm. The grooves can, at least in part, define separation channels
14. A first face of plate 26 is substantially planar, and, when disposed
against the first face of plate 28 as shown in FIG. 1, further defines channels
14. In the illustrated arrangement, the grooves of plate 28 define
lower boundaries (a floor) and sidewalls of each channel 14, and the first
face of plate 26 provides an upper wall or ceiling for each channel 14.
According to various embodiments, both the upper and lower plates can be
provided with complimentary sets of grooves that can be aligned with one another
so that corresponding upper and lower grooves cooperate to define a plurality of
elongate channels.
Instead of providing grooves in a lower plate which are covered by a planar
first face of an upper plate, such as shown in FIG. 1, the device of the invention
can include an upper plate with grooves formed along a first surface, which can
be placed over a planar first surface of a lower plate (that is, essentially, the
reverse of what is shown in FIG. 1). Although the device of the invention is illustrated
as operating with the major planar surfaces of the plates disposed in a substantially
horizontal fashion, the device can be configured to operate with the plates disposed
substantially vertically, or tilted at a desired angle.
While the channels depicted in FIG. 1 are parallel to one another, it should
be appreciated that other configurations are possible. The channels can converge
toward one end of the device such that the distance separating adjacent channels
(i.e., the pitch) becomes smaller along a direction towards the outlet ends. The
central longitudinal axes of the channels can be straight, curved, or a combination
thereof. Each channel can diverge to form two or more pathways having a common
inlet end, a common outlet end, one or more common portion of a pathway, or a combination
thereof. The channel can diverge horizontally, vertically, at an angle, or a combination thereof.
In the embodiment shown in FIG. 1, the flow cross-area, which is the cross-section
taken perpendicular to the direction of sample migration, can be substantially
the same for each channel. The channels shown in FIG. 1 all can be of a uniform
depth as measured from the first face of the upper plate bounding a top region
of the channel to the lowermost point, or floor, of the channel groove. Such uniformity
can be achieved as the ordinary result of common microfabrication methods employed
in constructing the device, such as etching. However, the invention additionally
contemplates channels of varying depths, which can be made, for example, by use
of a two stage etching process with multiple masks, or by other methods known to
those skilled in the art of microfabrication. Each channel can have the same or
a different depth as at least one other channel. The depth of each channel can
vary along the length of the channel.
In practice, a separation medium can be injected by pressure or vacuum aspiration,
or can be otherwise provided, in one or more separation channels to effect electrophoretic
separation of the components of the sample(s). Any suitable injection technique
can be used without limitation, including electrokinetic injection, hydrodynamic
injection, injection by cross tee injector or double tee injector, or other injection
method known to those skilled in the art of injection. According to various embodiments,
the separation medium can be a flowable, non-crosslinked polymer solution.
As shown in FIG. 1, an excitation-beam source 34 can be provided for stimulating
emission from sample zones located in detection zone 30. In an embodiment,
the light source is a laser, for example, an argon ion laser, or a solid-state
laser. Any suitable beam source can be used. As described in more detail below,
according to an embodiment of the present invention, an excitation-beam pathway
or path can extend through detection zone 30. An energy beam 48 generated
by the beam source can pass along the excitation beam pathway. The beam pathway
can be located between the inlet and outlet ends. The beam pathway can extend along
a plane defined by the channels such that the beam pathway is co-planar with the
plane of the channels. The beam pathway can be perpendicular to the direction of
sample migration across the detection chamber. The beam pathway can approach the
detection chamber at an angle with respect to the direction of sample migration.
The beam passing along the beam pathway can be capable of simultaneously exciting
plural sample zones in respective (different) channels.
According to various embodiments and as shown in of FIG. 1, the beam can
enter a lateral side 28
b of lower plate 28, pass through plate
28 including each of channels 14, and exit at an opposite lateral
side 28
d of plate 28. A laser dump or sink can be incorporated
into plate 28, for example, proximate a region of side 28
d,
to terminate the beam after the beam passes through the channels.
As previously mentioned, a first electrode (not visible in FIG. 1) is in electrical
communication with inlet ends 16 of separation channels 14. During
operation of device 12, the first electrode can be maintained at a first
voltage V using power source 20. Electrical communication between the first
electrode and the inlet ends 16 of the separation channels 14 can
be established, for example, by providing an electrically conductive solution in
a reservoir/loading region 35 of device 14 so that both the inlet
ends 16 of the channels 14 and the first electrode are in contact
with the conductive solution.
As shown in FIG. 1, each channel 14 communicates with a second reservoir
37 through a respective outlet end 18. The reservoir can be located
proximate the outlet ends 18.
The second electrode (not visible in FIG. 1) can be in electrical communication
with outlet ends 18 of separation channels 14. During operation of
device 12, the second electrode can be maintained at a second voltage V
also using power supply 20. Electrical communication between the second
electrode and second reservoir 37 can be established by providing an electrically
conductive solution in second reservoir 37, such that the second electrode
and outlet ends 18 are in contact with the conductive solution.
The electrodes used in the device can be formed from any electrically conducting
materials. The electrodes can be made from a chemically inert material, for example,
platinum, gold, stainless steel, or other relatively inert conductive materials.
According to various embodiments, electrodes, for example, platinum electrodes,
can be fabricated on the top or bottom plate by RF sputtering and/or photolithography
before the top plate is bonded to the bottom plate.
The electrically conductive solution used to establish electrical continuity
throughout the system can be any fluid capable of transporting an electrical current.
For example, the conductive solution can be an ionic solution, for example, an
aqueous solution containing a dissolved salt. In various embodiments, the conductive
solution includes a buffer for stabilizing the pH of the solution. According to
various embodiments, the ionic composition of the conductive solution can be the
same in each of the separation channels, each of the electrode reservoirs, the
detection chamber, or any combination thereof.
To facilitate optical detection of sample zones in the detection zone 30,
part or all of a region in upper plate 26 covering detection zone 30
can be formed from a material which efficiently transmits light, such as an optically
clear material, for example, glass, quartz, clear plastic, or an optically clear
polymer film. To facilitate the introduction of an excitation light beam 48
into the detection zone to excite fluorescence of sample zones therein, part or
all of the lower plate 28 along a region between the beam source 34
and the endmost channel 14 closest to the beam source 34 can be formed
from a material which efficiently transmits light, for example, glass, quartz,
clear plastic, or an optically clear polymer. According to various embodiments,
the light-transmitting material does not significantly scatter light, and/or has
little intrinsic fluorescence.
As shown in FIG. 1, a detector 38 is provided for detecting sample zones
passing through the detection zone 30. The detector can be any type of detector
for detecting emission of any type of radiation, for example, radioactivity, fluorescence,
phosphorescence, chemi-luminescence, or a combination thereof. Detector 38
is capable of detecting fluorescence from a plurality of locations independently
and/or simultaneously. Detector 38 can be, for example, a CCD camera, an
array of photomultiplier tubes, a diode array, another detector means as known
to those skilled in the art, or a combination thereof. As illustrated in FIG. 1,
detector 38 can be connected to a computer 42 to store, analyze,
and display data collected by the detector and/or to control the operation of the
detector and other aspects of the device, as desired. For example, computer 42
can be programmed to control power source 20 and/or beam source 34.
In regions of the device where it is not required and/or desired for radiative
emissions to be able to pass through, non-optically clear materials can be used.
For example, non-optically clear regions can include polymeric materials, for example,
Teflon or silicone.
The detection zone, as previously mentioned, can permit light to pass from each
channel to the next channel, and from each channel to the detector. As previously
mentioned, the detection zone can include a substantially or partially transparent
region of one or more channels. According to various embodiments, the detection
zone can include a substantially transparent region of each channel sidewall, wherein
the transparent regions form a substantially linear path for passage of the excitation
beam therethrough. Alternatively, the detection zone and/or excitation beam pathway
can include a channel transverse to each of the plurality of channels and passing
therethrough, such that the beam is substantially linear and passes through each
channel without passing through sidewalls between channels.
The device can include one or more additional elements capable of conducting
capillary electrophoresis. For example, the device can include a temperature control
device for controlling the temperature of the separation channels. Details of these
and other common features of an operable capillary electrophoresis device can be
found in any number of available publications, including Capillary Electrophoresis
Theory and Practice, Grossman and Colburn, eds., Academic Press (1992), incorporated
herein in its entirety by reference.
Various embodiments can provide for reduced crosstalk between channels while
not inhibiting excitation of the one or more fluorescent dye in the sample, or
detection of the emitted light therefrom. For example, bandpass filters that transmit
light only within a defined spectral band can be used. An excitation filter can
be employed such that only light capable of exciting a reporter of interest strikes
the sample. For example, an excitation filter can be coated on or fixed to one
or more desired regions of the lower plate. An emission filter can be employed
such that the fluorescence from the sample passes to a detector while stray light
from the light source or interfering components in the sample can be blocked. For
example, an excitation filter can be coated on or fixed to one or more desired
regions of the upper plate.
According to various embodiments and as depicted in the sectional views
of FIGS. 2A-C, a lower glass or plastic plate 28 can be provided with spaced-apart
etched channels 14. While each channel 14 is shown having vertical
sidewalls 14
a and a flat bottom or floor region 14
b,
which meet at ninety-degree angles, other channel geometries can be employed, as
shown, for example, in FIG. 3.
The sidewalls 14
a of the channels can have bandpass characteristics
that substantially only permit passage of the excitation (laser) beam 48
through the device. For example, a coating material 52 can be applied to
the channel sidewalls 14
a and, optionally, the floor regions 14
b
(see FIG. 2A); micro-optical elements 54 can be attached on each sidewall
14
a (see FIG. 2B); the whole lower etched plate, including the channels
in their entireties, can be coated with a bandpass coating 52 permitting
only excitation light to pass through (see FIG. 2C); or a combination thereof.
An upper or cover plate 26, overlaid over the channels 14, can be
provided with bandpass characteristics that allow only sample emission (for example,
fluorescence) wavelengths to pass through, and can prevent the passage of excitation
beam wavelengths. For example, a bandpass coating material 56, as shown
in FIG. 2A, can be applied to one or more face regions of the upper plate 26
that face the lower plate 28 and channels 14, at least along regions
along the detection zone. A micro-optical bandpass element 55 as shown in
FIG. 2B can be attached to the face of upper plate 26 confronting lower
plate. Optical elements and coatings useful in connection with the present teachings
are described, for example, in U.S. Pat. Nos., 3,466,120; 6,112,005; 5
