EP1019712A4 - Vorrichtung und verfahren zur kapillarströmung mit einem elektrischen feld - Google Patents

Vorrichtung und verfahren zur kapillarströmung mit einem elektrischen feld

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Publication number
EP1019712A4
EP1019712A4 EP98948072A EP98948072A EP1019712A4 EP 1019712 A4 EP1019712 A4 EP 1019712A4 EP 98948072 A EP98948072 A EP 98948072A EP 98948072 A EP98948072 A EP 98948072A EP 1019712 A4 EP1019712 A4 EP 1019712A4
Authority
EP
European Patent Office
Prior art keywords
sample
array
samples
plate
microfluidic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98948072A
Other languages
English (en)
French (fr)
Other versions
EP1019712A1 (de
Inventor
Torleif Ove Bjornson
Randy M Mccormick
David S Soane
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Monogram Biosciences Inc
Original Assignee
Aclara Biosciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aclara Biosciences Inc filed Critical Aclara Biosciences Inc
Publication of EP1019712A1 publication Critical patent/EP1019712A1/de
Publication of EP1019712A4 publication Critical patent/EP1019712A4/de
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • B01J2219/00783—Laminate assemblies, i.e. the reactor comprising a stack of plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • B01J2219/00891—Feeding or evacuation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • B01J2219/00905—Separation
    • B01J2219/00912—Separation by electrophoresis
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178—Special arrangements of analysers
    • G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks

Definitions

  • This invention relates generally to the receiving and dispensing of samples such as in the field of separation of biomolecules and, in particular, separations by capillary electrophoresis and the use of the capillary electrophoresis to detect such biomolecules.
  • combinatorial libraries complement the large numbers of synthetic compounds available from the more traditional drug discovery programs based, in part, on identifying lead compounds through natural product screening.
  • Robotic-based high-throughput tools are now routinely used for screening libraries of compounds for the purpose of identifying lead molecules for their therapeutic potential.
  • considerable art has emerged.
  • PerSeptive Biosystems • screening method for characterizing ligand binding to a given target employs a variety of separation techniques and is described filer in the PCT application WO 97/01755. Another related method is described in U.S. Patent No. 5,585,277 (Scriptgen Pharmaceuticals) .
  • Highly parallel and automated methods for DNA synthesis and sequencing have also contributed significantly to the success of the human genome project to date.
  • DNA synthesis instrumentation see, e.g., PE/Applied Biosystems (ABI) , PerSeptive BioSystems, Pharmacia Biotech, and Beckman Instruments.
  • microfluidics technology embodied in the form of analytical devices has many attractive features for pharmaceutical high throughput screening. Advantages of miniaturization include greatly increased throughput and reduced costs, in addition to low consumption of both sample and reagents and system portability. Implementation of these developments in microfluidics and laboratory automation holds great promise for contributing to advancements in life sciences research and development.
  • microtiter plate has and still is the pharmaceutical industry standard for carrying out bioanalytical assays despite the recent advances in miniaturization and microfluidics. Because an enormous number of synthetic libraries have and continue to be generated using this particular multiwell format, the microtiter plate will remain entrenched within the industry.
  • Automated workstations for drug discovery and genomics applications are not capable of incorporating microfluidic multi-assay cards into existing robotic based high throughput microtiter plate handling and assay systems.
  • new methods for enabling fluid transfer between multi-well plates and microassay cassettes would be beneficial.
  • a critical factor currently limiting such a microfluidic HTS hybrid device is a means for reproducible liquid communication between the disparate dimensions of the two systems. More specifically, integration of microfluidics technology with existing robotic-based methods currently used in automated workstations is constrained by differences in volume size of samples used. For these reasons, new automated methods for multiplexing common lab tasks such as sample handling and dispensing on the microscale is required.
  • Capillary-based separations are widely used for analysis of a variety of analyte species. Numerous subtechniques, all based on electrokinetic-driven separations, have been developed. Capillary electrophoresis is one of the more popular of these techniques and can be considered to encompass a number of related separation techniques such as capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic chromatography. In the context used throughout this application, the phrase "capillary electrophoresis" is used to refer to any and all of the aforementioned electrokinetic separation subtechniques.
  • Electrophoresis is a separation process in which molecules with a net charge migrate through a medium under the influence of an electric field.
  • slab gel electrophoresis has been a widely used tool in the analysis of genetic materials. See, for example, G. L. Trainor, Anal. Chem. (1990) 62:418 ⁇ 26.
  • Capillary electrophoresis has emerged as a powerful separation technique with applicability to a wide range of molecules from simple atomic ions to large DNA fragments.
  • capillary electrophoresis has become an attractive alternative to slab electrophoresis for biomolecule analysis, including DNA sequencing. See, for example, Y. Baba, et al., Trends in Anal. Chem. (1992) 11:280-287. This is generally because the small size of the capillary greatly reduces Joule heating associated with the applied electrical potential.
  • capillary electrophoresis requires less sample and produces faster and better separations than slab gels.
  • Capillary electrophoresis possesses several characteristics which makes it amenable to this application.
  • the substantial reduction of Joule heating per lane makes the overall cooling and electrical requirements 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, as well as focused broad band sources, and for imaging onto array detectors or discrete spot detectors.
  • the concentration of analyte into such small bands results in high sensitivity.
  • electromigration injection i.e., applying the sample to the capillary by an electrical field, provides reproducible sample introduction with little band spreading, minimal sample consumption, and little labor.
  • fluorescence detection has been used for the detection of a variety of analyses, especially macromolecules, in capillary electrophoresis.
  • analyses especially macromolecules
  • capillary electrophoresis 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.
  • S. Takahashi, et al. Proceedings of Capillary Electrophoresis Symposium, December, 1992, referred to a multicapillary 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. 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 due to cross- talk between adjacent capillaries.
  • 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 of the capillary 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 ⁇ 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.
  • long capillaries provide slow separation, foul easily, and are difficult to replace.
  • U.S. Patent No. 5,332,480 (Datta, et al.) describes a multiple capillary electrophoresis device for continuous batch electrophoresis.
  • U.S. Patent No. 5,277,780 (Kambara) describes a two dimensional capillary electrophoresis apparatus for use with a two dimensional array of capillaries for measuring samples, such as DNA samples, in an array of test wells.
  • U.S. Patent 5,413,686 (Klein and Miller) describes a multi-channel automated capillary electrophoresis analyzer in which multiple individual separation capillaries are installed in a instrumental analyzer which serves to flush and fill the capillaries and associated buffer reservoirs from supplies of buffer situated within the instrument.
  • U.S. Patent 5,439,578 (Dovichi and Zhang) describes a multiple capillary biochemical analyzer based on an array of separation capillaries terminating in a sheath flow cuvette.
  • the use of the sheath flow cuvette facilitates detection of the analyte bands by reducing the magnitude of scattered radiation from the detection zone.
  • U.S. Patent No. 5,338,427 (Shartle, et al.) describes a single use capillary cartridge having electrically conductive films as electrodes; the system does not provide for multiplexed sampling, sample handling, and electrophoresis .
  • U.S. Patent 5,372,695 (Demorest) describes a system for delivering reagents to serve a fix capillary scanner system. Numerous examples of sample handling for capillary electrophoresis are known. For example, James in U.S. Patent No. 5,286,652 and Christianson in U.S. Patent No. 5,171,531 are based on presenting a single vial of sample to a single separation capillary for a sequential series of analyses.
  • the present invention provides short disposable capillaries mounted in a frame that is integral with a liquid handling system. This system permits a rapid multiplexed approach to capillary electrophoresis.
  • Zare, et al. discusses a fluoroassay method for the detection of macromolecules such as genetic materials and proteins by capillary electrophoresis . 99/15888
  • Yeung, et al. (U.S. Patent No. 5,006,210) presented a system for capillary zone electrophoresis with indirect laser-induced fluorescence detection of macromolecules, including proteins, a ino acids, and genetic materials. Systems such as these generally involve only one capillary.
  • U.S. Patent 5,463,910 discloses a multi-function aspirating device (AVL Scientific Corp.)
  • U.S. Patent No. 5,384,093 discusses an apparatus for aspirating and discharging a liquid sample (Toa Medical Electronics Co., Ltd.).
  • U.S. Patent No.5, 525, 302 discloses a method and device for simultaneously transferring plural samples.
  • a multiwell plate is disclosed in PCT WO 97/15394 published May 1, 1997 (SmithKline Beecham Corporation) .
  • the wells have a large opening at the top and small nozzle hole in the base. The opening is chosen so that a jet of liquid is emitted when a pressure pulse is applied to the surface such that by selecting a time for the pressure pulse a precise amount of volume in the well can be dispensed.
  • the apparatus comprises integral first and second plates.
  • the first plate comprises an array of sample receiving elements for receiving and/or dispensing a plurality of samples from an array of sample containers.
  • the second plate comprises a planar array of microfluidic networks of cavity structures and channels for conducting a microfluidic process.
  • the apparatus for conducting a microfluidic process comprises a first plate comprising an array of sample receiving elements adapted for receiving and/or dispensing a plurality of samples from an array of sample wells.
  • the apparatus also comprises a second plate integral with the first plate.
  • the second plate comprises a planar array of microfluidic networks of cavity structures and channels for conducting a microfluidic process. Each of the microfluidic networks is adapted for fluid communication with a corresponding sample receiving element.
  • Another aspect of the present invention is a method for processing an array of samples. At least a portion of each sample in an array of sample wells is simultaneously transferred to a corresponding array of microfluidic networks of cavity structures and channels by means of a corresponding array of sample receiving elements that are adapted for fluid communication with a corresponding sample receiving element of said first plate. The samples are then processed.
  • Another aspect of the present invention is a method for processing an array of samples. At least a portion of each sample in an array of sample wells is simultaneously transferred to a corresponding array of sample receiving elements. At least a portion of each sample is simultaneously transferred from the sample receiving elements to a corresponding array of microfluidic networks that are adapted for fluid communication with a corresponding sample receiving element. The array of samples is then processed.
  • Another aspect of the present invention is a method for processing an array of samples.
  • at least a portion of each sample in an array of sample wells is simultaneously transferred to a corresponding array of sample receiving elements that are part of a first plate comprising an array of sample receiving elements adapted for receiving a plurality of samples from an array of sample wells.
  • At least a portion of each sample from the sample receiving elements is simultaneously transferred to a corresponding array of microfluidic networks that is part of a second plate integral with the first plate.
  • the second plate comprises a planar array of microfluidic networks of cavity structures and channels for conducting a microfluidic process.
  • Each of the microfluidic networks is adapted for fluid communication with a corresponding sample receiving element .
  • the samples are then processed.
  • kits for processing a sample comprises an apparatus as described above and reagents, other than reagents within the apparatus, for processing a sample.
  • Another aspect of the present invention is a method for analysis of an array of samples in an array of sample containers by capillary electrophoresis.
  • An array of samples in an array of sample containers is provided. At least a portion of each sample in the array of sample containers is simultaneously transferred to a corresponding array of capillary electrophoresis columns. Separation of the transferred samples is simultaneously conducted by capillary electrophoresis. The capillary electrophoresis separations are then analyzed.
  • Another aspect of the present invention is a method for analysis of an array of samples in an array of sample containers by capillary electrophoresis.
  • An array of aliquots of sample is acquired simultaneously from an array of samples in sample containers.
  • the array of samples is simultaneously processed to provide an array of processed samples.
  • the array of processed samples is simultaneously transferred for capillary electrophoresis to an array of capillary electrophoresis columns.
  • Capillary electrophoresis is simultaneously conducted on the array of the capillary electrophoresis columns and the columns are analyzed.
  • the invention encompasses a system for multiplexing capillary electrophoresis analysis of multiple samples comprising: a) means for simultaneously acquiring an array of aliquots of sample from an array of samples in sample containers; b) means, in combination with means (a) , for simultaneously processing the array of samples to provide an array of processed samples and presenting the array of processed samples for capillary electrophoresis; c) means for simultaneously transferring an array of processed samples to an array of capillary electrophoresis columns; d) means for simultaneously conducting capillary electrophoresis on the array of the capillary electrophoresis columns from (c) ; and e) means for analyzing capillary electrophoresis columns from (d) .
  • Another aspect of the present invention is a device for conducting a microfluidic process wherein the device comprises a planar substrate having a planar array of microfluidic networks of cavity structures and channels for conducting a microfluidic process.
  • Another aspect of the present invention is a method for securing an array of samples. At least a portion of each sample in an array of sample wells of a multiwell plate is simultaneously transferred to a corresponding array of sample receiving elements. At least a portion of each sample from the sample receiving elements is simultaneously transferred to a corresponding array of sample handling wells.
  • Another aspect of the present invention is a method for securing an array of samples wherein at least a portion of each sample in an array of sample wells of a multiwell plate is simultaneously transferred to a corresponding array of sample receiving elements. At least a portion of each sample from the sample receiving elements is simultaneously transferred to a corresponding array of sample handling wells. The portion of each sample is expelled from the sample receiving elements by application of an electric field or application of pressure.
  • Another aspect of the present invention is a method for processing an array of samples that comprises:
  • FIG. 1 is a perspective view of one embodiment of an apparatus in accordance with the present invention.
  • Fig. 2 is an exploded view of the apparatus of Fig. 1.
  • Fig. 3 is a cross-sectional view of the apparatus of Fig. 1 taken along lines 3-3.
  • Fig. 4 is a perspective view of an embodiment of a microfluidic network.
  • Fig. 5 is a perspective view of one embodiment of a portion of a plate having a plurality of microfluidic networks.
  • Fig. 6 is a perspective view of another embodiment of a portion of a plate having a plurality of microfluidic networks.
  • Fig. 7 is a perspective view of another embodiment of a portion of a plate having a plurality of microfluidic networks.
  • Fig. 8 is a cross-sectional view of one embodiment of an assembly depicting means of transferring liquid from a sample container to a sample receiving element.
  • Fig. 9 is a cross-sectional view of another embodiment of an assembly depicting means of transferring liquid from a sample container to a sample receiving element.
  • Fig. 10 is a cross-sectional view of another embodiment of an assembly depicting means of transferring liquid from a sample container to a sample receiving element.
  • Fig. 11 is a cross-sectional view of another embodiment of an assembly depicting means of transferring liquid from a sample container to a sample receiving element.
  • Fig. 12 is a cross-sectional view of another embodiment of an assembly depicting means of transferring liquid from a sample container to a sample receiving element.
  • the present invention encompasses methods and apparatus for simultaneously transferring samples from an array of sample containers to an array of sample receiving elements, simultaneously transferring samples from said sample receiving elements to a planar array of microfluidic networks of interconnected cavity structures and channels of capillary dimensions and simultaneously conducting a microfluidic process. It is to be noted that simultaneous transfer may be from all or a portion of the array to a corresponding portion of the microfluidic networks.
  • the miniaturized system of enrichment trenches, reaction chambers and detection zones enable multiple laboratory processes to be integrated "on-board" a planar substrate, including sample preparation, incubation, electrophoretic separations, and analyses.
  • the present invention differs from known procedures in microfluidic technology such as microelectrophoresis in that it provides for simultaneously sampling an array of samples; simultaneously handling the samples and presenting an array of the samples for electrophoresis; simultaneously transferring the array of presented samples to an array of capillaries; and simultaneously conducting processing of the samples in the capillary columns.
  • the invention has many advantages over conventional microfluidic techniques such as microelectrophoresis technology.
  • the time period for conducting a microfluidic process is much shorter than with conventional techniques such as electrophoresis.
  • conventional separation on gels is manually intensive, the present invention only requires sample introduction since the remaining processing is automatic.
  • the invention system will perform confirmations in parallel.
  • the present invention system typically requires only 1 to 5% of the amount of sample used in standard electrophoresis.
  • the present invention system can perform a quantitative determination in a much simpler format and much shorter time period than with conventional electrophoresis. Since reactions are in the liquid phase, the invention system provides greater speed, greater specificity and less background biochemical noise and quantitation is achieved in tens of minutes instead of hours.
  • the present invention system only requires small amounts of sample.
  • Reagent reservoirs may have volumes ranging from 0.01 to 100 ⁇ l, more typically, 0.1 to 10 ⁇ l. Once drawn from the reservoir, sample volumes transported through the microchannels range from 1 to 1000 nanoliters, more typically, 10 to 100 nanoliters. Volumes of sample drawn for individual microinjected reaction or separation plugs are 0.01 to 10 nanoliters, more typically, 0.1 to 1 nanoliters.
  • simultaneous transfer may be carried out with respect to all of the wells in a multiwell plate or only with respect to some of the wells thereof. For example, one may wish to transfer samples with respect to only 8 or 16 or some other number of wells in a 96 well plate. Such transfer may be achieved by employing means for independently activating the present device to provide simultaneous transfer for less than the full number of wells in a multiwell plate.
  • Array an arrangement of a plurality of elements such as a plurality of wells in a multiwell source plate, a plurality of apertures or nozzles in a sample transfer plate, a plurality of microfluidic networks on the multi- assay card, and so forth.
  • Planar array an array that is arranged in a plane, which may be the plane of an object such as, for example, a planar substrate, comprising the array.
  • Cavity structure an unfilled space with a mass, preferably, a hollowed out space in an object, such as, e.g., a planar substrate, a plate, or the like in accordance with the present invention such as, for example, a well, a reservoir, an incubation chamber, a separation chamber, an enrichment chamber, a detection chamber, and the like.
  • an object such as, e.g., a planar substrate, a plate, or the like in accordance with the present invention such as, for example, a well, a reservoir, an incubation chamber, a separation chamber, an enrichment chamber, a detection chamber, and the like.
  • the cavity structures are usually present at one or both of the termini, i.e., either end, of a channel.
  • the cavity structures may serve a variety of purposes, such as, for example, means for introducing a buffer solution, elution solvent, reagent rinse and wash solutions, and so forth into a main channel or one or more interconnected auxiliary channels, receiving waste fluid from the main channel, and the like.
  • Channels a conduit or means of communication. usually fluid communication, more particularly, liquid communication, between elements of the present apparatus.
  • the elements in communication are, e.g., cavity structures, and the like.
  • Channels include capillaries, grooves, trenches, microflumes, and so forth.
  • the channels may be straight, curved, serpentine, labyrinth-like or other convenient configuration within the planar substrate.
  • the cross-sectional shape of the channel may be circular, ellipsoid, square, rectangular, triangular and the like so that it forms a microchannel within the planar substrate in which it is present.
  • the inside of the channel may be coated with a material for strength, for enhancing or reducing electrokinetic flow, for enhancing detection limits and sensitivity, and so forth.
  • a material for strength for enhancing or reducing electrokinetic flow, for enhancing detection limits and sensitivity, and so forth.
  • Exemplary of coatings is silylation, polyacrylamide (vinyl bound) , methylcellulose, polyether, polyvinylpyrrolidone, and polyethylene glycol, polypropylene.
  • TeflonTM (DuPont) , NafionTM (DuPont) , and the like may also be used.
  • Capillary dimension a cross-sectional area that provides for capillary flow through a channel.
  • At least one of the cross-sectional dimensions is at least about 1 ⁇ m, usually at least 10 ⁇ m, and is usually no more than 500 ⁇ m, preferably no more than 200 ⁇ m.
  • Channels of capillary dimension typically have an inside bore diameter (ID) of from about 1 to 200 microns, more typically from about 25 to 100 microns.
  • Microfluidic of or pertaining to fluids and being of a magnitude on the order consistent with capillary dimension.
  • Microfluidic network a system of interconnected cavity structures and capillary-size channels configured with a plurality of branches through which fluids may be manipulated and processed.
  • Well plate a plate comprising an array of wells. The plate may have any number of wells, which are usually in a pattern, and are usually 96, 192, 384 or 1536 well plates. Exemplary of such well plates is microtiter plates having a pattern of wells.
  • Integral a single unit or a group of parts formed as a unit. Although the unit may be formed from separate parts, the parts are generally non-separable by virtue of being permanently attached or rendered integral by interlocking means. Parts in such a unit may be rendered non-separable by such processes as welding such as ultrasonic welding, the use of adhesives, gluing, bonding, sealing and the like. The components are present in a single, compact readily handled unit.
  • Electroflow the manipulation of entities such as molecules, particles, cells and the like through a medium under the influence of an applied electric field by use of electrodes and the like to induce movement such as electrokinetic flow including, electroosmotic flow, electrophoretic flow, dielectrophoretic flow, and so forth.
  • 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.
  • electroflow can be carried out in conjunction with movement of material by gravity or by application of a magnetic field, centrifugal force, thermal gradients, pneumatic means including negative (vacuum) and positive (pumping) pressure, and the like.
  • Electroflow medium an electrically conductive medium; a medium generally utilized in carrying out electrophoretic processes.
  • the particular medium chosen is one that is suitable to a particular application of the present invention.
  • the medium should not interfere to any substantial degree with the electric fields used when utilizing an apparatus of the present invention.
  • Such media include, for example, a buffer solution, cross-linked and uncross-linked polymeric solution, solvents, detergents, surfactant micellular dispersion, a gel of the type generally used in connection with analytical separation techniques, and so forth.
  • polyacrylamide gel used in PAGE analytical procedures, cellulose derivatives, polyvinyl alcohols, polyethylene oxides and the like may be used.
  • Barron and Blanch "DNA Separations by Slab Gel and Capillary Electrophoresis: Theory and Practice, " Separation and Purification Methods (1995) 24:1-118.
  • the electroflow medium may be a conventional buffer such as, for example, the Good's buffers (HEPES, MOPS, MES, Tricine, etc.), and other organic buffers (Tris, acetate, citrate, and formate) , including standard inorganic compounds (phosphate, borate, etc.).
  • exemplary buffer systems include: i) 100 M sodium phosphate, pH 7.2ii) 89.5 mM tris-base, 89:5 mM Boric acid, 2 mM ETDA, pH 8.3.
  • Buffer additives include methanol, metal ions, urea, surfactants, and zwitterions, intercalating dyes and other labeling reagents.
  • Polymers can be added to create a sieving buffer for the differential separation of DNA based on fragment length.
  • examples of such polymers are polyacrylamide (cross-linked or linear) , agarose, methylcellulose and derivatives, dextrans, and polyethylene glycol.
  • Inert polymers can be added to the separation buffer to stabilize the separation matrix against factors such as convection mixing.
  • buffers containing micelles could be used for effecting separation of electrically neutral or hydrophobic substances of interest.
  • the micelles are formed in the buffer by addition of an appropriate surfactant at a concentration exceeding the critical micelle concentration of that detergent.
  • useful surfactants include but are not limited to sodium dodecyl sulfate, dodecyltrimethyl ammonium bromide, etc.
  • Weakly charged or apolar analyses partition into the micelles to different degrees depending upon their degree of hydrophobicity and thus can be separated. This subtechnique of capillary electrophoresis is termed micellar electrokinetic chromatography. Electrophoresis — separation of components in a liquid by electroflow.
  • electrophoresis include, by way of example and not limitation, free zone electrophoresis, gel electrophoresis, isotachophoresis, high performance CE, capillary zone electrophoresis, isoelectric focusing, micellar electrokinetic capillary chromatography, and the like.
  • Electrophoresis column in the context of the present invention, a channel for carrying out electrophoresis .
  • Processing samples may be processed by one or more of any number of procedures such as, for example, subjecting such sample to separation procedures for sample enrichment, isolation or purification, analyzing such sample such, e.g., as an assay, detection and the like, carrying out a chemical synthesis with such sample, such as those involved with combinatorial chemistry methods for small and large molecule synthesis, and so forth.
  • polynucleotides may be synthesized or sequenced. Different nucleotides can be reacted to form DNA and different amino acids can be reacted to form proteins. These reactions can be carried out at greatly increased speeds as compared with conventional mechanical technologies. In addition to increased speeds, the yield is vastly improved due to the precision with which the reactants can be moved in accordance with the present invention.
  • the present invention is useful for a variety of additional purposes.
  • a mixture of components can be separated into a variety of pure groups and moved along parallel tracks. Upon resolving the mixtures, the desired components can be guided by the electrical wave fields to appropriate spots within one or more channels.
  • selected components may be guided to channels filled with specific binding pair members, such as antigen-antibodies, reactive with given substances of interest being moved in the medium or moved into contact with complementary components having a label or other member of a signal producing system of other types of chemicals for any number of purposes such as various transformations that are either physical or chemical in nature.
  • specific binding pair members such as antigen-antibodies
  • complementary components having a label or other member of a signal producing system of other types of chemicals for any number of purposes such as various transformations that are either physical or chemical in nature.
  • bacterial or mammalian cells, or viruses may be sorted by complicated microfluidic networks in connection with a plurality of electrodes capable of generating electrical potentials of a variety of different strengths in order to move the cells or viruses through the fields based on the size, charge or shape of the particular material being moved. Separated cells or viruses may be analyzed or modified subsequently.
  • cell fractionation is possible by employing solid-phase extraction materials, including paramagnetic beads, nonmagnetic particles, or the like, to specifically bind with the desired cells such that the bead-cell complex can be separated from the other cells. Cell lysis is then possible for releasing the intracellular materials for further analysis.
  • solid-phase extraction materials including paramagnetic beads, nonmagnetic particles, or the like
  • Microfluidic processing processing carried out on a microfluidic scale.
  • the processing involves fluid handling, transport and manipulation within chambers and channels of capillary dimension.
  • Valveless sample injection is achieved by moving fluid from the reagent reservoirs into cross-channel injection zones, where plugs of buffer or test compounds are precisely metered and dispensed into a desired flowpath.
  • the rate and timing of movement of the fluids in the various microchannels can be controlled by electrokinetic, magnetic, pneumatic, and/or thermal-gradient driven transport, among others.
  • sample manipulation methods enable the profile and volume of the fluid plug to be controlled over a range of sizes with high reproducibility.
  • microfluidic processing may include sample preparation and isolation where enrichment microchannels containing separation media are employed for target capture and purification.
  • Microfluidic processing may also include reagent mixing, reaction/incubation, separations and sample detection and analyses.
  • Sample - a medium containing a substance of interest, synthetic or natural, to be examined, treated, determined or otherwise processed.
  • Typical sources for biological samples include body fluids such as, for example, whole blood, blood fractions such as serum and plasma, synovial fluid, cerebro-spinal fluid, amniotic fluid, semen, cervical mucus, sputum, saliva, gingival fluid, urine, and the like.
  • sample includes combinatorial chemistry generated libraries of compounds, usually small molecules, oligonucleotides and peptides.
  • Other sources of samples are aqueous or water soluble solutions of natural or synthetic compounds, particularly, compounds that are potential therapeutic drugs where it is desired to determine if the compound binds to a specific receptor.
  • the amount of the sample depends on the nature of the sample and the nature of the processing to be conducted. For fluid samples such as whole blood, saliva, urine and the like the amount of the sample is usually about 1 to 1000 nanoliters, more usually, about 10 to 100 nanoliters.
  • the sample can be pretreated and can be prepared in any convenient medium, which does not interfere with a microfluidic process in accordance with the present invention. An aqueous medium is preferred.
  • the substance can be comprised of a member of a specific binding pair (sbp) and may be a ligand, which is monovalent (monoepitopic) or polyvalent (polyepitopic) , synthetic or natural, antigenic or haptenic, and is a single compound or plurality of compounds which share at least one common epitopic or determinant site.
  • the substance of interest can be a part of a cell such as bacteria or a cell bearing a blood group antigen such as A, B, D, etc., or an HLA antigen, or cell membrane receptors, or a microorganism, e.g., bacterium, fungus, protozoan, or virus.
  • the monoepitopic ligands will generally be from about 100 to 2,000 molecular weight, more usually from 125 to 1,000 molecular weight.
  • the substances of interest include drugs, potential drug candidates, metabolites, pesticides, pollutants, and the like.
  • the polyvalent ligands will normally be poly(amino acids), i.e., polypeptides and proteins, polysaccharides, nucleic acids, and combinations thereof. Such combinations include components of bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes and the like.
  • the polyepitopic ligands to which the subject invention can be applied will have a molecular weight of at least about 5,000, more usually at least about 10,000.
  • the poly (amino acids) of interest will generally be from about 5,000 to 5,000,000 molecular weight, more usually from about 20,000 to 1,000,000 molecular weight; among the hormones of interest, the molecular weights will usually range from about 5,000 to 60,000 molecular weight.
  • the molecular weights will generally range from 10,000 to 2X108, more usually from 10,000 to 106.
  • immunoglobulins IgA, IgG, IgE and IgM
  • the molecular weights will generally vary from about 160,000 to about 106.
  • Enzymes will normally range from about 10,000 to 1,000,000 in molecular weight.
  • Natural receptors vary widely, generally being at least about 25,000 molecular weight and may be 106 or higher molecular weight, including such materials as avidin, DNA, RNA, thyroxine binding globulin, thyroxine binding prealbumin, transcortin, etc.
  • polynucleotides such as -RNA, r-RNA
  • analyte also includes receptors that are polynucleotide binding agents, such as, for example, restriction enzymes, activators, repressors, nucleases, polymerases, histones, repair enzymes, chemotherapeutic agents, and the like.
  • polynucleotide binding agents such as, for example, restriction enzymes, activators, repressors, nucleases, polymerases, histones, repair enzymes, chemotherapeutic agents, and the like.
  • sbp member one of two different molecules having an area on the surface or in a cavity that specifically binds to and is therefore defined as complementary with a particular spatial and polar organization of the other molecule.
  • the members of the sbp can be referred to as ligand and receptor such as members of an immunological pair, e.g., antigen-antibody.
  • Complementary sbp members bind to one another, as for example, a ligand and its complementary receptor. With respect to two complementary sbp members, one may be referred to as the "binding partner" for the other.
  • Sbp members can be immunological pairs such as antigen and antibody, or non-immunological pairs such as avidin and biotin.
  • Sbp members can also be small molecules or residues of small molecules and their receptors.
  • Small molecules have a molecular weight of from 100-2000, preferably 150-1000, and a receptor for the small molecule either exists or can be prepared.
  • Examples of small molecules include derivatives of biotin, lysergic acid, fluorescein or a fluorescein derivative, and vitamin B12, with the corresponding receptors being avidin or streptavidin, anti-lysergic acid, anti-fluorescein and intrinsic factor, respectively.
  • Ligand any organic compound for which a receptor naturally exists or can be prepared.
  • Receptor any compound or composition capable of recognizing a particular spatial and polar organization of a molecule, e.g., epitopic or determinant site.
  • Illustrative receptors include membrane bound receptors such as G-protein receptors (e.g., muscarinic, adrenergic, prostaglandin and dopamine such as the D2 receptor) , tyrosine kinase (insulin-like IGF, epidermal EGF, nerve NGF, fibroblast FGF growth factors) , ion channels, T-cell receptors, the interleukins , and other naturally occurring receptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A, complement component Clq, and the like.
  • G-protein receptors e.g., muscarinic, adrenergic, prostaglandin and dopamine such as the D2 receptor
  • tyrosine kinase insulin-like IGF, epidermal EGF, nerve NGF, fibroblast FGF growth factors
  • ion channels e.g.,
  • Label or reporter molecule a chemical entity capable of being detected by a suitable detection means, including, but not limited to, spectrophotometric, chemiluminescent, electrochemical or radiochemical means.
  • the reporter molecule can be conjugated to another molecule such as an sbp member, e.g., a ligand or an antibody, by procedures well known in the art.
  • the reporter molecule contains a functional group suitable for attachment to the sbp member.
  • the functional groups suitable for attaching the reporter group are usually activated esters or alkylating agents. Details of techniques for attaching reporter groups are well known in the art. See, for example, Matthews, et al., Anal. Biochem.
  • Reporter molecules are members of a signal producing system capable of being detected directly or through a specific binding reaction to produce a detectable signal.
  • the reporter molecule can be isotopic or nonisotopic, usually nonisotopic, and can be a catalyst, dye, fluorescent molecule, chemiluminescent molecule, coenzyme, enzyme, substrate, radioactive group, certain particles such as carbon and the like.
  • Antibody an immunoglobulin that specifically binds to, and is thereby defined as complementary with, a particular spatial and polar organization of another molecule.
  • the antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal) , or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies.
  • Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3 , IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular molecule is maintained.
  • Signal producing system one or more components, at least one component being a detectable label or reporter molecule, which generate a detectable signal that relates to the amount of bound and/or unbound label, i.e., the amount of label bound or not bound to the compound being detected.
  • the label and optionally other sps members are bound to an sbp member.
  • the label is an enzyme, electroluminescent group such as a transition metal complex (see, e.g., U.S. Patent Nos. 5,541,113, 5,610,017, 5,52 7 , 7 10, 5,591,581, the relevant disclosures of which are incorporated herein by reference, chemilu inescer, fluorescer, radiolabel, or the like.
  • the signal is preferably detected and/or measured by detecting enzyme activity, luminescence, light emissions, or radioactivity, respectively.
  • the labels and other reagents of the signal producing system must be stable at the temperatures used in the electroseparation method and subsequent assay.
  • suitable labels include, by way of illustration and not limitation, enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH”) and horseradish peroxidase; promoters; dyes; fluorescers, such as fluorescein, isothiocyanate, rhodamine compounds, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde, and fluorescamine; electroluminescent labels such as ruthenium chelates; chemiluminescers such as isoluminol; sensitizers; coenzymes; enzyme substrates; radiolabels such as 1251, 1311, 14C, 3H, 57Co and 75Se.
  • enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase (“G6PDH”) and horseradish peroxidase
  • promoters dyes
  • fluorescers such as fluorescein
  • Suitable enzymes and coenzymes are disclosed in Litman, et al., U.S. Patent No. 4,275,149, columns 19-28, and Boguslaski, et al. , U.S. Patent No. 4,318,980, columns 10- 14; suitable fluorescers and chemiluminescers are disclosed in Litman, et al., U.S. Patent No. 4,275,149, at columns 30 and 31; which are incorporated herein by reference.
  • Some labels can directly produce a signal, and therefore, additional components are not required to produce a signal.
  • Numerous organic molecules for example fluorescers, are able to absorb ultraviolet and visible light, where the light absorption excites these molecules to an excited energy state. This absorbed energy is then dissipated by emission of light at a second wavelength.
  • Other labels that directly produce a signal include radioactive isotopes and dyes. Alternately, the label may need other components to produce a signal. in this situation the signal producing system would then include all the components required to produce a measurable signal.
  • These components may include substrates, electron transfer agents, coenzymes, enhancers, additional enzymes, substances that react with enzymic products, catalysts, activators, cofactors, inhibitors, scavengers, metal ions, and a specific binding substance required for binding of signal generating substances.
  • suitable signal producing systems can be found in Ullman, et al. U.S. Patent No. 5,185,243, columns 11-13, incorporated herein by reference.
  • the label can be bound covalently to numerous sbp members: an antibody; a receptor for an antibody; a receptor that is capable of binding to a small molecule conjugated to an antibody, a ligand analog, an oligonucleotide and the like.
  • Bonding of the label to the sbp member may be accomplished by chemical reactions that result in replacing a hydrogen atom of the label with a bond to the sbp member or may include a linking group between the label and the sbp member.
  • Other sps members may also be bound covalently to sbp members such as avidin- biotin, fluorescein-anti-fluorescein, and the like.
  • Two sps members such as a fluorescer and quencher can each be bound to a different antibody that forms a specific complex with the analyte. Formation of the complex brings the fluorescer and quencher in close proximity, thus permitting the quencher to interact with the fluorescer to produce a signal. Methods of conjugation are well known in the art.
  • one label may be bound to a particle and a second label bound to a sbp member that binds to the sbp member attached to the particle.
  • Assay a method for determining a substance capable of binding to a specific binding pair member, for example, for determining an analyte or detecting the degree of binding of a compound to a receptor.
  • the determination may be qualitative or quantitative.
  • Such assays depend on specific binding of a ligand to its receptor and include receptor binding assays, immunoassays , ligand/binding assays, polynucleotide assays, particularly polynucleotide hybridization assays, and cell surface binding assays.
  • the assays may be utilized for drug discovery and screening, studies of receptors, detection of drugs and other substances, DNA detection, DNA sequencing, genetic analysis, monitoring of gene expression, and so forth.
  • Receptor-ligand binding competitive binding assays are a useful preliminary means for screening a large number of compounds for their therapeutic potential. Improved high throughput bioanalytical techniques are needed for characterizing the functional properties of receptor- mediated signaling and other cell transduction mechanisms.
  • the receptor binding assays routinely arise in the fields of pharmacology, neurobiology, cardiology, immunology, microbiology and oncology, among others.
  • Heterogeneous assay an assay wherein free labeled species is separated from a labeled species that is bound to another species such as an sbp member.
  • the separation may be carried out by physical separation, e.g., by transferring one of the species to another reaction vessel, filtration, centrifugation, chromatography, solid phase capture, magnetic separation, and so forth and may include one or more washing steps.
  • the separation may be nonphysical in that no transfer of one or both of the species is conducted, but the species are separated from one another in situ.
  • the activity of a label is not affected by the reaction of specific binding pair members with one another. Regardless of the means of separation, the signal from the label may be measured from one or both of the separated species.
  • Homogeneous assay an assay wherein free labeled species is not separated from a labeled species that is bound to another species such as an sbp member. The signal from the label is significantly different between the free labeled species and that which is bound and, thus, can be measured without separation.
  • Immunoassay a specific binding assay in which the reagents include an antibody.
  • the apparatus comprises first plate 100 that has an array of sample receiving elements 102 for receiving a plurality of samples from an array of sample containers.
  • each of sample receiving elements 102 is capillary.
  • the array of sample receiving elements 102 may be aligned so that they correspond with wells of a multiwell plate that contain samples. When the elements 102 are in the sample, an aliquot of sample is transferred to the element. For capillaries, transfer is conveniently by capillary action.
  • the samples in sample receiving elements 102 can be manipulated and presented for simultaneous transfer to input reservoir 142 for microfluidic processing.
  • the volume of sample drawn into the capillary is controlled by the length and the bore diameter of the capillary.
  • the bore diameter will be several hundred microns and the length of the capillary could range up to 10-20 mm.
  • Such capillaries of these dimensions can fill with sample volumes in the 1 to 10 ⁇ L range.
  • the volume of fluid drawn into the capillary can be controlled by carefully defining the bore diameter of the capillary and by defining the length of the capillary. The latter dimension can be defined by positioning a stop junction along the inner wall of the capillary. This stop junction could be an abrupt increase in the bore diameter of the capillary.
  • the configuration of the capillaries conforms to the spacing format of the wells in the well plate.
  • the capillaries can be constructed by any number of means. A major requirement is that the capillaries must be sufficiently hydrophilic to draw in several microliters of liquid sample by capillary action.
  • a suitable capillary can be constructed from glass or silica tubing of appropriate dimensions.
  • a suitable capillary can be constructed from a plastic material such as polyethylene, polypropylene, polycarbonate, polysulfone, polymethylmethacrylate, etc.
  • the inner bore of the plastic capillary must be treated in some way to make the inner walls of the capillary sufficiently hydrophilic to draw in the sample by capillary action.
  • Appropriate treatments for altering the normally hydrophobic surface of the plastic and imparting hydrophilicity to the inner walls of the capillaries include coating the walls with a surfactant or wetting agent, grafting a layer of hydrophilic polymer onto the wall of the hydrophobic capillary or treating the walls of the capillary by plasma etching.
  • sample receiving elements 102 are sipper capillaries as disclosed in U.S. Patent No. 5,560,811, at column 9, line 53, to column 10, line 45, the disclosure of which is incorporated herein by reference.
  • first plate 100 has an array of sample receiving elements that comprise sample handling wells with a corresponding array of sipper capillaries.
  • the array of sipper capillaries is aligned with wells of a multiwell plate containing the samples.
  • an aliquot of sample is transferred to the sipper capillary by wicking action.
  • the samples in the capillaries can be manipulated to be presented to the microfluidic networks in second plate 110.
  • first plate 100 may also comprise a matrix element 104 , which is typically made of a wide variety of porous matrix materials.
  • the porous matrix materials should have little or no affinity for sample.
  • Useful porous matrix materials include membrane materials such as regenerated cellulose, cellulose acetate, polysulfone, polyvinylidine fluoride, polycarbonate and the like.
  • membrane materials such as regenerated cellulose, cellulose acetate, polysulfone, polyvinylidine fluoride, polycarbonate and the like.
  • a cellulose acetate membrane such as that available from Amicon is useful.
  • a membrane composed of polysulfone such as those available from Amicon or Gelman is useful.
  • porous matrix 104 could be a porous cylindrical or spherical plug of sintered polymer particles.
  • porous materials are available from Porex or Interflow and are typically comprised of a bed of small polymeric particles that have been fused together by heat and pressure (sintering) to form a porous plug of predefined geometry.
  • porous matrix 104 may comprise an ultrafiltration membrane with a defined molecular weight cut off.
  • porous matrix 104 could be derivatized with some biochemical agent to impart a selective binding capability to matrix 104.
  • the apparatus shown in Figs. 1-3 also comprises a second plate 110 that is integral with the first plate.
  • Second plate 110 comprises a planar array of microfluidic networks 108 having interconnected cavity structures 142 and channels 120 and 124 (see Fig. 4) .
  • Each of the microfluidic networks corresponds to a respective sample receiving element 102.
  • the capillaries are adapted for fluid communication with cavity structure 142. Liquid is transferred from sample receiving element 102 to cavity structure 142 by, for example, application of negative pressure, thermal gradient and the like.
  • the capillary may have a fritted element disposed therein such that capillary flow will continue until the fritted element is saturated whereupon capillary draw ceases.
  • the apparatus also includes a means of fluid communication between plates 100 and 110.
  • Such means of fluid communication includes, for example, a capillary between the two plates to provide for flow from the sample receiving well to the microfluidic networks of second plate 110.
  • the capillary may extend from the sample receiving well to a cavity structure of the corresponding microfluidic network.
  • the means of fluid communication may also be an opening in a cover plate for the second plate 110 where the opening permits liquid from the sample receiving well to be transferred mechanically, electrically, including electrostatically and piezoelectrically, or the like into a corresponding microfluidic network of second plate 110.
  • the microfluidic network has interconnected cavity structures and channels, the latter forming one or more flowpaths resulting in an interconnected system.
  • a desired microfluidic process may be carried out in the main flowpath or in one of the secondary flowpaths.
  • the additional flowpaths may be employed for a variety of purposes such as, for example, enrichment of a sample, isolation, purification, dilution, mixing, metering, and the like.
  • a variety of configurations are possible, such as a branched configuration in which a plurality of flowpaths is in fluid communication with the main flowpath. See, for example, U.S. Patent No. 5,126,022.
  • the main flowpath has associated with it at least one pair of electrodes for applying an electric field to the medium present in the flowpath.
  • a single pair of electrodes typically one member of the pair is present at each end of the pathway.
  • a plurality of electrodes may be associated with the flowpath, as described in U.S. Patent No. 5,126,022, the relevant disclosure of which is herein incorporated by reference, where the plurality of electrodes can provide for precise movement of entities along the flowpath.
  • the electrodes employed in the subject invention may be any convenient type capable of applying an appropriate electric field to the medium present in the flowpath with which they are associated.
  • Plate 110 is comprised of a plurality of microfluidic networks 108. Each network comprises main flowpath 120 and secondary flowpath 122, which intersect at 124. Electrode 130 is connected to reservoir 132 and electrode 134 is connected to reservoir 136. An electric potential can be applied to flowpath 122 by means of electrodes 130 and 134. Electrode 140 is connected to sample introduction port and reservoir 142 and electrode 144 is connected to reservoir 146. An electric potential can be applied to main flowpath 120 by means of electrodes 140 and 144.
  • the main flowpath 120 has optional portion 150 that is tortuous to provide an appropriate path length and residence time to achieve mixing by diffusion, incubation, and so forth.
  • Secondary flowpath 122 has detection zone 148 where the result of a microfluidic process may be detected.
  • the detection zone permits the detection of a signal produced during the assay.
  • the detection zone may be used to detect the presence of the synthesized compound. It is, of course, within the purview of the present invention to utilize several detection zones depending on the nature of the microfluidic process. There may be any number of detection zones associated with a single channel or with multiple channels.
  • Suitable detectors for use in the detection zones include, by way of example, photomultiplier tubes, photodiodes, photodiode arrays, avalanche photodiodes, linear and array charge coupled device (CCD) chips, CCD camera modules, spectrophotometers, spectrofluorometers, and the like.
  • Excitation sources include, for example, filtered lamps, LED's, laser diodes, gas, liquid and solid state lasers, and so forth.
  • the detection may be laser scanned excitation, CCD camera detection, coaxial fiber optics, confocal back or forward fluorescence detection in single or array configurations, and the like.
  • Detection may be by any of the known methods associated with the analysis of capillary electrophoresis columns including the methods shown in U.S. Patent Nos. 5,560,811 (column 11, lines 19-30), 4,675,300 and 5,324,401, the relevant disclosures of which are incorporated herein by reference.
  • An example of an optical system for reading the channels in the detection zones comprises a power supply, which energizes a photomultiplier tube.
  • a power supply energizes a 75 watt Xenon lamp.
  • Light from the lamp is condensed by focusing lens, which passes light to an excitation filter.
  • a dichroic mirror directs excitation light to a microscope.
  • the apparatus is mounted on a so that light passes over the channels.
  • Fluorescent emission light is collected by the microscope, passed through a dichroic mirror, emission filter, or spatial filter before reaching the photomultiplier (PMT) .
  • the output signal of PMT is fed to an analog-to-digital converter, which in turn is connected to computer.
  • a static detection system in which a stationary detection point some distance from the injection end of the capillary is monitored as bands to be analyzed traverse the length of the capillary and pass by the detection zone could be used.
  • This type of detection could be implemented using optical fibers and lenses to deliver the excitation radiation to the capillary and to collect the fluorescent emission radiation from the detection zone in the capillary.
  • Appropriate multiplexing and demultiplexing protocols might be used to sequentially irradiate and monitor a large array of capillaries using a single source and a single or a small number of photodetectors. Using this approach, each capillary in the array is sequentially polled to detect any analyte band in the detection zone of that capillary.
  • the detectors may be part of an instrument into which the present apparatus is inserted.
  • the instrument may be the same instrument that comprises the electrode leads and other components necessary for utilizing the present apparatus.
  • separate instruments may be used for housing a sample container plate, incubation of sample and reagents, detection of a result, electrical field application, and other operations such as temperature and humidity control, and so forth.
  • Humidity control may be achieved in a number of ways such as, for example, the use of humidistats, water vapor sources confined in the device in fluid communication with other areas thereof, and so forth. Other methods of humidity control will be evident to those skilled in the art.
  • a suitable electroflow medium as described above is introduced into the flowpaths defined by the channels in the secondary plate.
  • the medium may be conveniently introduced through one of the reservoirs at the termini of each of the channels or directly into the channels themselves prior to sealing of a cover plate to the planar substrate.
  • a microfluidic network is next discussed with reference to Fig. 4.
  • Sample is introduced into sample introduction port and reservoir 142 together with appropriate reagents for carrying out a microfluidic process.
  • An electric potential is applied across electrodes 140 and 144 causing medium containing the sample and other reagents to move through flowpath 120 and, in particular, portion 150 of 120. Mixing of sample and reagents, as well as incubation, take place in portion 150.
  • the electric potential applied between electrodes 140 and 144 is discontinued and an electric potential is applied between electrodes 130 and 134.
  • the point at which the sample and other reagents reach intersection 124 may be determined by detecting the presence of the sample or one of the reagents directly or by empirically determining the time at which the sample and reagents should reach the intersection 124, based on the particular nature of the sample, the medium employed, the strength of the electric potential and so forth.
  • Application of the electrical potential to electrodes 130 and 134 causes a plug of medium of precise amount (determined by the dimensions of the channel) to move along secondary flowpath 122 towards reservoir 136 and through detection zone 148 where detection is conducted.
  • This is the basic manner in which an exemplary microfluidic network operates.
  • the precise manner of operation of microfluidic networks in an apparatus in accordance with the present invention is dependent on the construction of the apparatus.
  • the upper voltage limit for commercial systems is 30 kV, with a capillary length of 40-60 cm, giving a maximum field of about 600 V/cm.
  • Normal polarity is to have the injection end of the capillary at a positive potential.
  • the electroosmotic flow is normally toward the cathode.
  • all positive ions and many negative ions will run away from the injection end.
  • the "end capillary" detector will be near the cathode.
  • the polarity may be reversed for strongly negative ions so that they run against the electroosmotic flow.
  • DNA typically the capillary is coated to reduce electroosmotic flow, and the injection end of the capillary is maintained at a negative potential.
  • Figs. 5-7 Examples of devices that are suitable for the second plate in the above-integrated apparatus are provided in Figs. 5-7. Only a portion of the microfluidic network plates is shown in Figs. 5-7. It is to be understood that the microfluidic network plates may have any number of separate networks including more than or less than 96. The number of microfluidic networks may be multiples of 96 where the number is greater than 96 or multiples of 8 where the number is less than 96. In addition, some of the features of the microfluidic networks are not shown in all of the networks depicted in Fig. 5-7.
  • a portion of a plate 210 is shown where the plate may have up to ninety-six (96) microfluidic networks 208.
  • Each network comprises main flowpath 220 and secondary flowpath 222, which intersect at 224.
  • Electrode 230 is connected to reservoir 232 and electrode 234 is connected to reservoir 236.
  • An electric potential can be applied to secondary flowpath 222 by means of electrodes 230 and 234.
  • Electrode 240 is connected to sample introduction port and reservoir 242 and electrode 244 is connected to reservoir 246.
  • An electric potential can be applied to main flowpath 220 by means of electrodes 240 and 244.
  • the main flowpath 220 has a portion 250 that is in the form of a linear reciprocating coil to provide a tortuous path.
  • a portion of a plate 310 is shown where the plate may have up to ninety-six (96) microfluidic networks 308.
  • Each network comprises main flowpath 320 and secondary flowpath 322, which intersect at 324.
  • Electrode 330 is connected to reservoir 332 and electrode 334 is connected to reservoir 336.
  • An electric potential can be applied to secondary flowpath 322 by means of electrodes 330 and 334.
  • Electrode 340 is connected to sample introduction port and reservoir 342 and electrode 344 is connected to reservoir 346.
  • An electric potential can be applied to main flowpath 320 by means of electrodes 340 and 344.
  • the main flowpath 320 is a circular coil to provide a tortuous path.
  • a portion of a plate 410 is shown where the plate may have up to ninety-six (96) microfluidic networks 408.
  • Each network comprises main flowpath 420 and secondary flowpath 422, which intersect at 424.
  • Electrode 430 is connected to reservoir 432 and electrode 434 is connected to reservoir 436.
  • An electric potential can be applied to secondary flowpath 422 by means of electrodes 430 and 434.
  • Electrode 440 is connected to sample introduction port and reservoir 442 and electrode 444 is connected to reservoir 446.
  • An electric potential can be applied to main flowpath 420 by means of electrodes 440 and 444.
  • the main flowpath 420 has a portion 450 that is in the form of a linear reciprocating coil to provide a tortuous path.
  • the microfluidic networks of plate of Fig. 6 also comprise set of reagent reservoirs 452, 454, 456 and 458.
  • Each of the reagent reservoirs has a channel providing communication between the reagent reservoir and each of the main flowpaths of the microfluidic networks.
  • reagent reservoir 452 has a channel 470 that intersects main flowpath 420 at 460 for each of the microfluidic networks in row 462 of plate 410.
  • reagent reservoir 454 has a channel 472 that intersects main flowpath 420 at 464 for each of the microfluidic networks in row 464 of plate 410.
  • Reagents are moved through channels 470 and 472 by means of application of electric potential at electrodes 480 and 482, respectively.
  • electric potential at electrodes 480 and 482 By appropriate alternation of electric potential in channels 470 and 472 on the one hand and main channel 420 on the other, precise amounts of reagents can be metered into main flowpath 420.
  • some or all of the electrodes may be within the second plate with external connections to power supplies that may be part of an instrument into which the present apparatus is inserted.
  • some or all of the electrodes might be on a separate part (e.g. built into an instrument into which the present apparatus is inserted) , such that the electrodes can be immersed into the appropriate fluid reservoirs at the time of use.
  • the electrodes in the separate instrument may be adapted to make contact with an appropriate lead from each of the reservoirs forming a part of the microfluidic networks in the subject apparatus.
  • the electrodes may be strip metal electrodes formed in a stamping process or chemical etching process.
  • the electrodes may be wires or strips either soldered or glued with epoxy and can be made of conductive materials such as platinum, gold, carbon fibers and the like.
  • the electrodes could be deposited, coated or plated onto a section of the exterior wall of a capillary near each end of the capillary. Controlled vapor deposition of gold, platinum, or palladium metal onto the exterior wall of the capillary is one method of forming the electrodes. This technique can be used to produce an electrode layer with a thickness up to several microns. Thicker electrodes could be subsequently formed by electrochemically plating gold, palladium or platinum onto the thin electrode formed by the vapor deposition process. Electrodes could be integral with the second plate formed by silk screening process, printing, vapor position, electrode-less plating process, etc. Carbon paste, conductive ink, and the like could be used to form the electrode.
  • the electrodes it is preferable for the electrodes to be connected to an electronic computer.
  • the computer has programmed software dedicated to providing the moving waves or voltage profile along the channel.
  • Various different types of software can be provided so as to obtain the best possible results in the particular microfluidic processing conducted.
  • the computer software that is connected to the electrodes be made interactive with an optical detection device such as ultraviolet or fluorescence spectrometer.
  • the spectrometer can be focused singly or at various points along the medium in the channels.
  • the information can be sent to the computer, which can adjust the speed of the waves or voltage distribution profiles being generated in order to more precisely fine tune the resolution of the substances being moved through the medium.
  • the channels can be in any shape. More specifically the channels can be fashioned so that it has a plurality of branches. Each of the branches along with the channel itself can be filled with a desired medium.
  • the integrated apparatus of the present invention may have any convenient configuration capable of comprising the first and second plates and their respective component parts.
  • the cavities and channels of the second plate are usually present on the surface of a planar substrate where the substrate will usually, though not necessarily be covered with a cover plate to seal the microfluidic networks present on the surface of the planar substrate from the environment.
  • the cover plate will have appropriate communication means for establishing communication between each of the sample receiving elements of the first plate and the corresponding microfluidic network of the second plate.
  • Such means include, for example, through-holes, capillaries, porous wicks and the like.
  • the apparatus may have a variety of configurations such as, for example, rectangular, circular, or other convenient configuration.
  • apparatus in accordance with the present invention are of a size that is readily handled and manipulated.
  • a rectangular apparatus has dimensions of about 3 inches by 5 inches;
  • a circular apparatus has a diameter of about 4 to 16 inches; and each would have a thickness of about 0.60 to 1.5 inches (including all of the elements of the apparatus).
  • the size of the present devices and apparatus is not critical and is in general a function of the particular multiwell plate with which the present device may be used.
  • the apparatus may be fabricated from a wide variety of materials, including glass, silica, quartz, ceramics and polymers, including elastomeric material, thermosets and thermoplastics, e.g., acrylics, and the like.
  • the various components of the apparatus may be fabricated from the same or different materials, depending on a number of factors such as, e.g., the particular use of the device, the economic concerns, solvent compatibility, optical clarity, color, mechanical strength, dielectric properties, e.g., dielectric strength greater than 100 V/cm, and so forth.
  • the planar substrate of the second plate may be fabricated from the same material as the cover plate, e.g., poly ethylmethacrylate, or from different materials such as, e.g., polymethylacrylate for the substrate and glass for the cover plate.
  • the first plate may be fabricated from the same material as the second plate, or one of the components of the second plate, e.g., glass bottom, glass top; plastic bottom, plastic cover, or from different materials such as, e.g., glass for the first plate and plastic for the second plate.
  • the device typically is fabricated from a plastic.
  • the entire apparatus may be fabricated from a plastic material that is optically transparent, which generally allows light of wavelengths ranging from 180 to 1500 nm, usually 220 to 800 nm, more usually 450 to 700nm, to have low transmission losses. Suitable materials include fused silica, plastics, quartz, glass, and so forth.
  • plastics having low surface charge under conditions or electroflow are plastics having low surface charge under conditions or electroflow.
  • plastics finding use include polymethyl methacrylate, polymethyl acrylate, polycarbonate, polyethylene terephthlate, polystyrene or styrene copolymers, polyesters, and the like.
  • the apparatus may be fabricated using any convenient means, including conventional molding and casting techniques, extrusion sheet forming, calendaring, thermoforming, and the like.
  • a silica mold master which is negative for the network structure in the planar substrate of the second plate, can be prepared by etching or laser micromachining.
  • the silica mold may have a raised area that provides for one or more cavity structures in the planar substrate.
  • a polymer precursor formulation can be thermally cured or photopolymerized between the silica master and support planar plate, such as a glass plate.
  • support planar plate such as a glass plate.
  • electrodes may be introduced where desired.
  • cavity structures or reservoirs may be formed by boring holes only part way through the substrate at the ends of the channels, so that the cavity structures are not open on the opposite surface of the second plate.
  • the substrate for the second plate may take a variety of shapes such as, for example, disk-like, card-like, and may be a layered or laminated sandwich structure.
  • the substrate for the second plate is usually about 1 ⁇ m thick, usually at least about 5 ⁇ m , and more usually at least about 50 ⁇ m thick, where the thickness may be as great as 5 mm or greater.
  • the second plate may be constructed from two or more parts, usually two parts, e.g., a base plate and a cover plate. Each part generally has a planar surface and the parts are sealed together so that the planar surfaces are opposed.
  • the planar surface of the base plate usually includes one or more cavity structures and channels, while the planar surface of the cover plate may or may not include one or more cavity structures and channels.
  • the cover plate is usually placed over, and sealed to, the surface of the substrate of the base plate.
  • the cover plate may be sealed to the substrate using any convenient means, including ultrasonic welding, adhesives, etc.
  • the cover may be a more or less rigid plate, or it may be a film, and the thickness of the cover may be different for materials having different mechanical properties. Usually the cover ranges in thickness from at least about 200 ⁇ , more usually at least about 500 ⁇ m, to as thick as usually about 5 mm or thicker, more usually about 2 mm.
  • the cover substrate may be fabricated from a single material or be fabricated as a composite material. In some instances the cover is of a plastic material, and it may be rigid or elastomeric.
  • the apparatus may have multiple layers that are sandwiched together similar to multiple layer electronic printed circuit boards.
  • the apparatus may be made in a manner similar to the printed circuit boards.
  • Each layer contains cavities, channels and through-holes.
  • the channels and through-holes in each layer can interconnect forming three dimensional fluid circuits. This approach allows significantly greater circuit complexity and circuit density than the single layer approach.
  • Another approach for the transfer of liquids from the first plate to the second plate of the present apparatus involves a plurality of active liquid transfer elements corresponding to each well of a multiwell plate. Upon activation of the active liquid transfer elements, an amount of liquid from the well of the well plate is actively transferred to a microfluidic network of the second plate through a corresponding through hole in the second plate.
  • active liquid transfer elements include capillary droplet ejectors that are driven mechanically, electrically, pneumatically, thermally, and so forth, and capillary forces and surface tension, hydrodynamics, and the like.
  • the apparatus in Fig. 8 comprises first plate 700 and second plate 710.
  • the first plate 700 that has an array of sample receiving elements 702 in the form of capillaries for taking up a plurality of samples from an array of sample containers.
  • the array of capillaries is aligned so that they correspond with wells 750 of a multiwell plate 752 that contain samples.
  • an aliquot of sample is transferred to capillary 702 by means of capillary action. Transfer of this aliquot to a microfluidic network 712 of the second plate is realized by applying an electrical potential using electrodes 720 and 722.
  • a pulse of liquid 714 which is a precise amount of the sample, is sprayed into a sample receiving reservoir 730 of the microfluidic network.
  • the electrical potential applied is on the same order of magnitude as that described above to achieve electroflow.
  • the electrical potential is about 100 V/cm to 5 kV/cm, preferably about 250 V/cm to 500 V/cm.
  • second plate 710 is secured above first plate 700 so that a space generally designated 740 lies in between.
  • the space 740 is usually about 0.5 to 3mm.
  • the apparatus of Fig. 8 may be constructed so as to achieve the spatial relationship between plates 700 and 710. Spacer elements may be employed in the manufacture of the apparatus.
  • the apparatus may also be designed to be assembled by the user into an integral device prior to contact with a multiwell plate.
  • Various interlocking means may be utilized to secure second plate 710 to first plate 700.
  • Such means include, for example, tongue and groove, or post and pit alignment with elastomeric gaskets or clamps, integral molded spring clips, and the like.
  • plate 710 can be separate from plate 700 in the device of Fig. 8.
  • plate 710 is positioned, by some mechanical or electromechanical means, above plate 700, which is first positioned above multiwell plate 752. Liquid is then transferred from multiwell plate 752 to sample receiving reservoir 730. After the transfer is complete, plate 710 can be moved away and further processing of the liquid can be carried out in the microfluidic networks of plate 710.
  • FIGs. 9-12 Other examples of various means for achieving liquid transfer from a capillary to a microfluidic network in the second plate are depicted in Figs. 9-12.
  • such means may be provided as a separate part, along with other parts, of an apparatus in accordance with the present invention. These parts may be assembled by the user.
  • intermediate plates may also be included between the first and second plates.
  • Such intermediate plates include, for example, a plate with filters spaced apart corresponding to the sample receiving elements.
  • the user would have the ability to carry out manipulations other than sample transfer such as a filtration of the sample and the like.
  • first plate 700 as a separate plate from second plate 710 can be used to transfer liquids to a plate other than one containing microfluidic networks. Separate first plate 700 can be employed to transfer samples to a planar surface in the form of an array of spots on which chemical synthesis or analysis can be conducted. As will be appreciated, first plates in Figs. 9-12 below can also be used separately in this fashion.
  • the apparatus of Fig. 9 is similar to that in Fig. 8 except that piezoelectric collar 790 is present in place of the electrodes of Fig. 8.
  • the pulse of liquid is generated by piezoelectric collar 790.
  • the piezoelectric collar 790 when actuated, rapidly constricts the capillary near the upper end resulting in the release of liquid in the capillary.
  • the apparatus comprises first plate 800 and second plate 810.
  • First plate 800 includes sealing membrane 804 through which a plurality of capillaries 802 extends.
  • First plate 800 also includes a plurality of plungers 806 that are slidably positioned in first plate 800. Each of the plungers are situated near a respective capillary.
  • Second plate 810 is secured above first plate 800 in a biased relationship so that second plate 810 may be reciprocally moved in the direction of first plate 800. In this way plungers 806 may be simultaneously depressed by depressing plate 810.
  • the space 840 between the first and second plates may contain suitable biasing elements such as springs, elastomeric substances, e.g., polydimethyl siloxane (PDMS) , and so forth.
  • PDMS polydimethyl siloxane
  • Figs. 11 and 12 utilize heat from heater 876 and compressed gas from tube 886, respectively, in place of the pressure achieved with plunger 806 of Fig. 10.
  • kits for processing a sample comprises an apparatus as described above and reagents, other than reagents within the apparatus, for processing a sample.
  • the reagents for the kits may be packaged in the same or separate containers, so that the concentration of the reagents provides for substantial optimization of the method and assay.
  • the reagents may each be in separate containers or various reagents can be combined in one or more containers depending on the cross-reactivity and stability of the reagents.
  • the reagents in the kit can be provided as a dry powder, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for performing a method or assay in accordance with the present invention.
  • the kit can also include additional reagents depending on the nature of the method for which the kit is used.
  • the kit may include solid phase extraction materials including paramagnetic beads and non-magnetic particles, lysis solutions, wash and elution and running buffers, biomolecular recognition elements including receptors, enzymes, antibodies and other specific binding pair members, labeling solutions, substrates, reporter molecules, sample purification materials including membranes, beads, and the like, and so forth.

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  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
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  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP98948072A 1997-09-19 1998-09-15 Vorrichtung und verfahren zur kapillarströmung mit einem elektrischen feld Withdrawn EP1019712A4 (de)

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US6758097P 1997-09-19 1997-09-19
US67580P 1997-09-19
PCT/US1998/018249 WO1999015888A1 (en) 1997-09-19 1998-09-15 Capillary electroflow apparatus and method

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AU9472198A (en) 1999-04-12
EP1019712A1 (de) 2000-07-19
AU753307B2 (en) 2002-10-17

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