WO2000069560A1 - Systeme microfluidique a fonctionnement centripete destine a effectuer une hybridation in vitro et une amplification des acides nucleiques - Google Patents

Systeme microfluidique a fonctionnement centripete destine a effectuer une hybridation in vitro et une amplification des acides nucleiques Download PDF

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Publication number
WO2000069560A1
WO2000069560A1 PCT/US2000/013131 US0013131W WO0069560A1 WO 2000069560 A1 WO2000069560 A1 WO 2000069560A1 US 0013131 W US0013131 W US 0013131W WO 0069560 A1 WO0069560 A1 WO 0069560A1
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WO
WIPO (PCT)
Prior art keywords
platform
microchannels
heating elements
microsystem
fluid
Prior art date
Application number
PCT/US2000/013131
Other languages
English (en)
Inventor
Gregory Kellogg
Bruce L. Carvalho
Norman F. Sheppard, Jr.
Kevin E. Noonan
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Gamera Bioscience Corporation
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Filing date
Publication date
Application filed by Gamera Bioscience Corporation filed Critical Gamera Bioscience Corporation
Priority to AU47134/00A priority Critical patent/AU4713400A/en
Publication of WO2000069560A1 publication Critical patent/WO2000069560A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • 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
    • 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
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00356—Holding samples at elevated temperature (incubation)
    • G01N2035/00366—Several different temperatures used

Definitions

  • This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures.
  • the invention relates to microminiaturization of genetic, biochemical and bioanalytic processes.
  • the invention relates to performing bioanalytic and biotechnological processes that are dependent on incubating samples and solutions at different temperatures, and particularly reactions that require sequential sample incubation at alternating temperatures, including in vitro amplification reactions and most particularly the polymerase chain reaction.
  • Methods for performing any of a wide variety of such microanalytical or microsynthetic processes using the microsystems apparatus of the invention are also provided.
  • PCR polymerase chain reaction
  • the polymerase chain reaction comprises repeated rounds, or cycles, of target denaturation, primer annealing, and polymerase-mediated extension; the reaction process yields an exponential amplification of a specific target sequence.
  • Methods for miniaturizing and automating PCR are desirable in a wide variety of analytical contexts, particularly under conditions where a large multiplicity of samples must be analyzed simultaneously or when there is a small amount of sample to be analyzed.
  • PCR and other temperature-dependent bioanalytic reactions has been the difficulty in designing systems for moving fluids on the microchips through channels and reservoirs having diameters in the 10-100 ⁇ m range. This is due in part to the need for high-pressure pumping means for moving fluid through the small sizes of the components of these microchips. These disabilities of the prior art microchips limits the usefulness of these devices for miniaturizing and automating PCR and other bioanalytic processes.
  • Some of the present inventors have developed a microsystem platform and a micromanipulation device to manipulate said platform by rotation, thereby utilizing the centripetal forces resulting from rotation of the platform to motivate fluid movement through microchannels embedded in the microplatform, as disclosed in co-owned U.S. Patent No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Serial Nos. 08/761,063, filed December 5, 1996; 08/768,990, filed December 18, 1996; 08/910,726, filed August 12, 1997; 08/995,056, filed December 19, 1997; and 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
  • Patent No. 6,063,589 issued May 16, 2000, and co-owned and co-pending patent applications U.S. Serial Nos. 08/761,063, filed December 5, 1996; 08/768,990, filed December 18, 1996; 08/910,726, filed August 12, 1997; 08/995,056, filed December 19, 1997; and 09/315,1 14, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein.
  • the invention provides apparatus and methods for performing microscale processes on a microplatform, whereby fluid is moved on the platform in defined channels motivated by centripetal force arising from rotation of the platform.
  • the first element of the apparatus of the invention is a microplatform that is a rotatable structure, most preferably a disk, the disk comprising fluid (sample) inlet ports, fluidic microchannels, reagent reservoirs, reaction chambers, detection chambers and sample outlet ports, generically termed "microfluidic structures,” and most preferably heating elements comprising a portion of the surface area of the platform for heating fluids contained therein to temperatures greater than ambient temperature.
  • the disk is rotated at speeds from about 1-30,000 rpm for generating centripetal acceleration that enables fluid movement through the microfluidic structures of the platform.
  • the disks of the invention also preferably comprise air outlet ports and air displacement channels.
  • the air outlet ports and in particular the air displacement ports provide a means for fluids to displace air, thus ensuring uninhibited movement of fluids on the disk.
  • Specific sites on the disk also preferably comprise elements that allow fluids to be analyzed, as well as detectors for each of these effectors. Alternatively, some or all of these elements can be contained on a second disk that is placed in optical or direct physical contact, most preferably thermal contact, with the first platform disk.
  • the second element of the invention is a micromanipulation device that is a disk player/ reader device that controls the function of the disk.
  • This device comprises mechanisms and motors that enable the disk to be loaded and rotated.
  • the device provides means for a user to operate the microsystems in the disk and access and analyze data, preferably using a keypad and computer display.
  • the invention specifically provides microsystems platforms comprising regions of the platform surface that comprise or are in thermal contact with heating elements, wherein fluids contained in such regions, most preferably in microchannels as defined herein, are efficiently heated to and maintained at temperatures greater than ambient for a time that is proportional to the length of the longitudinal extent or path of the microchannel in the region, the viscosity of the fluid and inversely proportional to the product of the square of the hydraulic diameter of the microchannel, the square of the rotational speed of the platform, the average distance of the fluid in the channels from the center of the disk and the radial extent of the fluid subject to the centripetal force.
  • fluids with defined viscosities one combine the controlled rotational speed of the platform with engineered or designed microchannels to control the flow rate of fluids within the disk.
  • said regions of the platform comprising or in thermal contact with a heating element or multiplicity thereof comprise an array of heating elements that can be operated at different temperatures.
  • the proportion of time that a fluid contained in said region, most preferably in a microchannel, is maintained at a particular temperature is directly proportional to the longitudinal extent of the microchannel in the region.
  • the path of the microchannel on the surface of the platform in the region comprising or in thermal contact with a heating element or thermal array thereof as described herein can be straight, curved, spiral, "zig-zag,” or meandering, and the path can repeatedly enter and leave any particular temperature region on the disk.
  • This arrangement of heating elements into thermal arrays and transit of microchannels through such arrays provides for incubating fluid contained in such microchannels at different temperatures without having to use the heating elements to actively change the temperature of the platform.
  • This arrangement permits the establishment of regions of the platform that are maintained at any particular temperature greater than ambient, whereby heating and incubating a fluid is dependent on the length of the portion of the microchannel in that region of the platform, and hence the amount of time the fluid spends in that region as it transits the microchannel under the influence of centripetal force from rotation of the platform.
  • thermal array comprises a region of the platform comprising or in thermal contact with a single heating element.
  • the temperature of fluids contained in such regions is alternated by movement of the fluid in the microchannel between an elevated temperature from operation of the heating element and the ambient temperature of the platform by traversal through a microchannel that enters and leaves the area comprising or in thermal contact with the heating element.
  • the proportion of the time that the fluid is maintained at the elevated temperature is directly proportional to the extent of the longitudinal path of the microchannel in the region comprising or in thermal contact with the heating element.
  • Heating elements on the platforms of the invention encompassed by this aspect can produce elevated temperatures from 37°C to about 95°C, and the absolute amount of time the fluid is kept at the elevated temperature can be varied with platform rotational speed.
  • the relative amount of time that the fluid is maintained at the elevated temperature is dependent on the geometry of the microchannel path on the platform, the average distance of the fluid in the channels from the center of the disk, the radial extent of the fluid subject to the centripetal force and the rotation rate of the platform and can be made invariant for any particular geometry in any particular embodiment of the platforms of this aspect of the invention by the judicious combination of these variables.
  • Another embodiment of such an thermal array comprises a region of the platform comprising or in thermal contact with two heating elements, which will be understood to encompass embodiments having a single heating element in thermal contact with two discrete regions of the platform.
  • the areas of the platform in contact with each of the two heating elements are immediately adjacent to one another.
  • the two different areas can be operated at two different temperatures greater than the ambient temperature of the platform.
  • the temperature of fluids contained in such regions most preferably in microchannels, is alternated between each of the two elevated temperatures produced by operation of the heating elements by traversal through a microchannel that enters and leaves the each area comprising or in thermal contact with each of the heating elements.
  • the proportion of the time that the fluid is maintained at each of the elevated temperatures is directly proportional to the extent of the longitudinal path of the microchannel in the region comprising or in thermal contact with each of the heating elements.
  • Platforms of the invention encompassed by this aspect can be used wherein each of the areas is independently maintained at elevated temperatures from 37°C to about 95 °C by the heating elements, and the amount absolute amount of time the fluid is kept at each of the elevated temperature can be varied with platform rotational speed.
  • the thermal array comprises an area of the platform comprising or in thermal contact with more than two, most preferably three, heating elements, wherein the temperatures of each of these areas can be independently maintained at a different temperature greater than the ambient temperature of the platform.
  • each of the areas of the platform in contact with each of the heating elements are immediately adj acent to one another.
  • the three different areas can be operated at three different temperatures greater than the ambient temperature of the platform.
  • the temperature of fluids contained in such regions, most preferably in microchannels is alternated between each of the elevated temperatures from operation of the heating elements by traversal through a microchannel that enters and leaves each area comprising or in thermal contact with each of the heating elements.
  • the proportion of the time that the fluid is maintained at each of the elevated temperatures is directly proportional to the extent of the longitudinal path of the microchannel in the region comprising or in thermal contact with each of the heating elements.
  • Platforms of the invention encompassed by this aspect can be used wherein each of the areas is independently maintained at elevated temperatures from 37°C to about 95°C, and the absolute amount of time the fluid is kept at each of the elevated temperature can be varied with platform rotational speed.
  • the relative amount of time that the fluid is maintained at each of the elevated temperature is dependent on the geometry of the microchannel path on the platform, the average distance of the fluid in the channels from the center of the disk, the radial extent of the fluid subject to the centripetal force and the rotation rate of the platform and can be made invariant for any particular geometry in any particular embodiment of the platforms of this aspect of the invention by the judicious combination of these variables.
  • the thermal array comprises two or more areas of the platform surface comprising or in thermal contact with a heating element, wherein at least one of said areas is separated from another area by an area that does not comprise and is not in thermal contact with a heating element. This intervening area of the array is maintained at the ambient temperature of the platform.
  • a microchannel is constructed in the thermal array to traverse both the areas comprising or in thermal contact with the heating elements, and the area or areas that do not comprise or are not in thermal contact with a heating element. The proportion of time that fluid contained in such microchannels is at an elevated temperature or ambient temperature is directly proportional to the extent of the longitudinal path of the microchannel in the areas that do or do not comprise or are or are not in thermal contact with each of the heating elements.
  • heat originates from a single face of the platform, most preferably from a position underneath the micro fluidics components.
  • a fluidic disk or manifold is positioned between two thermal regulation layers each comprising heating elements to provide heating on both surfaces of the microfluidic disk.
  • the microsystems platforms of the invention comprise a multiplicity of the same or different embodiments of the thermal arrays described herein.
  • each of the microchannels traversing said arrays is in fluid contact with one or a multiplicity of sample input ports or fluid holding chambers comprising a biological sample or reaction mixture.
  • each microchannel is also fluidly connected with a collection chamber, most preferably wherein each microchannel is uniquely in fluid contact with a particular collection chamber.
  • the thermal arrays and regions of elevated temperatures constructed in the surface of the platforms of the invention comprise a thermal heating element.
  • the thermal heating element is a resistive heater element.
  • the invention also provides methods for using the microsystems platform for performing any bioanalytical or biosynthetic reaction that requires or advantageously includes the step of incubating a biological sample or reaction mixture at one or a multiplicity of temperatures greater than ambient temperature, and more preferably wherein incubation of the biological sample or reaction mixture at one or a multiplicity of temperatures greater than ambient temperature is performed sequentially or repetitively or both.
  • the bioanalytical or biosynthetic reaction is nucleic acid hybridization, polymerase chain reaction or ligase chain reaction.
  • Figure 1 depicts an oblique view of a microsystems platform of the invention.
  • Figure 2 depicts a plan view of the microsystems platform shown in oblique view in Figure 1.
  • Figure 3 is a cross-sectional view of the microsystems platform shown in Figures 1 and 2.
  • Figure 4 shows a plan view of a microsystems platform comprising a multiplicity of fluidic and thermal regulatory structures for performing a nucleic acid hybridization reaction.
  • Figure 5 shows a plan view of a microsystems platform adapted for performing an in vitro amplification reaction.
  • Figure 6 is a cross-sectional view of the platform shown in Figure 5.
  • Figure 7 shows the arrangement of twelve fluidic structures as depicted in Figure 5 around the interior diameter of a disc.
  • This invention provides a microplatform and a micromanipulation device as disclosed in co-owned U.S. Patent No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Serial Nos. 08/761,063, filed December 5, 1996; 08/768,990, filed December 18, 1996; 08/910,726, filed August 12, 1997; 08/995,056, filed December 19, 1997; 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein, adapted for performing microanalytical and microsynthetic assays of biological samples.
  • sample will be understood to encompass any fluid, solution or mixture, either isolated or detected as a constituent of a more complex mixture, or synthesized from precursor species.
  • sample will be understood to encompass any fluid, solution or mixture, either isolated or detected as a constituent of a more complex mixture, or synthesized from precursor species.
  • sample will be understood to encompass any biological species of interest, and preferably nucleic acid, and most preferably a nucleic acid in a solution wherein detection or amplification of said nucleic acid is accomplished.
  • biological sample or “biological fluid sample” will be understood to mean any biologically-derived analytical sample, including but not limited to blood, plasma, serum, lymph, saliva, tears, cerebrospinal fluid, urine, sweat, plant and vegetable extracts, semen, and ascites fluid.
  • a centripetally motivated fluid micromanipulation apparatus is intended to include analytical centrifuges and rotors, microscale centrifugal separation apparatuses, and most particularly the microsystems platforms and disk handling apparatuses as described in co-owned U.S. Patent No.
  • microsystems platform is intended to include centripetally-motivated micro fluidics arrays as described in co-owned U.S. Patent
  • microchannel will be understood to be interchangeable and to be constructed of either wetting or non-wetting materials where appropriate.
  • fluid chamber for the purposes of this invention, the term "fluid chamber,” “fluid holding chamber” and “collection chamber” will be understood to mean a defined volume on a microsystems platform of the invention comprising a fluid.
  • the terms "entry port” and “fluid input port” will be understood to mean a defined volume on a microsystems platform of the invention comprising a means for applying a fluid to the platform.
  • the terms “exit port” and “fluid outlet port” will be understood to mean a defined volume on a microsystems platform of the invention comprising a means for removing a fluid from the platform.
  • the term “capillary junction” will be understood to mean a junction of two components wherein one or both of the lateral dimensions of the junction are larger than the corresponding dimensions the capillary. In wetting or wettable systems, such junctions are where capillary valving occurs, because fluid flow through the capillaries is stopped at such junctions. In non-wetting or non-wettable junctions, the exit from the chamber or reservoir is where the capillary junction occurs.
  • capillary junctions are formed when the dimensions of the components change from a small diameter (such as a capillary) to a larger diameter (such as a chamber) in wetting systems, in contrast to non-wettable systems, where capillary junctions form when the dimensions of the components change from a larger diameter (such as a chamber) to a small diameter (such as a capillary).
  • capillary action will be understood to mean fluid flow in the absence of rotational motion or centripetal force applied to a fluid on a rotor or platform of the invention.
  • capillary microvalve will be understood to mean a capillary microchannel comprising a capillary junction whereby fluid flow is impeded and can be motivated by the application of pressure on a fluid, typically by centripetal force created by rotation of the rotor or platform of the invention.
  • the term "sacrificial valve” will be understood to mean a valve preferably made of a fungible material that can be removed from the fluid flow path.
  • said sacrificial valves are wax valves and are removed from the fluid flow path by heating, using any of a variety of heating means including infrared illumination and most preferably by activation of heating elements on or embedded in the platform surface as described in co-owned U.S. Patent No. 6,063,589, incorporated by reference.
  • the term “in fluid communication” or “fluidly connected” is intended to define components that are operably interconnected to allow fluid flow between components.
  • the platform comprises a rotatable platform, more preferably a disk, whereby fluid movement on the disk is motivated by centripetal force upon rotation of the disk.
  • air displacement channels will be understood to include ports in the surface of the platform that are contiguous with the components (such as microchannels, chambers and reservoirs) on the platform, and that comprise vents and microchannels that permit displacement of air from components of the platforms and rotors by fluid movement.
  • microplatforms of the invention are provided to comprise one or a multiplicity of microsynthetic or microanalytic systems.
  • Such microsynthetic or microanalytic systems in turn comprise combinations of related components as described in further detail herein that are operably interconnected to allow fluid flow between components upon rotation of the disk.
  • These components can be microfabricated as described below either integral to the disk or as modules attached to, placed upon, in contact with or embedded in the disk.
  • microfabricated refers to processes that allow production of these structures on the sub-millimeter scale.
  • the invention also comprises a micromanipulation device for manipulating the disks of the invention, wherein the disk is rotated within the device to provide centripetal force to effect fluid flow on the disk. Accordingly, the device provides means for rotating the disk at a controlled rotational velocity, for stopping and starting disk rotation, and advantageously for changing the direction of rotation of the disk.
  • electromechanical means and control means are provided as components of the devices of the invention.
  • User interface means (such as a keypad and a display) are also provided, as further described in co-owned U.S. PatentNo.6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Serial Nos.
  • the invention provides a combination of specifically-adapted microplatforms that are rotatable, analytic/synthetic micro volume assay platforms (collectively referred to herein as a "disk” or “disc”), and a micromanipulation device for manipulating the platform to achieve fluid movement on the platform arising from centripetal force on the platform as result of rotation.
  • the platform of the invention is preferably and advantageously a circular disk; however, any platform capable of being rotated to impart centripetal for a fluid on the platform is intended to fall within the scope of the invention.
  • the disk incorporates microfabricated mechanical, optical, and fluidic control components on platforms made from, for example, plastic, silica, quartz, metal or ceramic. These structures are constructed on a sub-millimeter scale by molding, photolithography, etching, stamping or other appropriate means.
  • Platforms of the invention such as disks and the components comprising such platforms are advantageously provided having a variety of composition and surface coatings appropriate for a particular application.
  • Platform composition will be a function of structural requirements, manufacturing processes, and reagent compatibility/chemical resistance properties.
  • platforms are provided that are made from inorganic crystalline or amorphous materials, e.g. silicon, silica, quartz, inert metals, or from organic materials such as plastics, for example, poly(methyl methacrylate) (PMMA), acetonitrile-butadiene-styrene (ABS), polycarbonate, polyethylene, polystyrene, polyolefms, polypropylene and metallocene.
  • PMMA poly(methyl methacrylate)
  • ABS acetonitrile-butadiene-styrene
  • PCC polycarbonate
  • polyethylene polystyrene
  • polyolefms polypropylene and metallocene
  • platforms made of composites or combinations of these materials, for example, platforms manufactures of a plastic material having embedded therein an optically transparent glass surface comprising for example the detection chamber of the platform.
  • the surface properties of these materials may be modified for specific applications, as disclosed in co-owned U.S. PatentNo.6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Serial Nos. 08/761 ,063, filed December
  • the platforms of the invention can be used to perform any microanalytical and microsynthetic reaction requiring that the sample be incubated at one or a multiplicity of temperatures greater than the ambient temperature of the disk for a controlled amount of time.
  • the microsystems platforms of the invention are adapted for performing nucleic acid hybridization assays and in vitro amplification reactions, such as the polymerase chain reaction or ligase chain reaction, using centrifugal action to move a fluid reaction mixture between various temperature controlled regions on a microfluidics disc.
  • the reaction mixture remains at one location and the temperature of this location is typically cycled between a denaturation temperature (> 90°C), an annealing temperature (which varies with the sequence complexity, length and hybridization fidelity of the oligonucleotide primers) and an extension temperature (usually around 70°C for reactions performed with Taq polymerase).
  • a denaturation temperature > 90°C
  • an annealing temperature which varies with the sequence complexity, length and hybridization fidelity of the oligonucleotide primers
  • an extension temperature usually around 70°C for reactions performed with Taq polymerase.
  • Typical thermal cyclers are able to heat and cool at rates between 1 °C/sec and 2°C/sec, with a temperature accuracy and precision of approximately +/- 1°C.
  • microsystem platforms of the invention do not require active temperature changes of any particular region of the platform such as a reaction chamber.
  • thermal arrays comprising adjacent regions of different temperature are provide by the platforms of the invention, so that temperature changes are effected by transit of a fluid through a microchannel from one temperature region to another region at a different temperature.
  • the amount of time a fluid is maintained at a particular temperature is determined by the transit time of the fluid through any particular region of a thermal array at a particular temperature, which is proportional to the longitudinal extent or length of the microchannel in that temperature region.
  • the present invention provides components of the microsystems platform that can perform at levels sufficiently equivalent to these specifications as to enable microminiaturization of in vitro amplification reactions on such platforms.
  • Fluid (including reagents, samples and other liquid components) movement is controlled by centripetal acceleration due to rotation of the platform.
  • the magnitude of centripetal acceleration required for fluid to flow at a rate and under a pressure appropriate for a particular microsystem is determined by factors including but not limited to the effective radius of the platform, the interior diameter of microchannels, the position angle of the microchannels on the platform with respect to the direction of rotation, and the speed of rotation of the platform.
  • a metered amount of the fluid sample applied to the platform is transferred from a fluid holding chamber to a microchannel as described in co-owned U.S. Patent No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Serial Nos.
  • the metered amount of the fluid sample provided on an inventive platform is from about lO ⁇ L to about 500 ⁇ L.
  • microchannels comprising the surface of the platforms of the invention.
  • MicroChannel sizes are optimally determined by specific applications and by the amount of delivery rates required for each particular embodiment of the platforms and methods of the invention. MicroChannel sizes can range from 0.1 ⁇ m to a value close to the thickness of the disk (e.g., 1mm). In preferred embodiments, the interior dimension of the microchannel is from 1 to about 200 ⁇ m, more preferably about 50 ⁇ m for microchannels having a circular aspect and 50 ⁇ m x 50 ⁇ m for microchannels having a square aspect. MicroChannel and reservoir shapes can be trapezoid, circular or other geometric shapes as required.
  • Microchannels preferably are embedded in a microsystem platform having a thickness of about 0.1 to 100mm, wherein the cross-sectional dimension of the the microchannels across the thickness dimension of the platform is less than 500 ⁇ m and can be from 1 to 90 percent of said cross-sectional dimension of the platform.
  • Reagent reservoirs, reaction chambers, detections chambers and sample inlet and outlet ports preferably are embedded in a microsystem platform having a thickness of about 0.1 to 100mm, wherein the cross-sectional dimension of the the microchannels across the thickness dimension of the platform is from 1 to 75 percent of said cross-sectional dimension of the platform.
  • delivery of fluids through such channels is achieved by the coincident rotation of the platform for a time and at a rotational velocity sufficient to motivate fluid movement between the desired components.
  • the flow rate through a channel is inversely proportional to the length of the longitudinal extent or path of the microchannel in the region and the viscosity of the fluid and proportional to the product of the square of the hydraulic diameter of the microchannel, the square of the rotational speed of the platform, the average distance of the fluid in the channels from the center of the disk and the radial extent of the fluid subject to the centripetal force. Since the hydraulic diameter of a channel is proportional to the ratio of the cross-sectional area to cross-sectional perimeter of a channel, one can judiciously vary the depth and width of a channel to affect the flow.
  • Input and output (entry and exit) ports are components of the microplatforms of the invention that are used for the introduction of removal of a variety of fluid components.
  • Entry ports are provided to allow samples and reagents to be placed on or injected onto the disk; these types of ports are generally located towards the center of the disk. Exit ports are also provided to allow products to be removed from the disk. Port shape and design vary according specific applications. For example, sample input ports are designed, inter alia, to allow capillary action to efficiently draw the sample into the disk. In addition, ports can be configured to enable automated sample/reagent loading or product removal. Entry and exit ports are most advantageously provided in arrays, whereby multiple samples are applied to the disk or to effect product removal from the microplatform.
  • air displacement channels are also included in air handling systems on the disk, whereby the movement of fluids displaces air through channels that connect to the fluid- containing microchannels retrograde to the direction of movement of the fluid, thereby providing a positive pressure to further motivate movement of the fluid.
  • Temperature control elements are provided to control the temperature of the platform during incubation of a fluid thereupon.
  • the invention therefor provides heating elements, including heat lamps, direct laser heaters, Peltier heat pumps, resistive heaters, ultrasonication heaters and microwave excitation heaters, and cooling elements, including Peltier devices and heat sinks, radiative heat fins and other components to facilitate radiative heat loss.
  • Thermal devices are preferably arrayed to control the temperature of the platform over a specific area or multiplicity of areas.
  • heating and cooling elements comprise the platforms of the invention comprising a thermal regulation layer in the platform surface that is in thermal contact with the microfluidics components, most preferably microchannels as described herein.
  • the temperature of any particular area on the platform is monitored by resistive temperature devices (RTD), thermistors, liquid crystal birefringence sensors or by infrared interrogation using IR-specific detectors, and can be regulated by feedback control systems.
  • RTD resistive temperature devices
  • thermistors thermoelectric sensors
  • liquid crystal birefringence sensors liquid crystal birefringence sensors
  • infrared interrogation using IR-specific detectors can be regulated by feedback control systems.
  • Temperature control on the microsystems platforms of this invention is most preferably achieved using the methods and devices disclosed in co-owned U.S. Patent No. 6,063,589, incorporated by reference herein.
  • portions of the microsystems platform surface are adapted for providing regions of controlled temperature (termed “thermal regions” or “thermal arrays” herein) using integral heating elements as disclosed in U.S. Patent 6,063,589, incorporated by reference.
  • the portions of the microsystems platform surface are constituted in arrays of thermal control elements, most preferably wherein is produced adjacent regions of the platform surface having different temperatures.
  • the platform also comprises other components as disclosed in co-owned and co-pending patent applications U.S. SerialNos. 08/761,063, filed December 5, 1996; 08/768,990, filed December 18, 1996; 08/910,726, filed August 12, 1997; 08/995,056, filed December 19, 1997; 09/315,114, filed May 19, 1999, the disclosures of each of which are explicitly incorporated by reference herein, most preferably channels and microchannels, whereby fluid flow traverses each of the different regions having different temperatures at least once, or more preferably, several times.
  • the amount of time fluid is within any particular thermal region, and thus at any particular temperature is dependent on the path length of the channel in the region, the square of the hydraulic diameter of the channel, and the square of the rotational speed of the platform.
  • the arrays comprise at least 2 or 3 regions of different temperature adjacent to one another.
  • the thermal regions are rectangular in shape, while in other embodiments the thermal regions are wedge-shaped, having a broader annular diameter at positions distal to the axis of rotation than at positions proximal to the axis of rotation.
  • the thermal arrays and regions of elevated temperatures constructed in the surface of the platforms of the invention comprise a thermal heating element.
  • the thermal heating element is a resistive heater element or a thermofoil heater, which is an etched-foil heating element enclosed in an electrically insulating plastic (Kapton, obtained from Minco). Resistive heater elements comprising the platforms of the invention are as described in co-owned U.S. Patent 6,063,587.
  • said resistive heater elements comprise in combination an electrically inert substrate capable of being screen printed with a conductive ink and a resistive ink; a conductive ink screen-printed in a pattern; and a resistive ink screen-printed in a pattern over the conductive ink pattern wherein the resistive ink in electrical contact with the conductive ink and wherein an electrical potential applied across the conductive ink causes current to flow across the resistive ink wherein the resistive ink produces heat.
  • Such structures are defined as “electrically-resistive patches" herein.
  • the conductive ink is a silver conductive ink such as Dupont 5028, Dupont 5025, Acheson 423SS, Acheson 426SS and Acheson SS24890, and the resistive ink is, for example, Dupont 7082, Dupont
  • the resistive heater element can further comprise a dielectric ink screen-printed over the resistive ink pattern and conductive ink pattern.
  • the time for fluid to reach an equilibrium temperature can be fast.
  • the time for fluid to reach an equilibrium temperature can be approximated by the square of the channel depth divided by the thermal diffusivity of the fluid.
  • the thermal diffusivity is about 1.44 x 10 "4 cm 2 /sec and for a channel 50 ⁇ m deep (i.e., 5 x 10 "3 cm), the time required to reach thermal equilibrium is calculated to be about 0.2 sec.
  • Figure 1 shows an oblique view
  • Figure 2 shows a plan view
  • Figure 3 shows a cross- sectional view of a platform of the invention.
  • Fluidic manifold layer 100 comprises the microfluidic components of the platform including a fluid input port and input chamber 101, microchannel 102 and fluid collection chamber 103.
  • Thermal regulation layer 113 comprises the heater elements of the platform, including a pair of electrically conductive leads 107 and 108 connected to electrically resistive patch 106.
  • Fiducial marks 104, 105, 109 and 110 are use to align the manifold layer 100 and thermal regulation layer 113. When fiducial mark 104 is aligned with 109 and fiducial mark 105 is aligned with 110, the two layers are pressed together resulting in the plan view shown in Figure 2. When so assembled, the layers rotate together about a spindle inserted in the inner hole 111.
  • Fluid input port 200 is fluidly connected to fluid holding chamber 201 having a volumetric capacity of from about 1 to about 200 ⁇ L.
  • the fluid input port has a sufficient diameter to accommodate a standard 200 ⁇ L plastic pipette tip.
  • the fluid holding chamber 201 has a diameter of from about 0.3 to about 1cm and a depth in the platform surface of from about 0.02 to about 1cm.
  • Air displacement channel 202 having a cross-sectional dimension of from about 100 to about 500 ⁇ m is connected to fluid holding chamber 201 to permit displacement of air in fluid holding chamber 201 upon loading of a fluid sample into fluid input port 200.
  • the fluid holding chamber is fluidly connected through microchannel 203 having a cross-sectional dimension of from about 1 to about 200 ⁇ m to microchannel
  • MicroChannel 205 has a cross-sectional dimension of from about 1 to about 200 ⁇ m.
  • a capillary microvalve 204 or, optionally, a sacrificial valve 204 interrupts microchannel 203 and prevents fluid flow from fluid holding chamber 201 into microchannel 205 in the absence of platform rotation (i.e., at zero centripetal force).
  • MicroChannel 205 is fluidly connected to collection chamber 206 having volumetric capacity of from about 1 to about 200 ⁇ L.
  • Collection chamber 206 has a diameter of from about 0.3 to about 1 cm and a depth in the platform surface of from about 0.02 to about 1cm.
  • Air displacement channel 207 having a cross-sectional dimension of from about 100 to about 500 ⁇ m is connected to collection chamber 206 to permit displacement of air in collection chamber 206 upon rotation of the platform and fluid flow from fluid holding chamber 201 through microchannel 205 and into collection chamber 206.
  • Thermal control of the fluid flowing through microchannel 205 is accomplished by heating the fluid and microchannel using electrically-resistive patch 208. Electrically conductive leads 209 and 210 control heating by electrically-resistive patch 208. The time during which the fluid in microchannel 205 is maintained at a temperature greater than ambient is dependent on the rotational speed of the platform and the length and interior dimension of microchannel 205.
  • Electrically-resistive patch 305 is on the surface of thermal regulation layer substrate 306, and fluidic manifold layer 302 containing microfluidic structures including microchannel 303 in substrate layer
  • Insulation space 301 having a volumetric capacity of from about 0.1 to about 1 cm 3 is advantageously positioned in manifold layer 302 above the position of electrically-resistive patch 305 in thermal regulation layer 306. This insulation space is optionally enclosed by cover 300.
  • Insulation space 301 can be empty (i.e., containing only air) or can contain air trapped in fibrous material. Insulation space 301 supports the formation of a temperature gradient between the heater and the environment and permits the formation of a uniform temperature through the thickness of the capillary microchannel.
  • FIG. 1-3 The platform shown in Figures 1-3 is useful for binding assays at a controlled temperature greater than the ambient temperature of the platform.
  • hybridization probes deposited on the walls of the microchannel, or comprising carrier beads that are motivated into the capillary channel can be used to separate hybridizing from non- hybridizing material in a biological sample, particularly if the carrier beads are retained in the microchannel.
  • Figure 4 shows a microsystems platform according to the invention adapted for performing hybridization arrays.
  • fluid input port 400 is fluidly connected to fluid holding chamber 401 having a volumetric capacity of from about 10 to about 200 ⁇ L.
  • the fluid input port has a sufficient diameter to accommodate a standard 200 ⁇ L plastic pipette tip.
  • Fluid holding chamber 401 has a diameter of from about 1 to about 3 cm and a depth in the platform surface of from about 0.02 to about 1cm.
  • Air displacement channel 402 having a cross-sectional dimension of from about 100 to about 500 ⁇ m is connected to fluid holding chamber 401 to permit displacement of air in fluid holding chamber 401 upon loading of a fluid sample into fluid input port 400.
  • the fluid holding chamber is fluidly connected through a multiplicity of microchannels 403 having a cross-sectional dimension of from about 1 to about 200 ⁇ m to an equivalent multiplicity of microchannels 405.
  • Microchannels 405 each have a cross-sectional dimension of from about 1 to about 200 ⁇ m.
  • Microchannels 405 are fluidly connected to hybridization chambers 406 having volumetric capacity of from about 1 to about 20 ⁇ L.
  • the hybridization chambers have a diameter of from about 0.1 to about 1 cm and a depth in the platform surface of from about 10 to about 200 ⁇ m.
  • Heating elements 407 Positioned in thermal contact with each of hybridization chambers 406 are heating elements 407, most preferably electrically-resistive patches contained in a thermal regulation layer as shown in Figures 1 -3.
  • Hybridization chambers 406 are optionally and advantageously fluidly connected to detection chambers 410.
  • Detection chambers 410 enable detection of hybridization reaction products, most preferably using optical detectors and detection methods, as described more completely in 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Serial Nos. 08/761,063, filed December 5, 1996; 08/768,990, filed
  • Detection chambers 410 are fluidly connected through capillaries 411 having an interior dimension of from about 1 to about 200 ⁇ m to common waste chamber 412, that has a diameter of from about 1 to about 3 cm and a depth in the platform surface of from about 0.02 to about 0.2cm.
  • Air displacement channel 413 having a cross-sectional dimension of from about 100 to about 500 ⁇ m is connected to common waste chamber 412 to permit displacement of air in common waste chamber 412 upon rotation of the platform.
  • Figures 5 and 6 depict plan and cross-sectional views of a microfluidics platform of the invention adapted for performing polymerase chain reaction or ligase chain reaction.
  • a fluid input port 500 is fluidly connected to fluid holding chamber 501 having a volumetric capacity of from about 10 to about 200 ⁇ L.
  • the fluid input port has a sufficient diameter to accommodate a standard 200 ⁇ L plastic pipette tip.
  • Fluid holding chamber 501 has a diameter of from about 1 to about 3cm and a depth in the platform surface of from about 0.02 to about 0.3cm.
  • Air displacement channel 502 having a cross-sectional dimension of from about 100 to about 500 ⁇ m is connected to fluid holding chamber 501 to permit displacement of air in fluid holding chamber 501 upon loading of a fluid sample into fluid input port 500.
  • the fluid holding chamber is fluidly connected through microchannel
  • Thermal array 520 comprises a fluid microchannel 505 that traverses the array across three thermal zones comprising individually-addressable heating elements 508, 509 and 510, preferably comprising a thermal regulation layer as described above and in Figure 6.
  • Fluid microchannel 505 has a cross-sectional dimension of from about 1 to about 200 ⁇ m. The path of microchannel 505 is shown in the Figure to represent different transit times across the different thermal zones.
  • the transit time in the middle thermal zone is longer than in either of the flanking thermal zones, because the microchannel path traverses this zone several times for each time the path traverses either of the flanking zones.
  • MicroChannel paths traversing the thermal zones having equal transit times or any combination of differential transit times for each zone are within the scope of this invention.
  • Heating elements 508, 509 and 510 are constructed to be operated at separate and distinct temperatures greater than ambient; such heating elements are described more fully in co- owned U.S. Patent 6,063,589 issued May 16, 2000 and in co-owned and co-pending patent applications U.S. Serial Nos.
  • the length of the microchannel is chosen to be equivalent to 20-35 cycles of conventional PCR, wherein each cycle consists of a time interval at a denaturing temperature (from about 80°C to about 95°C), a time interval at an oligonucleotide primer annealing temperature (from about 40°C to about 65 °C) and a time interval at a polymerase extension temperature (from about 60°C to about 75°C).
  • the middle thermal zone is provided without a heating element, so that transit through this zone is at ambient temperature. It will be recognized that other configurations of the thermal arrays of the invention, as described herein, are also useful in constructing alternative platforms of the invention based on the platform explicitly disclosed in Figure 5.
  • platforms comprising a multiplicity of the microfluidic and thermal structures shown in Figure 5 are also encompassed by the invention, wherein individual fluid input and holding chambers, or such chambers shared in common, are provided fluidly connected to the thermal arrays of the invention.
  • An example of such a platform is shown in Figure 7.
  • Figure 6 shows a cross-sectional view of the platform of Figure 5.
  • Heating element 604 is shown on the surface of thermal regulation layer substrate 607, and fluidic manifold layer 602 containing microfluidic structures including microchannels 605 in substrate layer
  • Thermal conductive layer 606 is optionally included between the fluidic manifold layer and the thermal regulation layer to increase thermal conductivity and heat transfer to the fluid in microchannels 605.
  • An insulation space 601 having a volumetric capacity of from about 0.3 to about 3cm 3 is advantageously positioned in fluidic manifold layer 602 above the position of the heating element 604 in thermal regulation layer 607. This insulation space is optionally enclosed by cover 600.
  • the platform comprises a single thermal array. It will be recognized by those with skill in the art that such embodiments are useful for multiplex assay of a single sample or assays of multiple samples. Platforms of the invention are provided having, for example, multiple embodiments of the thermal arrays as described herein, in fluid communication with one or a multiplicity of sample entry ports. Thus, in more preferred embodiments, the platforms of the invention comprise a multiplicity of such arrays laid out on the platform surface as exemplified in Figure 7. In alternative embodiments, the fluidic manifold layer and thermal regulation layer are integrally micromachined onto a single substrate layer.
  • the thermal regulation layer comprises a platen that is in thermal contact with a fluidic microsystems platform of the invention, wherein the fluidic components such as microchannels are positioned to be in thermal contact with heating elements on the platen, most preferably using fiducials or other mechanical components that orient the platform and platen with one another.
  • the thermal regulating layer is a mechanical platen, in which the microfluidic components are positioned in thermal contact with heating elements on the platen.
  • a platen as the term is used herein is in certain embodiments a circuit board having electrical components mounted and soldered thereupon.
  • the electrical components are passive and consist primarily of resistors in defined locations on the platen. Heat is produced using such resistors because power is dissipated as heat when a voltage is applied across a resistor. The heat dissipation is proportional to the square of the applied voltage divided by the resistance of the resistor.
  • the platen comprises a multiplicity of resistors that share a common ground but that have different input wires so that different voltages can be applied and different temperatures obtained in microfluidics components placed proximate to each of the resistors.
  • the electrical signal is distributed from the micromanipulation device to the platen through a slip-ring assembly, as described more fully in co-owned U.S. Patent No. 6,063,589, incorporated by reference.
  • platens comprising integrated circuits, microprocessors and other "active" components are advantageously utilized.
  • EXAMPLE 1 Microplatform Disk for DNA Hybridization A series of hybridization reactions performed to determine the optimum hybridization stringency conditions between a probe molecule and a specific gene sequence is performed using the microsystems platform shown in Figure 4. A denatured hybridization mixture comprising the probe, the specific gene sequence, salts, buffers, detergents and hybridization facilitating agents such as dimethylformamide is applied to fluid holding chamber 401 at rest. The platform is then rotated at a first rotation speed sufficient to overcome capillary microvalve 404 and the hybridization solution traverses microchannels 405 and is deposited in substantially equal volumes into hybridization chambers 406.
  • Heating elements 407 are independently operated at a series of incrementally different and increasing temperatures, so that hybridization is achieved over a range of temperatures from ambient to a temperature at which no hybridization occurs.
  • the platform is rotated at a second rotation speed higher than the first rotation speed, and the hybridization reaction mixtures from each hybridization chamber are transferred to detection chambers 410.
  • the extent of hybridization is analyzed in each detection chamber using conventional methods, for example, absorbance at 260nm; in this embodiment, the portion of the platform comprising detection chambers 410 is transparent to ultraviolet light. The temperature conditions yielding the greatest extent of hybridization are determined thereby.
  • EXAMPLE 2 Microplatform Disk for DNA Amplification
  • An amplification reaction is performed on the microsystems platform disclosed in Figures 5 and 6 as follows. While the disk is stationary, lO ⁇ L of a fluid reaction mixture containing template DNA, PCR amplification primers, deoxynucleotide triphosphates, and thermostable polymerase in a buffer solution in which the reaction may proceed are applied to fluid input port 500. As the fluid fills fluid holding chamber 501 air is displaced through air displacement channels 502 and 507; in this example, 501 has a diameter of 0.4 cm and a depth of 0.3 cm and lO ⁇ L fills approximately 25% of the volumetric capacity of fluid holding chamber 501. Fluid also flows into microchannel 503 until it reaches the capillary microvalve 504 or optional sacrificial valve 504.
  • the platform is briefly rotated at about 3000rpm to overcome the capillary microvalve, or optionally the sacrificial valve is opened, and then the rotational speed is lowered to about 590 rpm.
  • fluid flows through fluid microchannel 505 at a rate of about 6nL/sec, as the microchannel has a hydraulic diameter of 50 ⁇ m and a overall length of approximately

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Abstract

L'invention concerne des procédés et un appareil permettant de mener à bien des analyses et des procédés microsynthétiques et microanalytiques. L'invention concerne une plate-forme de microsystème et un dispositif de micromanipulation servant à manipuler la plate-forme exploitant la force centripète résultant de la rotation de la plate-forme, favorisant de ce fait le mouvement fluidique à travers les microcanaux. Les plates-formes de microsystème de l'invention présentent des réseaux de zones de commande thermique, dans lesquels le fluide acheminé vers la plate-forme peut être soumis à une température et maintenu à cette température pendant un laps de temps tributaire de la longueur du chemin du canal dans la zone, de la dimension transversale du canal et de la vitesse de rotation de la plate-forme. Des procédés spécifiques permettent de mettre en oeuvre un large éventail de procédés microanalytiques ou microsynthétiques.
PCT/US2000/013131 1999-05-14 2000-05-12 Systeme microfluidique a fonctionnement centripete destine a effectuer une hybridation in vitro et une amplification des acides nucleiques WO2000069560A1 (fr)

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AU47134/00A AU4713400A (en) 1999-05-14 2000-05-12 A centripetally-motivated microfluidics system for performing in vitro hybridization and amplification of nucleic acids

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