US20040259237A1 - Devices and methods for the performance of miniaturized in vitro amplification assays - Google Patents

Devices and methods for the performance of miniaturized in vitro amplification assays Download PDF

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US20040259237A1
US20040259237A1 US10/801,460 US80146004A US2004259237A1 US 20040259237 A1 US20040259237 A1 US 20040259237A1 US 80146004 A US80146004 A US 80146004A US 2004259237 A1 US2004259237 A1 US 2004259237A1
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platform
fluid
chamber
temperature
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Gregory Kellogg
Charles Able
Todd Arnold
Bruce Carvalho
Hsin-Chiang Lin
Stephen Kieffer-Higgins
Norman Sheppard
Mikayla Kob
Shari Ommert
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TECAN TRADING AG TECAN GROUP Ltd
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Kellogg Gregory J.
Charles Able
Todd Arnold
Carvalho Bruce L.
Hsin-Chiang Lin
Stephen Kieffer-Higgins
Sheppard Norman F.
Mikayla Kob
Shari Ommert
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Priority to US14047799P priority Critical
Priority to US09/602,394 priority patent/US6706519B1/en
Application filed by Kellogg Gregory J., Charles Able, Todd Arnold, Carvalho Bruce L., Hsin-Chiang Lin, Stephen Kieffer-Higgins, Sheppard Norman F., Mikayla Kob, Shari Ommert filed Critical Kellogg Gregory J.
Priority to US10/801,460 priority patent/US20040259237A1/en
Publication of US20040259237A1 publication Critical patent/US20040259237A1/en
Assigned to TECAN TRADING, AG, TECAN GROUP, LTD. reassignment TECAN TRADING, AG, TECAN GROUP, LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ABLE, CHARLES, ARNOLD, TODD, CARVALHO, BRUCE L, KELLOGG, GREGORY J, KIEFFER-HIGGINS, STEPHEN, KOB, MIKAYLA, LIN, HSIN-CHIANG, OMMERT, SHARI, SHEPPARD, NORMAN F
Assigned to TECAN TRADING AG reassignment TECAN TRADING AG CORRECTIVE ASSIGNMENT TO CORRECT THE ASSIGNEE'S NAME, PREVIOUSLY RECORDED AT REEL 019442 FRAME 0001. Assignors: KIEFFER-HIGGINS, STEPHEN, KOB, MIKAYLA, LIN, HSIN-CHIANG, OMMERT, SHARI, SHEPPARD, NORMAN F., ABLE, CHARLES, ARNOLD, TODD, CARVALHO, BRUCE L., KELLOGG, GREGORY J.
Application status is Abandoned legal-status Critical

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING, DISPERSING
    • B01F13/00Other mixers; Mixing plant, including combinations of mixers, e.g. of dissimilar mixers
    • B01F13/0059Micromixers
    • B01F13/0061Micromixers using specific means for arranging the streams to be mixed
    • B01F13/0064Mixing chamber
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING, DISPERSING
    • B01F13/00Other mixers; Mixing plant, including combinations of mixers, e.g. of dissimilar mixers
    • B01F13/0059Micromixers
    • B01F13/0074Micromixers using mixing means not otherwise provided for
    • B01F13/0094Micromixers using mixing means not otherwise provided for the mixing being performed in a mixing chamber where the products are brought into contact
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING, DISPERSING
    • B01F15/00Accessories for mixers ; Auxiliary operations or auxiliary devices; Parts or details of general application
    • B01F15/02Feed or discharge mechanisms
    • B01F15/0201Feed mechanisms
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING, DISPERSING
    • B01F15/00Accessories for mixers ; Auxiliary operations or auxiliary devices; Parts or details of general application
    • B01F15/02Feed or discharge mechanisms
    • B01F15/0201Feed mechanisms
    • B01F15/0227Feed mechanisms characterized by the means for feeding the components to the mixer
    • B01F15/0233Feed mechanisms characterized by the means for feeding the components to the mixer using centrifugal forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers 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
    • B01L3/50273Containers 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 characterised by the means or forces applied to move the fluids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47Scattering, i.e. diffuse reflection
    • G01N21/4795Scattering, i.e. diffuse reflection spatially resolved investigating of object in scattering medium
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N35/00069Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING, DISPERSING
    • B01F2215/00Auxiliary or complementary information in relation with mixing
    • B01F2215/04Technical information in relation with mixing
    • B01F2215/0413Numerical information
    • B01F2215/0418Geometrical information
    • B01F2215/0431Numerical size values, e.g. diameter of a hole or conduit, area, volume, length, width, or ratios thereof
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01FMIXING, e.g. DISSOLVING, EMULSIFYING, DISPERSING
    • B01F2215/00Auxiliary or complementary information in relation with mixing
    • B01F2215/04Technical information in relation with mixing
    • B01F2215/0413Numerical information
    • B01F2215/0436Operational information
    • B01F2215/0477Numerical time values
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0621Control of the sequence of chambers filled or emptied
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0803Disc shape
    • B01L2300/0806Standardised forms, e.g. compact disc [CD] format
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0861Configuration of multiple channels and/or chambers in a single devices
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1805Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/06Valves, specific forms thereof
    • B01L2400/0677Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178Special arrangements of analysers
    • G01N2035/00237Handling microquantities of analyte, e.g. microvalves, capillary networks

Abstract

This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. The invention provides a microsystem platform and a micromanipulation device for manipulating the platform that utilizes the centripetal force resulting from rotation of the platform to motivate fluid movement through microchannels. The invention specifically provides devices and methods for performing miniaturized in vitro amplification assays such as the polymerase chain reaction. Methods specific for the apparatus of the invention for performing PCR are provided.

Description

    BACKGROUND OF THE INVENTION
  • This application claims priority to U.S. Provisional Application Ser. No. 60/140,477, filed Jun. 22, 1999, the disclosure of which is explicitly incorporated by reference herein.[0001]
  • 1. FIELD OF THE INVENTION
  • This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. In particular, the invention relates to microminiaturization of genetic, biochemical and bioanalytic processes. Specifically, the present invention provides devices and methods for the performance of integrated and miniaturized sample preparation, nucleic acid amplification, and nucleic acid detection assays. These assays may be performed for a variety of purposes, including but not limited to forensics, life sciences research, and clinical and molecular diagnostics. The invention may be used on a variety of liquid samples of interest, including bacterial and cell cultures as well as whole blood and processed tissues. Methods for performing any of a wide variety of such microanalytical or microsynthetic processes using the microsystems apparatus of the invention are also provided. [0002]
  • 2. BACKGROUND OF THE RELATED ART
  • Extraction and isolation of DNA from host cells is a cornerstone of modern molecular biology. One type of DNA, bacterial plasmid DNA has been particularly useful as a convenient vector for the insertion of genetic material into bacterial, yeast and mammalian cells. DNA isolated from an organism is inserted by being contiguously and covalently linked to plasmid DNA and is then introduced into a cell, such as a bacterial cell, and allowed to multiply, thereby creating large copy numbers of the plasmid in each cell. These plasmids may advantageously be harvested to provide a sufficient amount of DNA (typically on the order of several micrograms, although up to milligram quantities can be produced on an industrial scale) for a variety of experimental or therapeutic purposes. The harvesting of plasmid DNA, defined as its removal from cells and isolation from the genomic DNA content of the cells, has growing utility in life sciences research, diagnostics, therapeutics and other applications. [0003]
  • Currently, the extraction and isolation of DNA is either performed manually or through the use of robotic sample preparation stations. In either case, a variety of technologies and materials are used (see, for example, [0004] QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit Handbook, 1999, Qiagen GmbH, Max-Volmer-Strasse 4, 40724 Hildren, Germany; Bimboim & Doly, 1979, Nucl. Acids Res. 1: 1513-1522). Typically, cells are first incubated in a surfactant (detergent) solution, in some cases containing protein digesting enzymes such as Protease or Proteinase K. These lyse the cells, thereby releasing the DNA into solution. This is frequently performed under alkaline conditions, to destabilize nucleases and hydrolyze contaminating RNA. The DNA must then be separated from other cell constituents, which is performed using a number of different separation protocols, including, for example, selective precipitation of proteins and other cell debris, organic chemical extraction (using phenol and chloroform), and DNA affinity column chromatography. Plasmid DNA must also be isolated from contaminating cellular (bacterial genomic DNA). Filtration methods can produce a plasmid DNA solution, but the solutions required to solvate DNA are usually inappropriate for the desired final application of the DNA. As a consequence, plasmid DNA is removed from these solutions by ethanol precipitation, or solid-phase separation is used, which often requires further changes in solvent pH and salt concentration (especially for affinity binding methods using glass or silica). The technologies required for these steps include pipetting, pumping, filtration, washing, and centrifugation, requiring an expensive suite of devices and skilled operators thereof. The additional requirements of automated systems include sample transfer and robotics for the handling of sample containers.
  • This discussion illustrates the need in the art for more efficient, rapid, inexpensive automated methods and devices for performing DNA sample preparation, particularly plasmid DNA preparation. [0005]
  • In the field of integrated genetic analysis, some progress has been made in the integration of sample preparation, PCR, and detection via real-time fluorescence or hybridization methods (Anderson et al., 1998, “Advances in Integrated Genetic Analysis,” in [0006] Proc. Micro Total Analysis '98, Harrison & van den Berg, eds., Kluwer: Amsterdam, pp.11-16). These systems rely on macroscopic fluid handling systems such as pumps and valves that must be interfaced with the microfluidic devices within which fluids are processed.
  • However, there exists a need for devices and methods capable of processing cell cultures for harvesting DNA, particularly plasmid DNA. [0007]
  • In the biological and biochemical arts, analytical procedures frequently require incubation of biological samples and reaction mixtures at temperatures greater than ambient temperature. Moreover, many bioanalytical and biosynthetic techniques require incubation at more than one temperature, either sequentially or over the course of a reaction scheme or protocol. [0008]
  • One example of such a bioanalytical reaction is the polymerase chain reaction. The polymerase chain reaction (PCR) is a technique that permits amplification and detection of nucleic acid sequences. See U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis. This technique has a wide variety of biological applications, including for example, DNA sequence analysis, probe generation, cloning of nucleic acid sequences, site-directed mutagenesis, detection of genetic mutations, diagnoses of viral infections, molecular “fingerprinting,” and the monitoring of contaminating microorganisms in biological fluids and other sources. 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. [0009]
  • 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. [0010]
  • In addition to PCR, other in vitro amplification procedures, including ligase chain reaction as disclosed in U.S. Pat. No. 4,988,617 to Landegren and Hood, are known and advantageously used in the prior art. More generally, several important methods known in the biotechnology arts, such as nucleic acid hybridization and sequencing, are dependent upon changing the temperature of solutions containing sample molecules in a controlled fashion. Automation and miniaturization of the performance of these methods are desirable goals in the art. [0011]
  • Mechanical and automated fluid handling systems and instruments produced to perform automated PCR have been disclosed in the prior art. [0012]
  • U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et al. teach fluid handling on microscale analytical devices. [0013]
  • International Application, Publication No. WO93/22053, published 11 Nov. 1993 to University of Pennsylvania disclose microfabricated detection structures. [0014]
  • International Application, Publication No. WO93/22058, published 11 Nov. 1993 to University of Pennsylvania disclose microfabricated structures for performing polynucleotide amplification. [0015]
  • Wilding et al., 1994, [0016] Clin. Chem. 40: 43-47 disclose manipulation of fluids on straight channels micromachined into silicon.
  • Kopp et al., 1998, [0017] Science 21M: 1046 discloses microchips for performing in vitro amplification reactions using alternating regions of different temperature.
  • One drawback of the synthetic microchips disclosed in the prior art for performing 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 μm 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. [0018]
  • Thus, there exists a need in the art for devices and methods that provide integrated sample preparation and analysis, particularly of DNA samples. This need is particularly acute for high throughput analyses, which are currently burdened by the high costs and complexity of automated, typically robotic, systems. Integration of DNA sample preparation and analysis would be particularly useful if it reduced the current need in the art for need for multiple, complex technologies that demand highly-skilled operators. Importantly, for DNA analysis integration of sample preparation and in vitro amplification methods would minimize the possibility of contamination and sample carry-over, which is particularly important in high-sensitivity techniques such as various in vitro amplification reactions used in the art. [0019]
  • 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. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and 09/filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. [0020]
  • SUMMARY OF THE INVENTION
  • This invention provides Microsystems platforms as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. [0021]
  • 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 Microsystems platform is provided to perform integrated and miniaturized sample preparation, nucleic acid amplification, and nucleic acid detection assays. A 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, collection chambers, detection chambers and sample outlet ports, generically termed “microfluidic structures”. The disk is rotated at speeds from about 1 to about 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. The disk, and most preferably a face of the platform, may also contain heating elements for raising the temperature of fluids contained therein to temperatures greater than ambient temperatures. Specific sites on the disk also preferably comprise elements that allow fluids to be analyzed. [0022]
  • A preferred embodiment of the platforms of the invention is a platen that rotates with the microfluidics disk. The platen is most preferably a printed circuit board comprising resistive heating elements, thermoelectric (Peltier) elements, temperature sensors, assay optics and microprocessor and other electronic components. Electrical communication between a rotating platen and stationary power sources, motor controllers, temperature controllers, and computers is most preferably accomplished through a slip-ring assembly. By mounting the microfluidic disk on the platen and rotating both disk and platen together, the distribution and flow rate of fluid throughout the microfluidic structures as well as the temperature of fluid within localized regions of the microfluidics disc can be controlled. [0023]
  • In a preferred embodiment, one face of the microfluidics disk is mounted onto a face of the platen and the temperature of fluids at particular positions within the microfluidics disk is controlled through temperature exchange between the platen and disk. In alternative embodiments, a microfluidic disk is positioned between two platens, each comprising elements that effect temperature exchange between the disk and thermal regulation elements comprising the platens. In a preferred embodiment, the platen is a printed circuit board with resistive heating elements, Peltier elements and temperature sensors embedded therein or affixed thereto. In an alternative embodiment, thermal regulation within the microfluidic disk is achieved by permanently bonding a layer comprising resistive heaters directly to the disk; in this case, fluids within the disk are heated to temperatures greater than ambient temperature with resistive heating elements and cooled to temperatures above or equal to ambient temperature by spinning the disk and through the loss of heat to the environment. As with the platen, electrical communication between this composite disk and power supplies, temperature controllers and computers is most preferably accomplished through a slip-ring assembly. [0024]
  • In a first aspect, the present invention provides devices and methods for the performance of integrated and miniaturized sample preparation for the extraction, isolation, and purification of DNA from cells. In preferred embodiments, the devices and methods of the invention are particularly provided to isolate plasmid DNA from bacterial cells. [0025]
  • The plasmid DNA sample preparation platforms of the invention are provided to perform the following functions: sample processing to free DNA from the bacterial cell; filtration of the resultant solution to remove bacterial cell fragments; application of the solution to a binding matrix using solvent conditions that promote DNA binding to the matrix; washing of bound DNA and replacement of the original solution by a solution that is compatible with further analytical methods; and elution of the DNA from the binding matrix in a suitable solvent. The DNA thus eluted can be isolated, amplified in vitro or sequenced using methods known in the art. The platforms of the invention are provided comprising microfluidic structures that perform plasmid DNA sample preparation as described in further detail below. These microstructures are illustrated for clarity with regard to a single microstructure. However, platforms comprising a multiplicity of such plasmid DNA preparation microfluidic structures are provided by the invention, wherein the microfluidics structures are arrayed on the surface of the platform with a density determined by the size of the platform and the volumetric capacity of the chambers and reservoirs comprising the microfluidic structures as disclosed herein. [0026]
  • In a second aspect, the invention is provided having microfluidics structures as described herein for performing an integrated suite of biochemical processes for accomplishing in vitro amplification reactions. These include sample processing to isolate DNA from bacterial or mammalian cells; sample conditioning to adjust the solution conditions to those appropriate for PCR; mixing of the conditioned sample with PCR reagents, including deoxyribosenuclotides, polymerase enzyme, primers, and appropriate salts, buffers and additives; and thermal cycling to effect PCR. [0027]
  • In certain preferred embodiments, the discs of the invention are provided with a multiplicity of microfluidics structures that enable to platform to process and amplify several samples simultaneously. In these embodiments, multiple copies of an arrangement of microfluidics structures for performing the biochemical reaction suite are arrayed on the disc, and sample input ports or reservoirs provided for each copy, thereby permitting processing of multiple samples. In addition, the portion of the sample DNA to be amplified can be independently, by the choice of amplification primers provided in each of the individual copies of the microfluidics structures arrayed on the disc, thereby permitting amplification “multiplexing” of a particular sample. Alternatively, the same primers can be provided to process in parallel multiple samples for amplification of the same target fragment in the DNA of each sample. Independent thermal cycling profiles, including the temperature used for each step of the amplification cycle, temperature ramp-rates, and hold times, may be individually programmed into the instrument for each of the microfluidics structures or for each of the samples processed. [0028]
  • The invention advantageously permits simultaneous, independent thermal cycling of a multiplicity of different samples, independent amplification of different target fragments from a particular sample, or both. This feature also enables a user to optimize thermal cycling parameters for a single sample or amplicon quickly and in a single experiment, by varying reaction parameters on a plurality of the microfluidics structures arrayed in the disc, thereby simultaneously performing multiple experiments simultaneously. Since particular copies of the microfluidics structures can be arranged in microfluidic isolation from other copies on the platform, portions comprising less than all of the microfluidics structures can be discretely used and the remainder retained for future use. [0029]
  • In alternative embodiments of the platforms of the invention, metering structures as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000 and incorporated by reference herein, are used to distribute aliquots of reagent to each of a multiplicity of mixing structures, each mixing structure being fluidly connected to one of a multiplicity of sample reservoirs, thereby permitting parallel processing and mixing of the samples with a common reagent. This reduces the need for automated reagent distribution mechanisms, reduces the amount of time required for reagent dispensing (that can be performed in parallel with distribution of reagent to a multiplicity of reaction chambers), and permits delivery of small (nL-to-μL) volumes without using externally-applied electromotive means. [0030]
  • The assembly of a multiplicity of collection chambers on the platforms of the invention also permits simplified detectors to be used, whereby each individual collection/detection chamber can be scanned using mechanisms well-developed in the art for use with, for example, CD-ROM technology. Finally, the platforms of the invention are advantageously provided with sample and reagent entry ports for filling with samples and reagents, respectively, that can be adapted to liquid delivery means known in the art (such as micropipettors). [0031]
  • The discs of this invention have several advantages over those that exist in the centrifugal analyzer art. Foremost is the fact that flow is laminar due to the small dimensions of the fluid channels; this allows for better control of processes such as mixing and washing. Secondly, the small dimensions conferred by microfabrication enable the use of “passive” valving, dependent upon capillary forces, over much wider range of rotational velocities and with greater reliability than in more macroscopic systems. To this are added the already described advantages of miniaturization. [0032]
  • The present invention solves problems in the current art through the use of a microfluidic disc in which centripetal acceleration is used to move fluids. It is an advantage of the microfluidics platforms of the present invention that the fluid-containing components are constructed to contain a small volume, thus reducing reagent costs, reaction times and the amount of biological material required to perform an assay. It is also an advantage that the fluid-containing components are sealed, thus eliminating experimental error due to differential evaporation of different fluids and the resulting changes in reagent concentration. Because the microfluidic devices of the invention are completely enclosed, both evaporation and optical distortion are reduced to negligible levels. The platforms of the invention also advantageously permit “passive” mixing and valving, i.e., mixing and valving are performed as a consequence of the structural arrangements of the components on the platforms (such as shape, length, position on the platform surface relative to the axis of rotation, and surface properties of the interior surfaces of the components, such as wettability as discussed below), and the dynamics of platform rotation (speed, acceleration, direction and change-of-direction), and permit control of assay timing and reagent delivery. [0033]
  • The devices of the invention also implement simpler, more robust, and more economical sample preparation for performing in vitro amplification reactions such as PCR. All mechanical aspects of sample processing are carried out using a single motor that rotates the disc at prescribed velocities, thereby driving fluids on the disc through microchannels and other microfluidics structures. This is in advantageous over current sample preparation methods involving robotic pipetting stations or other fluid transfer mechanisms, automation for the delivery of processing plates to different “stations,” or both. [0034]
  • The invention advantageously integrates sample preparation with thermal cycling for PCR, thereby eliminating additional fluid transfer steps. This minimizes the potential for contamination or fluid loss. [0035]
  • The platforms of the invention reduce the demands on automation in at least three ways. First, the need for precise metering of delivered fluids is relaxed through the use of on-disc metering structures, as described more fully in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. By loading imprecise volumes, slightly in excess of those needed for the assay, and allowing the rotation of the disc and use of appropriate microfluidic structures to meter the fluids, much simpler (and less expensive) fluid delivery technology may be employed than is the conventionally required for high-density microtitre plate assays. [0036]
  • Second, the total number of fluid “delivery” events on the microfluidic platform is reduced relative to microtiter plates. By using microfluidic structures that sub-divide and aliquot common reagents (such as reagent solutions, buffers, and enzyme substrates) used in all assays performed on the platform, the number of manual or automated pipetting steps are reduced by at least half (depending on the complexity of the assay). A reduction in fluid transfers to the device can reduce total assay time. Examples of these structures have been disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and incorporated by reference herein. [0037]
  • The invention also provides on-platform means for mixing reagents with sample and washing the resulting reaction products, removing the need for transferring the assay collection chamber(s) to a separate “wash” station. This also reduces manipulation of the assay device as well as providing controlled and integrated fluid processing. [0038]
  • The invention disclosed herein is flexible as to sample and source, being capable of isolating nucleic acid from bacteria, whole animal blood, tissues and cellular sources. It is rapid, being about 50% more rapid than existing “automated” nucleic acid preparatory methods. The nucleic acid output of the system is of a quality higher than or equal to methods known in the art. The system is simple and easy to use, robust because it is not dependent on operator variability. In addition, the platforms and systems disclosed are self-contained and integrated, thereby minimizing both operator handling and error. [0039]
  • Certain preferred embodiments of the apparatus of the invention are described in greater detail in the following sections of this application and in the drawings.[0040]
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts an exploded, oblique view of a Microsystems platform of the invention. [0041]
  • FIG. 2 depicts a plan view of one component of the Microsystems platform shown in exploded, oblique view in FIG. 1, the microfluidics layer. [0042]
  • FIG. 3 is a detail of a section of the microfluidics layer illustrated in FIG. 2. [0043]
  • FIG. 4 shows a detail of a region of the structure illustrated in FIG. 3. [0044]
  • FIG. 5 is a cross-sectional view of the Microsystems platform of FIG. 1 in the vicinity of the thermal cycling chamber. [0045]
  • FIG. 6 depicts an explode, oblique view of a microfluidics disc and a printed circuit. [0046]
  • FIG. 7 illustrates a plan view of the microfluidics disc shown in FIG. 6. [0047]
  • FIG. 8 illustrates the velocity profile, rotational rate (rpm) vs. time, used to effect fluid motion through the Microsystems platform in Examples 1 and 2. [0048]
  • FIG. 9 illustrates the sequence of fluid motions motivated by the velocity profile of FIG. 8. [0049]
  • FIG. 10 is a photograph of gel electrophoretic analysis of PCR amplification of a target fragment contained in DNA isolated from [0050] E. coli.
  • FIG. 11 is a photograph of gel electrophoretic analysis of PCR amplification of a target fragment contained in DNA isolated from bovine blood. [0051]
  • FIG. 12 depicts an exploded, oblique view of the DNA sample preparation disk. [0052]
  • FIG. 13 is a plan view of this disk shown in FIG. 12. [0053]
  • FIG. 14 depicts a plan view of the microfluidics structure for a plasmid DNA preparation platform. [0054]
  • FIG. 15 depicts a plan view of the heating layer for a plasmid DNA preparation platform. [0055]
  • FIG. 16 depicts a plan view of a base layer for a plasmid DNA preparation platform. [0056]
  • FIGS. 17A through 17K illustrates fluid movement through the microfluidics structure for a plasmid DNA preparation platform. [0057]
  • FIG. 18 is a photograph of gel electrophoretic analysis of a restriction enzyme digestion profile of plasmid DNA prepared conventionally (control) or using a plasmid DNA preparation platform of the invention, wherein the outside lanes are size markers. [0058]
  • FIG. 19 is a photograph of gel electrophoretic analysis of an in vitro amplification reaction using primers specific for plasmid DNA or bacterial genomic DNA, wherein the amount of template DNA decreases in each set of amplification reactions moving from left to right, using a plasmid DNA preparation platform of the invention; the outside lanes are size markers. [0059]
  • FIG. 20 is a plan view diagram of the electric platen and controlling elements of the invention. [0060]
  • FIG. 21 is a plan view diagram of the temperature control elements on an electric platen of the invention. [0061]
  • FIG. 22 is a plan view diagram of the electrical contacts between the electrical leads on the printed circuit board of the platen and temperature control elements. [0062]
  • FIG. 23 is a cross-sectional view of the structure of a temperature control element comprising a Peltier element according to the invention. [0063]
  • FIG. 24 depicts a plan view of the electric circuit layer shown in FIG. 6. [0064]
  • FIG. 25 shows a cross-sectional view on the disk shown in FIG. 6. [0065]
  • FIG. 26 is a photograph of gel electrophoretic analysis of amplified DNA target from an [0066] E. coli sample using the disk pictured in FIG. 6.
  • FIG. 27 depicts an example of multiplexed PCR performed in the thermal cycling chamber. [0067]
  • DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
  • This invention provides a microplatform and a micromanipulation device as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein, adapted for performing microanalytical and microsynthetic assays of biological samples. [0068]
  • For the purposes of this invention, the term “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. In particular, the term “sample” will be understood to encompass any biological species of interest. The term “biological sample” or “biological fluid sample” will be understood to mean any biologically-derived sample, including but not limited to blood, plasma, serum, lymph, saliva, tears, cerebrospinal fluid, urine, sweat, plant and vegetable extracts, semen, and ascites fluid. [0069]
  • For the purposes of this invention, the term “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. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. [0070]
  • For the purposes of this invention, the term “microsystems platform” is intended to include centripetally-motivated microfluidics arrays as described in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. [0071]
  • For the purposes of this invention, the terms “capillary”, “microcapillary” and “microchannel” will be understood to be interchangeable and to be constructed of either wetting or non-wetting materials where appropriate. [0072]
  • For the purposes of this invention, the term “reagent reservoir,” “assay chamber,” “fluid holding chamber,” “collection chamber” and “detection chamber” will be understood to mean a defined volume on a Microsystems platform of the invention comprising a fluid. The volumetric capacity of these structures as provided herein is from about 2 nL to about 1000 μL. [0073]
  • For the purposes of this invention, the terms “entry port” and “fluid input port” will be understood to mean an opening on a microsystems platform of the invention comprising a means for applying a fluid to the platform. [0074]
  • For the purposes of this invention, 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. [0075]
  • For the purposes of this invention, the term “capillary junction” will be understood to mean a region in a capillary or other flow path where surface or capillary forces are exploited to retard or promote fluid flow. A capillary junction is provided as a pocket, depression or chamber in a hydrophilic substrate that has a greater depth (vertically within the platform layer) and/or a greater width (horizontally within the platform layer) that the fluidics component (such as a microchannel) to which it is fluidly connected. For liquids having a contact angle less than 90° (such as aqueous solutions on platforms made with most plastics, glass and silica), flow is impeded as the channel cross-section increases at the interface of the capillary junction. The force hindering flow is produced by capillary pressure, that is inversely proportional to the cross sectional dimensions of the channel and directly proportional to the surface tension of the liquid, multiplied by the cosine of the contact angle of the fluid in contact with the material comprising the channel. The factors relating to capillarity in microchannels according to this invention have been discussed in co-owned U.S. Pat. No. 6,063,589, issued May 12, 2000 and in co-owned and co-pending U.S. patent application Ser. No. 08/910,726, filed Aug. 12, 1997, incorporated by reference in its entirety herein. [0076]
  • Capillary junctions can be constructed in at least three ways. In one embodiment, a capillary junction is formed at the junction of two components wherein one or both of the lateral dimensions of one component is larger than the lateral dimension(s) of the other component. As an example, in microfluidics components made from “wetting” or “wettable” materials, such a junction occurs at an enlargement of a capillary as described in co-owned and co-pending U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; and Ser. No. 08/910,726, filed Aug. 12, 1997. Fluid flow through capillaries is inhibited at such junctions. At junctions of components made from non-wetting or non-wettable materials, on the other hand, a constriction in the fluid path, such as the exit from a chamber or reservoir into a capillary, produces a capillary junction that inhibits flow. In general, it will be understood that 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). [0077]
  • A second embodiment of a capillary junction is formed using a component having differential surface treatment of a capillary or flow-path. For example, a channel that is hydrophilic (that is, wettable) may be treated to have discrete regions of hydrophobicity (that is, non-wettable). A fluid flowing through such a channel will do so through the hydrophilic areas, while flow will be impeded as the fluid-vapor meniscus impinges upon the hydrophobic zone. [0078]
  • The third embodiment of a capillary junction according to the invention is provided for components having changes in both lateral dimension and surface properties. An example of such a junction is a microchannel opening into a hydrophobic component (microchannel or reservoir) having a larger lateral dimension. Those of ordinary skill will appreciate how capillary junctions according to the invention can be created at the juncture of components having different sizes in their lateral dimensions, different hydrophilic properties, or both. [0079]
  • For the purposes of this invention, the term “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 and is due to a partially or completely wettable surface. [0080]
  • For the purposes of this invention, the term “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. Capillary microvalves will be understood to comprise capillary junctions that can be overcome by increasing the hydrodynamic pressure on the fluid at the junction, most preferably by increasing the rotational speed of the platform. [0081]
  • For the purposes of this 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. In preferred embodiments, 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. Pat. No. 6,063,589, incorporated by reference. [0082]
  • For the purposes of this invention, the term “in fluid communication” or “fluidly connected” is intended to define components that are operably interconnected to allow fluid flow between components. In preferred embodiments, 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. [0083]
  • For the purposes of this invention, the term “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. [0084]
  • The microplatforms of the invention (preferably and hereinafter collectively referred to as “disks”; for the purposes of this invention, the terms “microplatform”, “Microsystems platform” and “disk” are considered to be interchangeable) are provided to comprise one or a multiplicity of microsynthetic or microanalytic systems (termed “microfluidics structures” herein). Such microfluidics structures 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. For the purposes of this invention, the term “microfabricated” refers to processes that allow production of these structures on the sub-millimeter scale. These processes include but are not restricted to molding, photolithography, etching, stamping and other means that are familiar to those skilled in the art. [0085]
  • 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. Both electromechanical means and control means, as further described herein, 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. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. [0086]
  • Temperature control elements are provided to control the temperature of the platform during incubation of a fluid thereupon. The invention therefore 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. Preferably, 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 (preferably, the microchannels at any particular thermally regulated area) 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. Temperature control on the microsystems platforms of this invention is most preferably achieved using the methods and devices disclosed in co-owned U.S. Pat. No. 6,063,589, incorporated by reference herein. [0087]
  • In preferred embodiments, 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. Pat. No. 6,063,589, incorporated by reference. In more preferred embodiments, 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. In preferred embodiments, the platform also comprises other components as disclosed in co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), 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. In these embodiments, 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. In preferred embodiments, the arrays comprise at least 2 or 3 regions of different temperature adjacent to one another. In certain embodiments, 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. [0088]
  • In preferred embodiments of the platforms of the invention, the thermal arrays and regions of elevated temperatures constructed in the surface of the platforms of the invention comprise a thermal heating element. In preferred embodiments, 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. Pat. No. 6,063,587. Briefly, 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. Preferably, 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 7102, Dupont 7271, Dupont 7278 or Dupont 7285, or a PTC (positive temperature coefficient) ink. In alternative embodiments, the resistive heater element can further comprise a dielectric ink screen-printed over the resistive ink pattern and conductive ink pattern. [0089]
  • The invention provides a combination of specifically adapted microplatforms that are rotatable, analytic/synthetic microvolume assay platforms, 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 micromanipulation devices of the invention are more fully described in co-owned and co-pending U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. [0090]
  • 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 microfluidics structure on the Microsystems platform 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. In certain embodiments of the methods of the invention an unmetered amount of a fluid (either a sample or reagent solution) is applied to the platform and a metered amount is transferred from a fluid reservoir to a microchannel, as described in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. In preferred embodiments, the metered amount of the fluid sample provided on an inventive platform is from about 1 nL to about 500 μL. In these embodiments, metering manifolds comprising one or a multiplicity of metering capillaries are provided to distribute the fluid to a plurality of components of the microfluidics structure. [0091]
  • The components of the platforms of the invention are in fluidic contract with one another. In preferred embodiments, fluidic contact is provided by microchannels comprising the surface of the platforms of the invention. Microchannel sizes are optimally determined by specific applications and by the amount of and delivery rates of fluids required for each particular embodiment of the platforms and methods of the invention. Microchannel sizes can range from 0.1 μm to a value close to the thickness of the disk (e.g., about 1 mm); in preferred embodiments, the interior dimension of the microchannel is from 0.5 μm to about 500 μm. 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 25 mm, wherein the cross-sectional dimension of the microchannels across the thickness dimension of the platform is less than 1 mm, and can be from 1 to 90 percent of said cross-sectional dimension of the platform. Sample reservoirs, reagent reservoirs, reaction chambers, collection chambers, detections chambers and sample inlet and outlet ports preferably are embedded in a microsystem platform having a thickness of about 0.1 to 25 mm, wherein the cross-sectional dimension of the microchannels across the thickness dimension of the platform is from 1 to 75 percent of said cross-sectional dimension of the platform. In preferred embodiments, 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. [0092]
  • Input and output (entry and exit) ports are components of the microplatforms of the invention that are used for the introduction or removal 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. [0093]
  • In some embodiments of the platforms of the invention, the inlet and outlet ports are adapted to the use of manual pipettors and other means of delivering fluids to the reservoirs of the platform. In alternative, advantageous embodiments, the platform is adapted to the use of automated fluid loading devices. One example of such an automated device is a single pipette head located on a robotic arm that moves in a direction radially along the surface of the platform. In this embodiment, the platform could be indexed upon the spindle of the rotary motor in the azimuthal direction beneath the pipette head, which would travel in the radial direction to address the appropriate reservoir. [0094]
  • Also included in air handling systems on the disk are air displacement channels, 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. [0095]
  • Platforms of the invention such as disks and the microfluidics components comprising such platforms are advantageously provided having a variety of composition and surface coatings appropriate for particular applications. Platform composition will be a function of structural requirements, manufacturing processes, and reagent compatibility/chemical resistance properties. Specifically, 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, polyolefins, polypropylene and metallocene. These may be used with unmodified or modified surfaces as described below. The platforms may also be made from thermoset materials such as polyurethane and poly(dimethyl siloxane) (PDMS). Also provided by the invention are 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 the detection chamber of the platform. Alternately, platforms composed of layers made from different materials may be made. The surface properties of these materials may be modified for specific applications, as disclosed in co-owned U.S. Pat. No. 6,063,589, issued May 16, 2000, and co-owned and co-pending patent applications U.S. Ser. No. 08/761,063, filed Dec. 5, 1996; Ser. No. 08/768,990, filed Dec. 18, 1996; Ser. No. 08/910,726, filed Aug. 12, 1997; Ser. No. 08/995,056, filed Dec. 19, 1997; Ser. No. 09/315,114, filed May 19, 1999; Ser. No. 09/579,492, filed May 12, 2000 and Ser. No. ______, filed Jun. 16, 2000 (Attorney Docket No. 95,1408-XX), the disclosures of each of which are explicitly incorporated by reference herein. [0096]
  • Preferably, 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, as described in more detail below. It will also be recognized that platforms comprising a multiplicity of the microfluidics structures are also encompassed by the invention, wherein individual combinations of microfluidics and reservoirs, or such reservoirs shared in common, are provided fluidly connected thereto. An example of such a platform is shown in FIG. 1. [0097]
  • Platform Manufacture and Assembly [0098]
  • Microfluidics structures are provided embedded in a substrate comprising the microsystems platform of the invention. The platform is preferably manufactured and assembled as layers containing separate components that are bonded together. As illustrated in FIG. 1, the exemplified embodiment of the platforms of the invention comprise two layers, a reservoir layer and a microfluidics layer. Platforms having additional layers are also within the scope of the invention. [0099]
  • The reservoir layer of the platform is manufactured from a thermoplastic material such as acrylic, polystyrene, polycarbonate, or polyethylene. For such materials, fabrication methods include machining and conventional injection molding. For injection molding, the mold inserts that are used to define the features of the platform can be created using standard methods of machining, electrical discharge machining, and other means known in the art. [0100]
  • The reservoir layer of the platform can also be manufactured from a thermoset material or other material that exists in a liquid form until subjected to heat, radiation, or other energy sources. Examples of thermoset materials include poly(dimethyl siloxane) (PDMS), polyurethane, or epoxy. [0101]
  • Typically, these materials are obtained from the manufacturer in two parts; the two parts are mixed together in a prescribed ratio, injected into or poured over a mold and subjected to heat to initiate and complete cross-linking of the monomers present in the pre-polymer fluid. The process of rapidly injecting a pre-polymer fluid into a mold and then cross-linking or curing the part is often referred to as reaction injection molding (RIM). The process of pouring a pre-polymer fluid over a mold and then allowing the part to cross-link or cure is often referred to as casting. Mold inserts for RIM or casting can be fabricated using standard methods of machining, electrical discharge machining, and other means known in the art. [0102]
  • The microfluidics layer of the platform can also be manufactured from a thermoplastic material such as acrylic, polystyrene, polycarbonate, or polyethylene. Because the dimensions of the channels and cuvettes may be much smaller than those found in the reservoir layer, typical fabrication methods with these materials may include not only machining and conventional injection molding but also compression/injection molding, and embossing or coining. For injection molding, the mold inserts that are used to define the features of this layer of the platform can be created using conventional methods such as machining or electrical discharge machining. For mold inserts with features too fine to be created in conventional ways, various microfabrication techniques are used. These include silicon micromachining, in which patterns are created on a silicon wafer substrate through the use of a photoresist and a photomask (Madou, 1997, [0103] Fundaments of Microfabrication, CRC Press: Boca Raton, Fla.). When the silicon wafer is subjected to an etching agent, the photoresist prevents penetration of the agent into the silicon beneath the photoresist, while allowing etching to occur in the exposed areas of the silicon. In this way patterns are etched into the silicon and can be used to create microfabricated plastic parts directly through embossing. In this process, the etched silicon is brought into contact with a flat thermoplastic sheet under high pressure and at a temperature near the glass transition temperature of the plastic. As a result, the pattern is transferred in negative into the plastic.
  • Etched silicon may also be used to create a metal mold insert through electroplating using, for example, metallic nickel. Silicon etched using any one of a variety of techniques such as anisotropoic or isotropic wet etching or deep reactive ion etching (DRIE) may serve as a basis for a metal mold. A seed layer of nickel is deposited through evaporation on the silicon; once such an electrically-conductie seed layer is formed, conventional electroplating techniques may be used to build a thick nickel layer. Typically, the silicon is then removed (Larsson, 1997, [0104] Micro Structure Bull. 1: 3). The insert is then used in conventional injection molding or compression/injection molding.
  • In addition to silicon micromachining for mold inserts, molds can alternatively be created using photolithography without etching the silicon. Photoresist patterns are created on silicon or other appropriate substrates. Rather than etching the silicon wafer as in silicon micromachining, the photoresist pattern and silicon are metallized through electroplating, thermal vapor deposition, or other means known in the art. The metal relief pattern then serves as a mold for coining, injection molding, or compression/injection molding as described above. [0105]
  • The microfluidic layer of the platform can also be manufactured using a thermoset material as described above for production of the reservoir layer, wherein the mold pattern for thermosets of the microfluidics layer is prepared as described above. Because reaction-injection molding and casting do not require the high pressures and temperatures of injection molding, a wider variety of mold patterns may be used. In addition to the use of a silicon or metal mold insert, the photoresist pattern as described can also be used as a mold relief itself. While the photoresist would not withstand the high pressures and temperatures of injection molding, the milder conditions of casting or RIM create no significant damage. [0106]
  • The assembly of the platform involves registration and attachment of the microfluidic layer to the reservoir layer. In order for the microfluidics structures on the platform to be useful for performing assays as described herein, certain microfluidics pathways in the reservoir layer must be connected to certain microfluidics pathways in the microfluidics layer. Registration of these microfluidics pathways may be accomplished through optical alignment of fiducial marks on the microfluidic and reservoir layers or through mechanical alignment of holes or depressions on the microfluidic layer with pins or raised features on the reservoir layer. The required registration tolerances may be relaxed by designing the microfluidics pathway in the reservoir layer to be much larger than the microfluidics pathway in the microfluidics layer, or vice versa to Attachment may be accomplished in a number of ways, including conformal sealing, heat sealing or fusion bonding, bonding with a double-sided adhesive tape or heat-sealable film, bonding with a ultraviolet (UV) curable adhesive or a heat-curable glue, chemical bonding or bonding with a solvent. [0107]
  • A requirement for conformal sealing is that one or both of the layers are made of an elastomeric material and that the surfaces to be bonded are free of dust or debris that could limit the physical contact of the two layers. In a preferred assembly approach, an elastomeric microfluidics layer is registered with respect to and then pressed onto a rigid reservoir layer. The elastomeric microfluidics layer may be advantageously made of silicone and the rigid reservoir layer may be advantageously made of acrylic or polycarbonate. Hand pressure allows the layers to adhere through van der Waals forces. [0108]
  • A requirement for heat sealing or fusion bonding is that both the reservoir and microfluidics layers are made of thermoplastic materials and that the sealing occurs at temperatures above the glass transition temperatures, in the case of amorphous polymers, or melting temperatures, in the case of semi-crystalline polymers, of both of the layer materials. In a preferred assembly approach, the microfluidics layer is registered with respect to and pressed onto the reservoir layer, this composite disk is then placed between two flat heated blocks and pressure is applied to the composite through the heated blocks. By adjusting the temperature versus time profile at each of the faces of the composite disk and by adjusting the pressure versus time profile that is applied to the composite system, one can determine the time-temperature-pressure profile that allows for bonding of the two layers yet minimizes variation of the features within each of the layers. For example, heating two acrylic disks from room temperature to a temperature just above the glass transition temperature of acrylic at a constant pressure of 250 psi over one hour is a recipe that allows for minimal variation of 250 μm wide fluidic channels. In another assembly approach, the bond surfaces of the microfluidics and reservoir layers are separately heated in a non-contact fashion with radiative lamp and when the bond surfaces have reached their glass transition temperatures the microfluidics layer is registered with respect to and pressed onto the reservoir layer. [0109]
  • A double-sided adhesive tape or heat sealable film may be used to bond the microfluidics and reservoir layers. Before bonding, holes are first cut into the tape (or film) to allow for fluid communication between the two layers, the tape (or film) is registered with respect to and applied onto the reservoir layer, and the microfluidics layer is registered with respect to and applied onto the tape (or film)/reservoir layer composite. In order to bond a heat-sealable film to a surface, it is necessary to raise the temperature of the film to above the glass transition temperature, in the case of an amorphous polymer, or the melting temperature, in the case of a semicrystalline polymer, of the film's adherent polymer material. For bonding with an adhesive tape or a heat-sealable film, an adequate bond can typically be achieved with hand pressure. [0110]
  • A photopolymerizable polymer (for example, a UV-curable glue) or a heat-curable polymer may be used to adhere the microfluidics and reservoir layers. In one approach, this glue is applied to one or both of the layers. Application methods include painting, spraying, dip-coating or spin coating. After the application of the glue the layers are assembled and exposed to ultraviolet radiation or heat to allow for the initiation and completion of cross-linking or setting of the glue. In another approach, the microfluidics and reservoir layers are each fabricated with a set of fluid channels that are to be used only for the glue. These channels may, for example, encircle the fluid channels and cuvettes used for the assay. The microfluidics layer is registered with respect to and pressed onto the reservoir layer. The glue is pipetted into the various designated channels and after the glue has filled these channels, the assembled system is exposed to ultraviolet radiation or heat to allow for the cross-linking or setting of the glue. [0111]
  • When polydimethylsiloxane (PDMS) or silicone is first exposed to an oxygen plasma and then pressed onto a similarly treated silicone surface in an ambient environment, the two surfaces adhere. It is thought that the plasma treatment converts the silicone surface to a silanol surface and that the silanol groups are converted to siloxane bonds when the surfaces are brought together (Duffy et al., 1998, [0112] Anal. Chem. 1: 4974-4984). This chemical bonding approach is used to adhere the silicone microfluidics and reservoir layer.
  • A requirement for solvent bonding is that the bond surfaces of both the microfluidics and reservoir layers can be solvated or plasticized with a volatile solvent. For solvent bonding, the bond surfaces are each painted with the appropriate solvating fluid or each exposed to the appropriate solvating vapor and then registered and pressed together. Plasticization allows the polymer molecules to become more mobile and when the surfaces are brought in contact the polymer molecules become entangled; once the solvent has evaporated the polymer molecules are no longer mobile and the molecules remain entangled, thereby allowing for a physical bond between the two surfaces. In another approach, the microfluidics and reservoir layers are each fabricated with a set of fluid channels that are to be used only for the solvent and the layers are bonding much like they are with the UV-curable or heat-curable glue as described above. [0113]
  • Once assembled, the internal surfaces of the microfluidic manifold may be passivated with a parylene coating. Parylene is a vapor-deposited conformal polymer coating that forms a barrier layer on the internal, fluid-contacting surfaces of a microfluidic device following construction. The coating forms an impermeable layer that prevents any exchange of matter between the fluids and materials used to construct the device. The use of a low temperature, vapor deposition method allows the device to be manufactured and then passivated in its final form. This passivation approach can be used to improve the performance of assays. In particular, when an adhesive is used in the disk construction, there is a potential for contamination of the fluids by the adhesive material (or the plastic substrate or cover). Interfering substances leaching from the adhesive, or adsorption and binding of substances by the adhesive, can interfere with chemical or biochemical reactions. This can be more of a problem at elevated temperatures or if solvents, strong acids or bases are required. [0114]
  • Construction of Electric or Electronic Platen Comprising Temperature Control Elements [0115]
  • The invention provides an electric or electronic platen containing temperature control elements positioned on the platen to correspond to microfluidics structures such as thermal cycling chambers and sacrificial valves. The platen and microfluidics structures are aligned using fiducials or other registers for proper positioning the components on each platform layer with each other. [0116]
  • The invention also provides a micromanipulation apparatus for rotating the platen and microfluidics platform, including most preferably a slip ring feature on a rotational spindle or axis that permits electrical contact to be maintained between the device and the rotating platen. Temperature controlling elements are provided in the device to maintain any particular temperature at a specific position on the disc surface using thermistors and heating elements, including resistive heaters and Peltier elements. The device controls rotation of the microfluidics disc and distributes and receives electrical signals to the platen rotating with the microfluidics disc in real time. [0117]
  • The relationship between the device and platen is illustrated in FIG. 20. With regard to the Figure, platen [0118] 509 is inserted on a spindle containing 24-channel slip ring 510, commercially available from Litton, (Part No. AC6023-24). Rotation of the platen about the spindle is controlled by drive motor 507, preferably also comprising an encoder such as one commercially available from Micromo, (Part No. 3557K012CR), via drive belt 508. Drive motor 507 is controlled by the device through drive motor power line 505 and where application encoder signal line 506.
  • The device is controlled by microprocessor [0119] 501, most preferably comprising a computer such as a PC. Platform rotation is controlled by servomotor controller 503, for example as commercially available from J. R. Kerr (Part No. PIC-SERVO). Servo motor 503 is equipped with a power supply 504, commercially available from Skynet Electronic (Part No. ARC-2133). The servo motor is controlled by the PC through an interface, for example, using a serial port converter connected to the COM port of the PC (Part No. Z238485, J. R. Kerr).
  • The device is also provided having a control system for controlling electric power to the platen. A multiline cable [0120] 511 connects the slip ring to a breakout board 517, which is connected to a proportional integral derivative (PID) circuit connected to a commercially-available AJD board in the PC (Computer Boards, Part No. CIO-DAS1600) by temperature sensor line 512. This circuit receives temperature data from thermistors on the platen surface, disclosed more extensively below, and controls current delivery to temperature control elements by programmable current source 515 and power source 516.
  • The platen itself is shown in plan view in FIG. 21. The platen most preferably is constructed from printed circuit board [0121] 551 onto which electronic elements (including electrical leads, thermistors, Peltier elements, brass blocks for providing thermal contact with the microfluidics disc, and radiative fins for heat dissipation have been affixed.
  • FIG. 21 shows the layout of the temperature control elements on the platen, illustrated in the Figure with Peltier elements [0122] 554. The platen has brass thermal contacts 552 and 553 positioned on the platen surface to correspond to microfluidics structure on the microfluidics disc. Brass contact 552 has a groove 555 embedded therein to accommodate a temperature sensing element. Positioned in between the thermal contacts in each combination is Peltier element 554. Also illustrated in the Figure is a second temperature control element, comprising brass thermal contacts 557 and 558, groove 559, and Peltier element 558. The positioning of these elements permits temperature control and heating of multiple components of a particular microfluidics structure (such as control of thermal cycling chambers and lysis chambers, for example).
  • Electric leads controlling the temperature control elements on the platen are more specifically depicted in FIG. 22. Peltier element [0123] 554 is controlled by leads 601 and 605 connected through 607 and 608. Thermistor 606 contained in groove 555 is controlled (that is, the temperature information in the form of changes in resistance to current flow in the thermistor upon heating or cooling is transmitted to the temperature control elements in the PC) through leads 600 and 602 connected through 609 and 610. Similarly, Peltier element 558 is controlled by leads 603 and 605 connected through 612 and 613. Thermistor 611 contained in groove 559 is controlled (that is, the temperature information in the form of changes in resistance to current flow in the thermistor upon heating or cooling is transmitted to the temperature control elements in the PC) through leads 602 and 604 connected through 614 and 615. In construction of the electric connections between the elements on the platen and the slip ring, the use of the same lead as a “ground” (see, for example, the common connection to lead 602 between thermistor 606 and thermistor 611) conserves the number of connections used per element and permits control of up to 8 elements per platen.
  • The structure of the temperature control element is displayed in cross section in FIG. 23. Peltier element [0124] 554 is positioned on platen surface 551 between brass contacts 552 and 553 and held together with bolts 653 and 654. Brass contacts 552 and 553 act as heat sources and sinks to transfer heat to and from the Peltier element 554. The microfluidic disk sits on brass contact 552. When heating the disk, the top surface of the Peltier element 554 heats brass contact 552, and brass contact 553 is cooled. When cooling the disk, the top surface of the Peltier element 554 cools brass contact 554 and heats brass contact 553. An additional aluminum heat sink 652 is positioned in thermal contact with brass contact 553, providing additional heat sink capacity, enhancing Peltier element 554 performance. Aluminum heat sink 652 is mounted to the platen 551 using screws 651 and 655. Brass contact 552 contains thermistor 606 in a cavity containing alumina-filled epoxy 650 that increases the temperature sensitivity of the thermistor. Thermal grease is applied between pieces 552, 554, 553, and 652 to increase thermal contact between the parts.
  • In the use of the platen of the invention, the platform of the invention is assembled using thermal grease between the brass contacts and t