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BACKGROUND
This invention relates to microfluidics, and particularly to microchannel devices in which fluids are manipulated at least in part by application of electrical fields.
Electrophoresis has become an indispensable tool of the biotechnology and other industries, as it is used extensively in a variety of applications, including the separation, identification and preparation of pure samples of nucleic acids,
proteins, carbohydrates, the identification of a particular analyte in a complex mixture, and the like. Of increasing interest in the broader field of electrophoresis is capillary electrophoresis (CE), where particular entities or species are moved
through a medium in an electrophoretic chamber of capillary dimensions under the influence of an applied electric field. Benefits of CE include rapid run times, high separation efficiency, small sample volumes, etc. Although CE was originally carried
out in capillary tubes, of increasing interest is the practice of using microchannels or trenches of capillary dimension on a planar substrate, known as microchannel electrophoresis (MCE). CE and MCE are increasingly finding use in a number of different
applications in both basic research and industrial processes, including analytical, biomedical, pharmaceutical, environmental, molecular, biological, food and clinical applications.
Despite the many advantages of CE and MCE, the potential benefits of these techniques have not yet been fully realized for a variety of reasons. Because of the nature of the electrophoretic chambers employed in CE and MCE, good results are not
generally obtainable with samples having analyte concentrations of less than about 10.sup.-6 M. This lower analyte concentration detection limit has significantly limited the potential applications for CE and MCE. For example, CE and MCE have not found
widespread use in clinical applications, where often an analyte of interest is present in femtomolar to nanomolar concentration in a complex sample, such as blood or urine.
In order to improve the detection limits of CE, different techniques have been developed, including improved sample injection procedures, such as analyte stacking (Beckers & Ackermans, "The Effect of Sample Stacking for High Performance Capillary
Electrophoresis," J. Chromatogr. (1993) 629: 371-378), field amplification (Chien & Burgi, "Field Amplified Sample Injection in High-Performance Capillary Electrophoresis," J. Chromatogr. (1991) 559: 141-152), and transient isotachophoresis (Stegehuis
et al., "Isotachophoresis as an On-Line Concentration Pretreatment Technique in Capillary Electrophoresis," J. Chromatogr. (1991) 538: 393-402), as well as improved sample detection procedures and "off-line" sample preparation procedures.
Another technique that has been developed to improve the detection limit achievable with CE has been to employ an analyte preconcentration device that is positioned directly upstream from the capillary, i.e., in an "on-line" or "single flow path"
relationship. As used herein, the term "on-line" and "single flow path" are used to refer to the relationship where all of the fluid introduced into the analyte preconcentration component, i.e., the enriched fraction and the remaining waste fraction of
the original sample volume, necessarily flows through the main electrophoretic portion of the device, i.e., the capillary tube comprising the separation medium. A review of the various configurations that have been employed is provided in Tomlinson et
al., "Enhancement of Concentration Limits of Detection in CE and CE-MS: A Review of On-Line Sample Extraction, Cleanup, Analyte Preconcentration, and Microreactor Technology," J. Cap. Elec. (1995) 2: 247-266, and the figures provided therein.
Although this latter approach can provide improved results with regard to analyte detection limits, particularly with respect to the concentration limit of detection, it can have a deleterious impact on other aspects of CE, and thereby reduce the
overall achievable performance. For example, analyte peak widths can be broader in on-line or single flow path devices comprising analyte preconcentrators.
Accordingly, there is continued interest in the development of improved CE devices capable of providing good results with samples having low concentrations of analyte, particularly analyte concentrations in the femtomolar to nanomolar range.
MCE devices are disclosed in U.S. Pat. No. 5,126,022; U.S. Pat. No. 5,296,114; U.S. Pat. No. 5,180,480; U.S. Pat. No. 5,132,012; and U.S. Pat. No. 4,908,112. Other references describing MCE devices include Harrison et al.,
"Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical Analysis System on a Chip," Science (1992) 261: 895; Jacobsen et al., "Precolumn Reactions with Electrophoretic Analysis Integrated on a Microchip," Anal. Chem. (1994) 66: 2949;
Effenhauser et al., "High-Speed Separation of Antisense Oligonucleotides on a Micromachined Capillary Electrophoresis Device," Anal. Chem. (1994) 66:2949; and Woolley & Mathies, "Ultra-High-Speed DNA Fragment Separations Using Capillary Array
Electrophoresis Chips," P.N.A.S. USA (1994) 91:11348.
Patents disclosing devices and methods for the preconcentration of analyte in a sample "on-line" prior to CE include U.S. Pat. No. 5,202,010; U.S. Pat. No. 5,246,577 and U.S. Pat. No. 5,340,452. A review of various methods of analyte
preconcentration employed in CE is provided in Tomlinson et al., "Enhancement of Concentration Limits of Detection in CE and CE-MS: A Review of On-Line Sample Extraction, Cleanup, Analyte Preconcentration, and Microreactor Technology," J. Cap. Elec.
(1995) 2: 247-266.
SUMMARY OF THE INVENTION
Integrated electrophoretic microdevices comprising at least an enrichment channel and a main electrophoretic flowpath, as well as methods for their use in electrophoretic applications, are provided. The enrichment channel serves to enrich a
particular fraction of a liquid sample for subsequent movement through the main electrophoretic flowpath. In the subject devices, the enrichment channel and electrophoretic flowpath are positioned such that waste fluid from the enrichment channel does
not flow through the main electrophoretic flowpath, but instead flows through a discharge outlet. The subject devices find use in a variety of electrophoretic applications, where entities are moved through a medium in response to an applied electric
field. The subject devices can be particularly useful in high throughput screening, for genomics and pharmaceutical applications such as gene discovery, drug discovery and development, and clinical development; for point-of-care in vitro diagnostics;
for molecular genetic analysis and nucleic acid diagnostics; for cell separations including cell isolation and capture; and for bioresearch generally.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a diagrammatic view of an enrichment channel for use in a device according to the subject invention.
FIG. 2 provides a diagrammatic view of an alternative embodiment of an enrichment channel also suitable for use in the subject device.
FIG. 3A provides a top diagrammatic view of a device according to the subject invention.
FIG. 3B provides a side view of the device of FIG. 3A.
FIG. 4 provides a diagrammatic top view of another embodiment of the subject invention.
FIG. 5 provides a diagrammatic view of an embodiment of the subject invention in which the enrichment channel comprises a single fluid inlet and outlet.
FIG. 6 provides a diagrammatic view of a device according to the subject invention in which the enrichment channel comprises an electrophoretic gel medium instead of the chromatographic material, as shown in FIGS. 1 and 2.
FIG. 7 provides a diagrammatic top view of disk shaped device according to the subject invention.
FIG. 8 is a flow diagram of a device as in FIGS. 1 or 2.
FIG. 9 is a flow diagram of a device as in FIGS. 3A, 3B.
FIG. 10 is a flow diagram of a device as in FIG. 4.
FIG. 11 is a flow diagram of a device as in FIG. 5.
FIG. 12 is a flow diagram of a device as in FIG. 6.
FIG. 13 is a flow diagram of a device as in FIG. 7.
FIG. 14 is a flow diagram of part of an embodiment of a device according to the invention, showing multiple inlets to the separation channel.
FIG. 15 is a flow diagram of an embodiment of a device according to the invention, showing an alternative configuration for the intersection between the main and secondary electrophoretic flowpaths.
FIG. 16 is a flow diagram of an embodiment of a device according to the invention, showing a plurality of analytical zones arranged in series downstream from the enrichment channel.
FIG. 17 is a flow diagram of an embodiment of a device according to the invention, showing a plurality of analytical zones arranged in parallel downstream from the enrichment channel.
FIG. 18 is a flow diagram of an embodiment of a device according to the invention, showing a plurality of main electrophoretic flowpaths downstream from the enrichment channel.
FIG. 19 is a flow diagram of an embodiment of a device according to the invention, showing a plurality of enrichment channels arranged in parallel.
FIGS. 20 and 21 are flow diagrams of embodiments of a device according to the invention, similar to those shown in FIGS. 15 and 16, respectively, and additionally having a reagent flowpath for carrying a reagent from a reservoir directly to the
main electrophoretic flowpath.
FIG. 22 is a flow diagram of an embodiment of a device according to the invention, similar to that shown in FIG. 17, respectively, and additionally having a plurality of reagent flowpaths for carrying a reagent from a reservoir directly to
downstream branches of the main electrophoretic flowpath.
FIG. 23 is a flow diagram of an embodiment of a device according to the invention, in which the enrichment medium includes coated magnetic beads.
DETAILED DESCRIPTION
Integrated electrophoretic microdevices comprising at least an enrichment channel and a main electrophoretic flowpath are provided. The enrichment channel serves to enrich a particular analyte comprising fraction of a liquid sample. The
enrichment channel and main electrophoretic flowpath are positioned in the device so that waste fluid from the enrichment channel does not flow through the main electrophoretic channel, but instead flows away from the main electrophoretic flowpath
through a discharge outlet. The subject devices may be used in a variety of electrophoretic applications, including clinical assay applications. In further describing the invention, the devices will first be described in general terms followed by a
discussion of representative specific embodiments of the subject devices with reference to the figures.
The subject device is an integrated electrophoretic microdevice. By integrated is meant that all of the components of the device, e.g., the enrichment channel, the main electrophoretic flowpath, etc., are present in a single, compact, readily
handled unit, such as a chip, disk or the like. As the devices are electrophoretic, they are useful in a wide variety of the applications in which entities, such as molecules, particles, cells and the like are moved through a medium under the influence
of an applied electric field. Depending on the nature of the entities, e.g., whether or not they carry an electrical charge, as well as the surface chemistry of the electrophoretic chamber in which the electrophoresis is carried out, the entities may be
moved through the medium under the direct influence of the applied electric field or as a result of bulk fluid flow through the pathway resulting from the application of the electric field, e.g., electroosmotic flow (EOF). The microdevices will comprise
a microchannel as the main electrophoretic flowpath. By microchannel is meant that the electrophoretic chamber of the main electrophoretic flowpath in which the medium is present is a conduit, e.g., trench or channel, having a cross sectional area which
provides for capillary flow through the chamber, where the chamber is present on a planar substrate, as will be described below in greater detail.
Critical to the subject device is an enrichment channel that comprises a sample inlet, a waste fluid outlet, an internal enrichment medium for enriching a particular fraction of a sample, and, optionally, an enriched fraction fluid outlet. The
purpose of the enrichment channel is to process the initial sample to enrich for a particular fraction thereof, where the particular fraction being enriched comprises the analyte or analytes of interest. The enrichment channel thus serves to selectively
retain and separate the target analyte comprising fraction from the remaining components or the waste portion of the initial sample volume. Depending on the particular application in which the device is employed, the enrichment channel can provide for a
number of different functions. The enrichment channel can serve to place the analyte of interest into a smaller volume than the initial sample volume, i.e., it can serve as an analyte concentrator. Furthermore, it can serve to prevent potentially
interfering sample components from entering and flowing through the main electrophoretic flowpath, i.e., it can serve as a sample "clean-up" means. In addition, the enrichment channel may serve as a microreactor for preparative processes on target
analyte present in a fluid sample, such as chemical, immunological, and enzymatic processes, e.g., labeling, protein digestion, DNA digestion or fragmentation, DNA synthesis, and the like.
The enrichment channel may be present in the device in a variety of configurations, depending on the particular enrichment medium housed therein. The internal volume of the channel will usually range from about 1 pl to 1 .mu.l, usually from
about 1 pl to 100 nl, where the length of the channel will generally range from about 1 .mu.m to 5 mm, usually 10 .mu.m to 1 mm, and the cross-sectional dimensions (e.g., width, height) will range from about 1 .mu.m to 200 .mu.m, usually from about 10
.mu.m to 100 .mu.m. The cross-sectional shape of the channel may be circular, ellipsoid, rectangular, trapezoidal, square, or other convenient configuration.
A variety of different enrichment media may be present in the enrichment channel. Representative enrichment medium or means include those means described in the analyte preconcentration devices disclosed in U.S. Pat. No. 5,202,010; U.S. Pat.
No. 5,246,577 and U.S. Pat. No. 5,340,452, as well as Tomlinson et al., supra, the disclosures of which are herein incorporated by reference. Specific enrichment means known in the art which may be adaptable for use in the subject integrated
microchannel electrophoretic devices include: those employed in protein preconcentration devices described in Kasicka & Prusik "Isotachophoretic Electrodesorption of Proteins from an Affinity Adsorbent on a Microscale," J. Chromatogr. (1983) 273:117128;
capillary bundles comprising an affinity adsorbent as described in U.S. Pat. No. 5,202,101 and WO 93/05390; octadodecylsilane coated solid phases as described in Cai & El Rassi, "On-Line Preconcentration of Triazine Herbicides with Tandem Octadecyl
Capillaries-Capillary Zone Electrophoresis," J. Liq. Chromatogr. (1992) 15:1179-1192; solid phases coated with a metal chelating layer as described in Cai & El Rassi, "Selective On-Line Preconcentration of Proteins by Tandem Metal Chelate
Capillaries-Capillary Zone Electrophoresis," J. Liq. Chromatogr. (1993) 16:2007-2024; reversed-phase HPLC solid packing materials as described in U.S. Pat. No. 5,246,577), Protein G coated solid phases as described in Cole & Kennedy, "Selective
Preconcentration for Capillary Zone Electrophoresis Using Protein G Immunoaffinity Capillary Chromatography," Electrophoresis (1995) 16:549-556; meltable agarose gels as described in U.S. Pat. No. 5,423,966; affinity adsorbent materials as described in
Guzman, "Biomedical Applications of On-Line Preconcentration--Capillary Electrophoresis Using an Analyte Concentrator: Investigation of Design Options," J. Liq. Chromatogr. (1995) 18:3751-3568); and solid phase reactor materials as described in U.S.
Pat. No. 5,318,680. The disclosures of each of the above-referenced patents and other publications are hereby incorporated by reference herein.
One class of enrichment media or materials that may find use as enrichment media are chromatographic media or materials, particularly sorptive phase materials. Such materials include: reverse phase materials, e.g., C8 or C18 compound coated
particles; ion-exchange materials; affinity chromatographic materials in which a binding member is covalently bound to an insoluble matrix, where the binding member may group specific, e.g., a lectin, enzyme cofactor, Protein A and the like, or substance
specific, e.g., antibody or binding fragment thereof, antigen for a particular antibody of interest, oligonucleotide and the like, where the insoluble matrix to which the binding member is bound may be particles, such as porous glass, polymeric beads,
magnetic beads, networks of glass strands or filaments, a plurality of narrow rods or capillaries, the wall of the channel and the like. Depending on the nature of the chromatographic material employed as the enrichment means, it may be necessary to
employ a retention means to keep the chromatographic material in the enrichment channel. Conveniently, glass frits or plugs of agarose gel may be employed to cover the fluid outlets or inlets of the chamber, where the frits or plugs allow for fluid flow
but not for particle or other insoluble matrix flow out of the enrichment channel. In embodiments where the enrichment means is a chromatographic material, typically sample will be introduced into, and allowed to flow through, the enrichment channel.
As the sample flows through the enrichment channel, the analyte comprising fraction will be retained in the enrichment channel by the chromatographic material and the remaining waste portion of the sample will flow out of the channel through the waste
outlet.
In embodiments where the enrichment means is a bed of polymeric beads or paramagnetic beads or particles, the beads may be coated with antibodies or other target-specific affinity binding moiety, including: affinity purified monoclonal antibodies
to any of a variety of mammalian cell markers, particularly human cell markers, including markers for T cells, T cell subsets, B cells, monocytes, stem cells, myeloid cells, leukocytes, and HLA Class II positive cells; secondary antibodies to any of a
variety of rodent cell markers, particularly mouse, rat or rabbit immunoglobulins, for isolation of B cells, T cells, and T cell subsets; uncoated or tosylactivated form for custom coating with any given biomolecule; and streptavidin-coated for use with
biotinylated antibodies. Paramagnetic beads or particles may be retained in the enrichment channel by application of a magnetic field.
Alternatively, or in addition to solid phase materials such as coated particles or other insoluble matrices as the enrichment means, one may employ a coated and/or impregnated membrane which provides for selective retention of the analyte
comprising fraction of the sample while allowing the remainder of the sample to flow through the membrane and out of the enrichment means through the waste outlet. A variety of hydrophilic, hydrophobic and ion-exchange membranes have been developed for
use in solid phase extraction which may find use in the subject invention. See, for example, Tomlinson et al., "Novel Modifications and Clinical Applications of Preconcentration-Capillary Electrophoresis-Mass Spectrometry," J. Cap. Elect. (1995) 2:
97-104; and Tomlinson et al., "Improved On-line Membrane Preconcentration-Capillary Electrophoresis (mPC-CE),"J. High Res. Chromatogr. (1995) 18:381-3.
Alternatively or additionally, the enrichment channel or the enrichment medium can include a porous membrane or filter. Suitable materials for capturing genomic DNAs and viral nucleic acids include those marketed by QIAGEN under the name QIAmp,
for analysis of blood, tissues, and viral RNAs; and suitable materials for capturing DNAs from plant cells and tissues include those marketed by QIAGEN under the name DNeasy.
Depending on the configuration of the device, the sample can be caused to flow through the enrichment channel by any of a number of different means, and combinations of means. In some device configurations, it may be sufficient to allow the
sample to flow through the device as a result of gravity forces on the sample; in some configurations, the device may be spun about a selected axis to impose a centrfuigal force in a desired direction. In other embodiments, active pumping means may be
employed to move sample through the enrichment channel and enrichment means housed therein. In other embodiments, magnetic forces may be applied to move the sample or to capture or immobilize a paramagnetic bead-target complex during wash and elution
steps. In yet other embodiments of the subject invention, electrodes may be employed to apply an electric field which causes fluid to move through the enrichment channel. An elution liquid will then be caused to flow through the enrichment medium to
release the enriched sample fraction from the material and carry it to the main electrophoretic flowpath. Generally, an applied electric field will be employed to move the elution liquid through the enrichment channel.
Electrophoretic gel media may also be employed as enrichment means in the subject applications. Gel media providing for a diversity of different sieving capabilities are known. By varying the pore size of the media, employing two or more gel
media of different porosity, and/or providing for a pore size gradient and selecting the appropriate relationship between the enrichment channel and the main electrophoretic flowpath, one can ensure that only the analyte comprising fraction of interest
of the initial sample enters the main electrophoretic flowpath. For example, one could have a device comprising an enrichment channel that intersects the main electrophoretic channel, where the enrichment channel comprises, in the direction of sample
flow, a stacking gel of large porosity and a second gel of fine porosity, where the boundary between the gels occurs in the intersection of the enrichment channel and the main electrophoretic flowpath. In this embodiment, after sample is introduced into
the stacking gel and an electric field applied to the gels in the enrichment channel, the sample components move through the stacking gel and condense into a narrow band at the gel interface in the intersection of the enrichment channel and main
electrophoretic flowpath. A second electric field can then be applied to the main electrophoretic flowpath so that the narrow band of the enriched sample fraction moves into and through the main electrophoretic flowpath. Alternatively, the enrichment
channel could comprise a gel of gradient porosity. In this embodiment, when the band(s) of interest reaches the intersection of the enrichment channel and electrophoretic flowpath, the band(s) of interest can then be moved into and along the main
electrophoretic flowpath.
Enrichment media that can be particularly useful for enrichment and/or purification of nucleic acids include sequence specific capture media as well as generic capture media. Generic capture media include, for example: ion exchange and silica
resins or membranes which nonspecifically bind nucleic acids, and which can be expected to retain substantially all the DNA in a sample; immobilized single-stranded DNA binding protein (SSB Protein), which can be expected to bind substantially all
single-stranded DNA in a sample; poly-dT modified beads, which can be expected to bind substantially all the mRNA in a sample. Sequence specific capture media include beads, membranes or surfaces on which are immobilized any of a variety of capture
molecules such as, for example: oligonucleotide probes, which can be expected to bind nucleic acids having complementary sequences in the sample; streptaviden, which can be expected to bind solution phase biotinylated probes which have hybridized with
complementary sequences in the sample. Suitable beads for immobilization of capture molecules include chemically or physically crosslinked gels and porous or non-porous resins such as polymeric or silica-based resins.
Suitable capture media for proteins include the following. Suitable capture media for proteins include: ion exchange resins, including anion (e.g., DEAE) and cation exchange; hydrophobic interaction compounds (e.g., C4, C8 and C18 compounds);
sulfhydryls; heparins; inherently active surfaces (e.g., plastics, nitrocellulose blotting papers); activated plastic surfaces; aromatic dyes such as Cibacron blue, Remazol orange, and Procion red. For carbohydrate moieties of proteins, lectins,
immobilized hydrophobic octyl and phenylalkane derivatives can be suitable. For enzymes, analogs of a specific enzyme substrate-product transition-state intermediate can be suitable; for kinases, calmodulin can be suitable. Suitable capture media for
receptors include receptor ligand affinity compounds.
As mentioned above, the enrichment channel will comprise at least one inlet and at least one outlet. Of course, where there is a single inlet, the inlet must serve to admit sample to the enrichment channel at an enrichment phase of the process,
and to admit an elution medium during an elution phase of the process. And where there is a single outlet, the outlet must serve to discharge the portion of the sample that has been depleted of the fraction retained by the enrichment media, and to pass
to the main electrophoretic microchannel the enriched fraction during the elution phase. Depending on the particular enrichment means housed in the enrichment channel, as well as the particular device configuration, the enrichment channel may have more
than one fluid inlet, serving as, e.g., sample inlet and elution buffer inlet; or the enrichment channel may have more than one outlet, serving as, e.g., waste outlet and enriched fraction fluid outlet. Where the enrichment channel is in direct fluid
communication with the main electrophoretic channel, i.e., the enrichment channel and main electrophoretic flowpath are joined so that fluid flows from the enrichment channel immediately into the main electrophoretic flowpath, the enrichment channel will
comprise, in addition to the waste outlet, an enriched fraction fluid outlet through which the enriched fraction of the sample flows into the main electrophoretic flowpath. When convenient, e.g., for the introduction of wash and/or elution solvent into
the enrichment channel, one or more additional fluid inlets may be provided to conduct such solvents into the enrichment channel from fluid reservoirs. To control bulk fluid flow through the enrichment channel, e.g., to prevent waste sample from flowing
into the main electrophoretic flowpath, fluid control means, e.g., valves, membranes, etc., may be associated with each of the inlets and outlets. Where desirable for moving fluid and entities through the enrichment channel, e.g., sample, elution
buffer, reagents, reactants, wash or rinse solutions, etc., electrodes may be provided capable of applying an electric field to the material and fluid present in the enrichment channel.
The next component of the subject devices is the main electrophoretic flowpath. The main electrophoretic flowpath may have a variety of configurations, including tube-like, trench-like or other convenient configuration, where the cross-sectional
shape of the flowpath may be circular, ellipsoid, square, rectangular, triangular and the like so that it forms a microchannel on the surface of the planar substrate in which it is present. The microchannel will have cross-sectional area which provides
for capillary fluid flow through the microchannel, where at least one of the cross-sectional dimensions, e.g., width, height, diameter, will be at least about 1 .mu.m, usually at least about 10 .mu.m, but will not exceed about 200 .mu.m, and will usually
not exceed about 100 .mu.m. Depending on the particular nature of the integrated device, the main electrophoretic flowpath may be straight, curved or another convenient configuration on the surface of the planar substrate.
The main electrophoretic flowpath, as well as any additional electrophoretic flowpaths, will have associated with it at least one pair of electrodes for applying an electric field to the medium present in the flowpath. Where a single pair of
electrodes is employed, typically one member of the pair will be present at each end of the pathway. Where convenient, a plurality of electrodes may be associated with the electrophoretic flowpath, as described in U.S. Pat. No. 5,126,022, the
disclosure of which is herein incorporated by reference, where the plurality of electrodes can provide for precise movement of entities along the electrophoretic flowpath. The electrodes employed in the subject device may be any convenient type capable
of applying an appropriate electric field to the medium present in the electrophoretic flowpath with which they are associated.
Critical to the subject invention is that the enrichment channel and the main electrophoretic flowpath are positioned in the device so that substantially only the enriched fraction of the sample flows through the main electrophoretic flowpath.
To this end, the device will further comprise a discharge outlet for discharging a portion of sample other than the enriched fraction, e.g., the waste portion, away from the main electrophoretic flowpath. Thus, where the enrichment channel is in direct
fluid communication with the main electrophoretic flowpath, the waste fluid flowpath through the enrichment channel will be in an intersecting relationship with the main electrophoretic flowpath. In other embodiments of the subject invention where the
enrichment channel and main electrophoretic flowpath are connected by a second electrophoretic flowpath so that they are in indirect fluid communication, the waste flowpath through the enrichment channel does not necessarily have to be in an intersecting
relationship with the main electrophoretic flowpath; the waste flowpath and main electrophoretic flowpath could be parallel to one another.
The subject devices will also comprise a means for transferring the enriched fraction from the enrichment channel to the main electrophoretic flowpath. Depending on the particular device configuration, the enriched fraction transfer means can be
an enriched fraction fluid outlet, a secondary electrophoretic pathway, or other suitable transfer means. By having a second electrophoretic flowpath in addition to the main electrophoretic flowpath, the possibility exists to employ the second
electrophoretic flowpath as a conduit for the enriched sample fraction from the enrichment channel to the main electrophoretic flowpath. In those embodiments where the waste outlet is the sole fluid outlet, the presence of a secondary electrophoretic
flowpath will be essential, such that the enrichment channel and the main electrophoretic flowpath are in indirect fluid communication.
In addition to the main and any secondary electrophoretic flowpath serving as an enriched sample transfer means, the subject devices may further comprise one or more additional electrophoretic flowpaths, which may or may not be of capillary
dimension and may serve a variety of purposes. With devices comprising a plurality of electrophoretic flowpaths, a variety of configurations are possible, such as a branched configuration in which a plurality of electrophoretic flowpaths are in fluid
communication with the main electrophoretic flowpath. See U.S. Pat. No. 5,126,022, the disclosure of which is herein incorporated by reference.
The main electrophoretic flowpath and/or any secondary electrophoretic flowpaths present in the device may optionally comprise, and usually will comprise, fluid reservoirs at one or both termini, i.e., either end, of the flowpaths. Where
reservoirs are provided, they may serve a variety of purposes, such as a means for introducing buffer, elution solvent, reagent, rinse and wash solutions, and the like into the main electrophoretic flowpath, receiving waste fluid from the electrophoretic
flowpath, and the like.
Another optional component that may be present in the subject devices is a waste fluid reservoir for receiving and storing the waste portion of the initial sample volume from the enrichment channel, where the waste reservoir will be in fluid
communication with the discharge outlet. Depending on the particular device configuration, the discharge outlet may be the same as, or distinct from, the waste outlet, and may open into a waste reservoir or provide an outlet from the device. The waste
reservoir may be present in the device as a channel, compartment, or other convenient configuration which does not interfere with the other components of the device.
The subject device may also optionally comprise an interface means for assisting in the introduction of sample into the sample preparation means. For example, where the sample is to be introduced by syringe into the device, the device may
comprise a syringe interface which serves as a guide for the syringe needle into the device, as a seal, and the like.
Depending on the particular configuration and the nature of the materials from which the device is fabricated, at least in association with the main electrophoretic flowpath will be a detection region for detecting the presence of a particular
species in the medium contained in the electrophoretic flowpath. At least one region of the main electrophoretic flowpath in the detection region will be fabricated from a material that is optically transparent, generally allowing light of wavelengths
ranging from 180 to 1500 nm, usually 220 to 800 nm, more usually 250 to 800 nm, to have low transmission losses. Suitable materials include fused silica, plastics, quartz glass, and the like.
The integrated device may have any convenient configuration capable of comprising the enrichment channel and main electrophoretic flowpath, as well as any additional components. Because the devices are microchannel electrophoretic devices, the
electrophoretic flowpaths will be present on the surface of a planar substrate, where the substrate wil | | |
