WO2006102396A2 - Plaque haute densite creusee d'une gorge - Google Patents

Plaque haute densite creusee d'une gorge Download PDF

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Publication number
WO2006102396A2
WO2006102396A2 PCT/US2006/010373 US2006010373W WO2006102396A2 WO 2006102396 A2 WO2006102396 A2 WO 2006102396A2 US 2006010373 W US2006010373 W US 2006010373W WO 2006102396 A2 WO2006102396 A2 WO 2006102396A2
Authority
WO
WIPO (PCT)
Prior art keywords
microplate
assay
wells
microplate according
main body
Prior art date
Application number
PCT/US2006/010373
Other languages
English (en)
Other versions
WO2006102396A3 (fr
Inventor
Donald Sandell
Ian A. Harding
Robin Li
Gary Lim
Original Assignee
Applera Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/087,101 external-priority patent/US20050225751A1/en
Priority claimed from US11/086,262 external-priority patent/US20050280811A1/en
Application filed by Applera Corporation filed Critical Applera Corporation
Publication of WO2006102396A2 publication Critical patent/WO2006102396A2/fr
Publication of WO2006102396A3 publication Critical patent/WO2006102396A3/fr

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Classifications

    • 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
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/028—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having reaction cells in the form of microtitration plates
    • 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
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B01L3/0262—Drop counters; Drop formers using touch-off at substrate or container
    • 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
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B01L3/0268—Drop counters; Drop formers using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
    • 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
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • 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
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50857—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using arrays or bundles of open capillaries for holding samples
    • 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
    • B01L2200/021—Adjust spacings in an array of wells, pipettes or holders, format transfer between arrays of different size or geometry
    • 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
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • 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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0642—Filling fluids into wells by specific techniques
    • 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
    • B01L2300/02—Identification, exchange or storage of information
    • B01L2300/021—Identification, e.g. bar codes
    • B01L2300/022—Transponder chips
    • 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
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • 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
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • 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
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • 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

Definitions

  • FIG. 3 is a top perspective view illustrating a microplate in accordance with some embodiments.
  • FIG. 15 is a cross-sectional view of the microplate of FIG. 14 taken along Line 15-15;
  • FIG. 23(b) is a top perspective view of a portion of a filling apparatus comprising a plurality of staging capillaries, microfluidic channels, and ramp features according to some embodiments;
  • FIG. 24 is a bottom perspective view of an output layer of a filling apparatus comprising spacer features according to some embodiments;
  • FIGS. 25(a)-(f) are top schematic views of a filling apparatus according to some embodiments.
  • FIG. 63 is an enlarged cross-sectional view of cap portion and main body portion of the multipiece microplate of FIG. 62;
  • FIG. 66(a) is a side view illustrating a loading distribution system according to some embodiments, comprising a dispensing device, a source plate and wash station, and a carriage;
  • FIG. 66(b) is a side view illustrating a loading distribution system according to some embodiments, comprising a dispensing device, a source plate station, a wash station, and a carriage;
  • FIG. 70 is a perspective view illustrating a carriage capable of holding a microplate according to some embodiments.
  • FIG. 77 is a perspective view illustrating a loading distribution system comprising the carriage, the table, and an alignment stage according to some embodiments;
  • FIG. 78 is a perspective view illustrating a lifting stage adapted to lift a carriage according to some embodiments.
  • FIG. 94 is a perspective view illustrating a source plate and wash station, wherein the source plate stays lidded and the washing tray can be accessed by a dispensing device according to some embodiments;
  • FIG. 105 is a top-plan view illustrating a mapping of fluid locations of a 384-well source plate into a dispensing device comprising 96 dispensers and further into a 6,144-well microplate according to some embodiments;
  • FIG. 108 is a cross-sectional view illustrating the filling apparatus comprising the intermediate layer according to some embodiments.
  • FIG. 110 is a top schematic view of the filling apparatus comprising the intermediate layer and nodules according to some embodiments.
  • FIG. 112 is a bottom perspective view of the intermediate layer of the filling apparatus according to some embodiments.
  • FIG. 113 is an exploded top perspective view illustrating a clamp system for a filling apparatus according to some embodiments.
  • FIG. 117 is a top schematic view of the filling apparatus comprising the vent layer and vent apertures positioned between staging capillaries according to some embodiments;
  • FIGS. 142(a)-(g) are cross-sectional views illustrating various possible positions and configurations microfluidic channels and staging capillaries according to some embodiments
  • FIG. 143 is an exploded perspective view illustrating a filling apparatus comprising a floating insert and cover according to some embodiments
  • FIG. 144 is a cross-sectional view illustrating the filling apparatus comprising the floating insert according to some embodiments.
  • FIG. 148 is a cross-sectional view illustrating a floating insert comprising tapered members according to some embodiments.
  • FIG. 151 is a cross-sectional view illustrating the floating insert comprising tapered members and the flanged base portion inserted into the corresponding depression and assay flow therebetween according to some embodiments;
  • FIG. 152 is a cross-sectional view illustrating the floating insert comprising tapered members and the flanged base portion being forced down onto the corresponding depression according to some embodiments;
  • FIG. 163 is a perspective view illustrating a filling apparatus comprising a surface wire assembly, a reservoir trough, and absorbent member according to some embodiments;
  • FIG. 170 is a cross-sectional view illustrating a funnel member comprising a tip portion according to some embodiments
  • FIG. 179 is an exploded top perspective view illustrating a multipiece funnel member comprising portions separated generally vertically according to some embodiments
  • FIG. 195 is a perspective view, with portions removed for clarity, illustrating a single cover cartridge according to some embodiments
  • FIG. 197 is an exploded perspective view illustrating the single cover cartridge according to some embodiments.
  • FIG. 207 is a pneumatic diagram illustrating a pneumatic system for a pressure chamber and a clamp mechanism according to some embodiments
  • a high density sequence detection system can further comprise a thermocycler.
  • a high density sequence system can further comprise microplate and components for, e.g., filling and handling the microplate, such as a pressure clamp system. It will be understood that, although high density sequence detection systems are described herein with respect to specific microplates, assays and other embodiments, such systems and components thereof are useful with a variety of analytical platforms, equipment, and procedures.
  • microplate 20 comprises a thickness of about 1.25 mm. In some embodiments, microplate 20 comprises a thickness of about 2.25 mm.
  • microplate 20 and skirt portion 30 can be formed in dimensions other than those specified herein.
  • each of the plurality of wells 26 comprises a generally square-shaped rim portion 38 with downwardly-extending sidewalls 40 that terminate at a bottom wall 42.
  • a draft angle of sidewalls 40 can be used. Again, the particular draft angle is determined, at least in part, by the manufacturing method and the size of each of the plurality of wells 26.
  • generally square-shaped rim portion 38 can have a side dimension of about 1.0 mm in length, a depth of about 0.9 mm, a draft angle of about 1° to 5° or greater, and a center-to-center distance of about 1.125mm, generally indicated at A (see FIG. 27).
  • microplate 20 comprises an alignment feature 58, such as a corner chamfer, a pin, a slot, a cut corner, an indentation, a graphic, or other unique feature that is capable of interfacing with a corresponding feature formed in a fixture, reagent dispensing equipment, and/or thermocycler.
  • alignment feature 58 comprises a nub or protrusion 60 as illustrated in FIG. 14.
  • alignment features 58 are placed such that they do not interfere with sealing cover 80 or at least one of the plurality of wells 26. However, locating alignment features 58 near at least one of the plurality of wells 26 can provide improved alignment with dispensing equipment and/or thermocycler block 102.
  • a radio frequency identification (RFID) tag 76 can be used to electronically identify microplate 20.
  • RFID tag 76 can be attached or molded within microplate 20.
  • An RFID reader (not illustrated) can be integrated into high-density sequence detection system 10 to automatically read a unique identification and/or data handling parameters of microplate 20. Further, RFID tag 76 does not require line-of-sight for readability. It should be appreciated that RFID tag 76 can be variously configured and used according to various techniques, such as those described in commonly-assigned U.S. Patent Application No. , entitled
  • the thermally conductive material comprises a powder.
  • the particle size used herein can be between 0.10 micron and 300 microns.
  • powders provide uniform (i.e. isotropic) thermal conductivity in all directions throughout the composition of the microplate.
  • microplate 20 comprises, at least in part, an electrically conductive material, which can improve reagent dispensing alignment.
  • electrically conductive material can reduce static build-up on microplate 20 so that the reagent droplets will not go astray during dispensing.
  • a voltage can be applied to microplate 20 to pull the reagent droplets into a predetermined position, particularly with a co-molded part where the bottom section can be electrically conductive and the sides of the plurality of wells 26 may not be electrically conductive.
  • a voltage field applied to the electrically conductive material under the well or wells of interest can pull assay 1000 into the appropriate wells.
  • microplate 20 can be made, at least in part, of non-electrically conductive materials.
  • non-electrically conductive materials can at least in part comprise one or more of crystalline silica (3.0 W/mK), aluminum oxide (42 VWmK), diamond (2000 VWmK), aluminum nitride (150-220 VWmK), crystalline boron nitride (1300 VWmK), and silicon carbide (85 VWmK).
  • microplate 20 can be molded by first receiving pellet material from a resin supplier; drying the pellet material in a resin dryer; transferring the dried pellet material with a vacuum system into a hopper of a mold press; molding microplate 20; trimming any resultant gates or flash; and packaging microplate 20.
  • the mold cavity can be centrally gated along the second surface 24 of microplate 20. In some embodiments, the mold cavity can be gated along a perimeter of main body 28 and/or skirt portion 30 of microplate 20.
  • the spotted microplate 720 can then be moved from spotting device 700, as indicated by 722. Spotted microplate 720 can then be moved to an optional quality-control station 724, as indicated by 726. After quality-control station 724, spotted microplate 720 can then be moved back to low-humidity storage unit 714, as indicated by 728. This procedure of spotting microplates 20 can continue until a desired number (e.g. all) of microplates in storage unit 714 have been spotted with reagents from storage plate 704. It should be noted that unspotted microplate 712 and spotted microplate 720 are each similar to microplate 20, however different numerals are used for simplicity in the above description.
  • this preloading process can vary as desired to accommodate user needs.
  • the reagents spotted in each of the plurality of wells 26 can be encapsulated with a material. Such encapsulation can prevent or reduce moisture at room temperature from interacting with the reagents.
  • each of the plurality of wells 26 can be spotted several times with reagents, such as for multiplex PCR. In some embodiments, these multiple spotted reagents can form layers.
  • primer sets and detection probes for a whole genome can be spotted from storage plates 704 onto spotted microplate 720. In other embodiments, a portion of a genome, or subsets of selected genes, can be spotted from source plates 704 onto spotted microplate 720.
  • spotted microplate 720 can be sealed with a protective cover, stored, and/or shipped to another location.
  • the protective cover is releasable from spotted microplate 720 in one piece without leaving adhesive residue on spotted microplate 720.
  • the protective cover is visibly different (e.g., a different color) from sealing cover 80 to aid in visual identification and for ease of handling.
  • the protective cover can be made of a material chosen to reduce static charge generation upon release from spotted microplate 720.
  • the package seal can be broken and the protective cover can be removed from spotted microplate 720.
  • the protective cover can be a pierceable film, a slitted film, or a duckbilled closure to, at least in part, reduce contamination and/or evaporation.
  • An analyte (such a biological sample comprising DNA) can then be added to spotted microplate 720, along with other materials such as PCR master mix, to form assay 1000 in at least some of the plurality of wells 26.
  • Spotted microplate 720 can then be sealed with sealing cover 80 as described above.
  • High-density sequence detection system 10 can then be actuated to collect and analyze data.
  • the filling apparatus comprises a device for depositing (e.g., spotting or spraying) of assay 1000 to specific wells, wherein one or more of the plurality of wells 26 of microplate 20 contains a different assay material than other wells 26 of microplate 20.
  • the device can include piezoelectric pumps, acoustic dispersion, liquid printers, or the like.
  • a pin spotter can be employed, such as described in PCT Publication No. WO 2004/018104.
  • a fiber and/or fiber-array spotter can be employed, such as described in U.S. Patent No. 6,849,127.
  • a loading distribution system 800 comprising a conveyer or a track 802 can be used to set up an expandable and flexible microplate loading distribution system.
  • FIG. 64 depicts four dispensing devices 814, 816, 818, and 820, disposed adjacent a corresponding source plate and wash station 814a, 816a, 818a, and 820a, respectively.
  • Dispensing devices 814, 816, 818, and 820 can each comprise a plurality of dispensers, for example, 24-dispensers, 48-dispensers, 96-dispensers, 384-dispensers.
  • the dispensing device can comprise, for example, at least 96 dispensing tips, at least 384 dispensing tips, at least 768 dispensing tips, at least 1536 dispensing tips, or more.
  • the dispensing device can comprise a plurality of dispensers and each dispenser can comprise a piezo-electric dispenser.
  • the dispensing device in loading distribution system 800 can comprise a plurality of dispensers and a respective plurality of storage reservoirs. Each dispenser can be designed to dispense a first volume of fluid per dispensing action, and each reservoir can be adapted to store many times the first volume, for example, at least 15 times the first volume, at least 25 times the first volume, at least 50 times the first volume, or at least 100 times the first volume.
  • each of the plurality of dispensers can be adapted to dispense about 100 nanoliters of liquid or fluid, per dispensing action.
  • the dispensing device can comprise a plurality of spotting devices.
  • the dispensing devices can comprise, for example, piezo-electric devices, acoustic devices, ink-jet devices, pump-action devices, pin spotters, or the like, or a combination thereof.
  • the number of dispensing devices 814, 816, 818, and 820 disposed around a conveyer 802 can be increased or decreased so as to address a desired throughput target.
  • conveyer 802 can expand (be lengthened) in an X-direction. This can allow more dispensing devices to be disposed around conveyer 802.
  • Conveyer 802 can comprise a track, for example, SuperTrakTM available from ATS Automation Tooling Systems Inc. However, it should be understood that other tracks can be used.
  • loading distribution system 800 can comprise a load position 806 on conveyer 802.
  • Loading distribution system 800 can comprise an unload position 808 on conveyer 802.
  • Load position 806 and unload position 808 can, according to some embodiments, be a same position along conveyer 802.
  • loading distribution system 800 can comprise a machine indicia reader 804 disposed along conveyer 802.
  • Machine indicia reader 804 can, according to some embodiments, comprise a plurality of machine indicia readers, one each disposed prior to every dispensing device along conveyer 802.
  • machine indicia reader 804 can be disposed past load position 806 along conveyer 802.
  • conveyer 802 transports, in serial fashion, empty microplates from a hotel or storage unit 828 to a position adjacent a load position 806.
  • Handling device 830 places the microplate on a table and carriage assembly for movement along conveyer 802.
  • the microplate is then moved by the table and carriage assembly along conveyer 802 to machine indicia reader 804.
  • the method of tracking can comprise scanning indicia on the bottom of the microplate.
  • the microplate can then be advanced to a position below an inspection station 810 that inspects each well of the microplate for the presence of spotted components of an assay. If the inspection operations indicate that the microplate has been properly loaded with components of an assay, the microplate is then moved along conveyer 802 to an unload position 808 where the microplate can be unloaded, for example, by handling device 830, and moved back to the storage unit 828. If a failure is indicated, on the other hand, unloading at unload position 808 can comprise depositing the microplate in a reject bin.
  • dispensing device 814 can comprise a plurality of dispensers 868.
  • a carriage 874 can be disposed on conveyer 802.
  • Carriage 874 can be positioned under dispensers 868, when dispensing of a fluid in or on microplate 20 is desired.
  • Microplate 20 can be disposed on a table 872.
  • Table 872 can comprise a vacuum chuck; see FIG. 80, adapted to hold microplate 20.
  • Table 872 can move to align microplate with dispensers 868.
  • Conveyer 802 can translate carriage 874 away from the dispensing position.
  • Carriage 874 can move along conveyer 802.
  • loading distribution system 800 can further comprise an accessory carriage configured to engage a source plate comprising a source of fluids to be loaded into the spotting or other dispensing station.
  • the accessory carriage can be adapted to move the source plate to the dispensing station for aspiration of the fluids from the source plate into the dispensing device.
  • Loading distribution system 800 can further comprise an incubator adapted to store the source plate, for example, to keep it in a cooler and more humid environment relative to the immediately surrounding atmosphere.
  • Loading distribution system 800 can comprise a source plate-handling device adapted to translate a source plate from the incubator to the dispensing station.
  • the incubator can comprise a de-lidder adapted to remove a lid from a source plate in loading distribution system 800.
  • the de-lidder in loading distribution system 800 can further be adapted to place a lid on a source plate.
  • Source plate and wash pallet 864 can be disposed on an elevator mechanism (not illustrated) to move source plate and wash pallet 864 within range of dispensers 868.
  • Source plate and wash pallet 864 can be in a rest position or a washing position. While in a rest position, washing tray 861 can be covered using a dust cover 866. Dust cover 866 can be hinged.
  • loading distribution system 800 can further comprise a plurality of source plates in the incubator, wherein the dispensing device comprises a plurality of multi-tip dispensing heads, and the source plate handling device can be adapted to translate one or more of the plurality of source plates from the incubator to each of the plurality of multi-tip dispensing heads. [0284] In FIG.
  • loading distribution system 800 can be adapted to convey the table along the X-axis direction.
  • the conveyance can be repeatably positionable to within about 100 micrometers of a predefined location.
  • a conveyer can be used that serially translates one or more of a plurality of tables, for example, with each table being disposed on a respective carriage.
  • the plurality of tables can be translated, for example, consecutively translated, to each of the plurality of processing stations.
  • a vacuum line supply 890 can provide communication from table 872 to a bellows 896. Bellows 896 can communicate with a vacuum connection shoe 907.
  • FIG. 71 illustrates a spring 908 that holds table 872 of carriage 874 against one corner.
  • FIG. 74 is a perspective view illustrating an embodiment of a pressure source 918 adapted to communicate with vacuum connection shoe 907.
  • Vacuum connection shoe 907 can comprise a port 920 on the opposite side that can engage with a vacuum supply port 916 disposed in a frame 914 attached to conveyer 902.
  • Bellows 896 or other means known in the art, can allow a flexible connection between vacuum connection shoe 907 and table 872 that can move up and down, and shift sideways.
  • vacuum connection shoe 907 can be disposed next to vacuum port 916 on frame 914.
  • Cam rails 922, 924 can be rising up, for example, by activating air-operated glide 921, to meet carriage 874 as it enters a station as long as cam rails 922, 924 are in position when roller 906, a Z-axis control roller, engages with slotted rail 926.
  • tripod 901 can drop.
  • table 872 rests on tripod 901
  • table 872 can drop down with tripod 901.
  • rollers 894 and 892 can engage cam rails 922, 924.
  • first cam 884 and second cam 886 can lift microplate 20 off table 872.
  • first cam 884 and second cam 886 can be dropped, by lowering air-operated slide 921 that in turn lowers cam rails 922, 924.
  • the lowering of cam rails 922, 924 can disengage rollers 894 and 892 from cam rails 922, 924, which in turn can open first cam 884 and second cam 886 releasing a gripped microplate 20.
  • the release can performed when, for example, a plate gripper robot 784 is ready to remove a microplate. Plate gripper robot 784 is illustrated in FIGS 82-90 described below.
  • Alignment stage 932 can comprise a long stroke actuator 935 for the X-axis since microplate 20 disposed on table 872 can index over a substantial distance for some kinds of dispensing, for example, dispensing of fluids for Focused Genome dispensing.
  • the X-axis carries two short stroke Y-axis actuators 934, 936.
  • the Y-axis actuators 934, 936 can operate independently from each other to compensate for skew.
  • alignment stage 932 works in cooperation with locating pins 882a, 882b, and 882c.
  • a location of microplate 20 can be offset in varying degrees from the center of dispensing device 814 to satisfy a need to interleave subsets of dot patterns or dispensing locations, and to form stripe pattern offsets for Focused Genome dispensing.
  • a system requiring operator intervention to mechanically align dispensing device 814 with the independent axes of motion, for example, X, Y, and Z-axis, can be very difficult to maintain.
  • loading distribution system 800 can work without a need for precision alignment by an operator after maintenance on loading distribution system 800 has been performed.
  • a periphery scan vision system or plate check vision system can be disposed upstream of a dispensing device to check the position and accuracy of microplate 20, prior to a dispensing by a dispensing device.
  • the periphery scan vision system can utilize a camera mount 941 to hold two cameras 946, 948. Cameras 946, 948 can be narrow focus cameras. Cameras 946, 948 can check the location of two or three dispensing locations.
  • the periphery scan vision system can comprise a carriage alignment 944 similar in functionality to carriage alignment device 945 described above.
  • the periphery scan vision system can comprise a marker indicia reader station.
  • a machine indicia reader for example, a bar code reader, can be used with a mirror to reflect a bar code on a microplate to separate reader assembly.
  • 50-micron repeatability can be desired for X, Y, and Z direction movements at a dispensing station.
  • the carriage can be driven on a conveyer or track by a linear stepper motor.
  • the dispensing device and dispensers therein can be held stationary.
  • Various components, for example, the conveyer, of loading distribution system 800 can be provided with EMI shielding.
  • FIG. 91 is a perspective view illustrating source plate and wash pallet 864 comprising washing tray 861 and source plate holder 863.
  • a source plate 862 can be disposed in source plate holder 863.
  • Washing tray 861 can comprise internal wash slots 878 and external wash slots 876. Washing tray 861 can be available from Aurora Discovery, Inc.
  • Source plate-handling device 822 can pick-up and deposit a source plate 862 from source plate holder 863 using a gripper 823.
  • Source plate 862 can be covered using a lid 860.
  • Lid 860 can be placed on source plate 862 by a de-lidding device 868.
  • De-lidding device 868 can comprise a lifting device 856 adapted to lift and hold lid 860.
  • Source plate and wash pallet 864 can be disposed on an elevator mechanism (not illustrated) to move source plate and wash pallet 864 within range of dispensers 868.
  • Source plate and wash station 814a can be in a rest position or a washing position, when an elevator mechanism is used. While in a rest position, washing tray 861 can be covered using a dust cover 866. Dust cover 866 can be hinged.
  • FIG. 99 is a side-plan view of source plate and wash station 814a in the wash position with respect to conveyer 802 and dispensing device 814.
  • a second slide 869 stays retracted.
  • First slide 867 slides crossways, and shifts to one-side so that source plate 872 is not under lid 860 holding mechanism and an external SCARA or 5-axis robot, like store plate-handling unit 822 can load and unload the source plate 872.
  • source plate and wash station 814a can extend on second slide 869 to position source plate 862 for aspiration by a dispensing device.
  • a robot arm can remove a correct source plate from an incubator and place it onto a source plate location.
  • the source plate can be moved to a de-lidder that can be mounted under a dust cover.
  • the lid of the source plate can be removed using the de-lidder.
  • FIG. 100 is a perspective view illustrating a hotel and a movable entry guide.
  • reliable insertion of microplates into shelves can be facilitated by adding an entry guide 974 that captures a leading edge of a microplate.
  • the vertical position of the edge can vary from microplate warping and/or variation in how a microplate can be gripped by a jaw of a plate gripper robot.
  • a shelf 970 can provide support for plate storage unit 828.
  • Entry guide 974 can be indexed using a linear motor 972.
  • system controller 982 can manage and track source plates and microplates at various locations in loading distribution system 800 (FIGS. 64 and 65).
  • Locations for a source plate can comprise, for example, in a source plate storage unit like an incubator, in one or more source plate holders, or in one or more grippers of one or more source plate handling devices.
  • Locations for a microplate can comprise, for example, in one or more plate storage units, in or on one or more tables, or in one or more jaws of one or more plate handling devices.
  • System controller 982 can be adapted to track and trace the contents of one or more dispensers, each disposed in one or more respective dispensing devices.
  • system controller 982 When processing a work order or manufacturing microplates, system controller 982 provides control, control, and communication for wash station assemblies module 984, a tip firing controller 986, a dispensing assemblies module 988, an incubator controller 990 also known as a source storage unit controller, an incubator robot controller 992 also known as a storage plate handling device controller, a fluidics controller 994, a hotel module 996 also known as a storage unit controller, a hotel robot controller 998 also known as a plate handling device controller, a bar code controller 976 also known as a marking indicia reader controller, a XYZ motion controller 978, and a quality control controller 929.
  • Wash station assemblies module 984, tip firing controller 986, dispensing assemblies module 988, incubator controller 990, incubator robot controller 992, fluidics controller 994, hotel module 996, hotel robot controller 998, and bar code controller 976 can be provided as part of one or more Original Equipment Manufacturer (OEM) packages including Application Protocol Interfaces (API) for all subassemblies.
  • System controller 982 and XYZ motion controller 978 can be provided using real-time manufacturing protocols, for example, Supervisory Control And Data Acquisition (SCADA), a computer system for gathering and analyzing real time data.
  • Quality control controller 929 can comprise a decision maker.
  • QC controller 929 can gather data and status from various systems comprising a loading distribution system, to render a decision for each microplate processed by loading distribution system.
  • the array of dispensers can be aligned to a microplate, in order to accomplish parallel dispensing of different reagents into different locations at the same time.
  • Dispensers can dispense spots of an assay reagent into one or more locations of a microplate by, for example, aspirating a volume of assay reagent sufficient for multiple spots. The aspirated volume can subsequently be dispersed as spots into multiple locations, where each location receives substantially the same mass of assay reagent.
  • a dilution problem can be observed using arrayed dispensers. Dilution can occur because a dispenser system fluid can dilute an assay reagent, as it is dispensed. Because a dispenser can dispense a volume of the reagent and system fluid, a reduced mass of assay reagent can be deposited into each location from dispensing action to dispensing action.
  • a dispenser can be programmed to compensate for the dilution affect.
  • the aspirate and dispense arrayed liquid handling technologies can dispense different amounts of assay reagents for each nozzle for each dispense action.
  • the level of dilution can be measured, and the measured curves can be used to calibrate the effect of dilution.
  • a method for calibrating the observed diffusion on a tip-by-tip basis, and compensating for the loss of dispensed assay reagent per nozzle from dilution by programming dispensing to dispense more solution per spot is provided.
  • a required increase in spot volumes can be calculated by mathematically integrating an area under a fluorescence-dispense calibration curve.
  • dynamic programming of the dispense volumes can provide microplate to microplate reproducibility of dispensed mass of assay reagents (spots), and can reduce assay reagent waste by allowing the use of highly diluted assay reagents from the dispensing device.
  • assay 1000 can be distributed on microplate 20 using a filling apparatus, such as filling apparatus 400, a robotic filler, or a manual filler to distribute one or more components of assay 1000 across microplate 20 in columns or bands, for example, as illustrated in FIG. 102.
  • a filling apparatus such as filling apparatus 400, a robotic filler, or a manual filler to distribute one or more components of assay 1000 across microplate 20 in columns or bands, for example, as illustrated in FIG. 102.
  • the sample distribution can map to this columnar or banded format.
  • FIG. 102 illustrates sample distribution in a banded format using a robotic or manual filler head.
  • the head comprises tips 746, 748, 750, 752, 754, 756, 758, and 760, respectively. Tips 746, 748, 750, 752, 754, 756, 758, and 760 can aspirate fluids from source plate 862.
  • Source plate 862 can comprise, for example, a 96 or a 384-location plate, including, for example, biological reagents or pre-amplified samples. Tips 746, 748, 750, 752, 754, 756, 758, and 760 can distribute the aspirated samples across microplate 20 to form bands or columns across microplate 20, for example, bands about 9 mm wide, bands about 4.5 mm wide, bands about 2.25 mm wide, or bands about 1.125 mm wide.
  • the microplate can include, for example, 6,144 wells. Tips 746, 748, 750, 752, 754, 756, 758, and 760 can dispense individual samples in bands across a plurality of rows of microplate 20. As illustrated in FIG.
  • FIG. 31 illustrates the use of a dead row between sample-loaded wells that can be used to avoid cross-contamination of two rows to be tested, taking advantage of a banded format.
  • FIG. 103 illustrates a microplate 764.
  • rows run from left to right.
  • Microplate 764 includes three rows, illustrated from left to right in the figure, including a first row into which a first sample is loaded and including sample wells 766.
  • a second row into which a second sample is loaded includes sample wells 770.
  • the row containing sample wells 768, located in between the rows respectively containing sample wells 766 and sample wells 770, can be used as a dead row and can be skipped during a sample loading process.
  • any of the first or second samples might stray from its intended row, it can be captured in the dead row. That is, if a sample deposited in well or location 766 or well or location 770 of microplate 764, carries over to an adjacent location 768, no problem arises because the results of any assays in wells 768 would not be analyzed. For example, when using a robotic or manual filler, any possible cross-contamination between samples can be prevented by leaving approximately one unused row (a "dead row") between each band of loaded samples in the microplate.
  • the dead row can comprise one or more rows.
  • a method of avoiding cross-contamination of a plurality of samples disposed in locations of a microplate can be provided.
  • the method can include loading a filling device that can include a plurality of dispensers, each dispenser can include a fluid; translating the filling device along a translation path traversing a microplate that can include rows of locations; and dispensing a band of a respective fluid from each of the dispensers along a portion of the translation path to load rows of the locations, where the bands do not contact one another and the rows include loaded rows and a dead row between otherwise adjacent loaded rows.
  • Bands can contain the same set of samples or assay reagents across the microplate. One row can be eliminated from each band on the microplate. Where one band or one sample is provided on the microplate, there can be no need for a dead row to prevent sample cross-contamination.
  • the microplate incubator 778 can store a microplate that is unspotted, partially spotted, or fully spotted.
  • the source incubator 776 can include circulated high humidity filtered air in order to prevent evaporation of the source assay reagents from the stored source plate.
  • Microplate incubator 778 can include circulated low humidity filtered air to dry the spotted assay reagents.
  • Microplate incubator 778 can maintain the spotted dried assay reagents in a dried state on the spotted microplate.
  • Microplate incubator 778 can prevent a post- batch drying step.
  • a system and method for manufacturing a microplate comprising a plurality of fluid samples, for example, about 768 or more samples, about 1536 or more fluids, about 3072 or more fluids, about 6,144 or more fluids, about 12,288 or more fluids, are described.
  • the plurality of fluids can all be the same fluid and in some embodiments each fluid can be different from all the other fluids.
  • the plurality of fluids can reside in or on a microplate.
  • fluids to loading distribution system 800 can be provided using a source plate, for example, a multiwell source plate.
  • the source plate can comprise 24 or more wells, for example, 48 or more wells, 96 or more wells, 192 or more wells, 384 or more wells, or 768 or more wells.
  • the method of dispensing can further involve loading from a plurality of source plates, for example, four, eight, 16, 32, 64, 96, 384, or more.
  • the first and second source plates can be the same and the first plurality of fluids can be a different plurality of fluids than the second plurality of fluids.
  • the first plurality of fluids can be the same plurality of fluids as the second plurality of fluids.
  • the first plurality of fluids can comprise a first plurality of mixtures, and each mixture can comprise two or more reagents for a nucleic acid sequence reaction.
  • the method can comprise spotting a microplate that comprises, for example, 6,144 or more wells.
  • control software and/or a dispensing device can be utilized that is configurable for a list of variables. Exemplary variables can be found herein in the EXAMPLE section.
  • Loading distribution system 800 can utilize, for example, a 96-dispenser dispensing device, or a 384-dispenser dispensing device. Loading distribution system 800 can utilize, for example, 1 , 2, 4, 8, 16, or more than 16 dispensing devices. Loading distribution system 800 can be designed to mitigate a throughput bottleneck at a dispensing device.
  • a filling apparatus 400 can be used to fill at least some of the plurality of wells 26 of microplate 20 with one or more components of assay 1000. It should be understood that filling apparatus 400 can comprise any one of a number of configurations.
  • output layer 408 can comprise a protrusion 450 formed on an outlet 434 of staging capillary 410.
  • protrusion 450 can be shaped to cooperate with a corresponding shape of each of the plurality of wells 26.
  • protrusion 450 can be conically shaped to be received within circular rim portion 32 of each of the plurality of wells 26.
  • protrusion 450 can be square- shaped to be received within square-shaped rim portion 38 of each of the plurality of wells 26.
  • Protrusion 450 in some embodiments, can define a sufficiently sharp surface such that the capillary force within staging capillary 410 can retain assay 1000 and protrusion 450 can inhibit movement of assay 1000 to adjacent wells 26.
  • protrusion 450 of output layer 408 can be positioned above microplate 20, flush with first surface 22 of microplate 20 (FIG. 22(a)), or disposed within well 26 of microplate 20 (FIG. 22(b)).
  • protrusion 450 can define a nozzle feature that comprises a diameter that is less than the diameter of the plurality of wells 26 to aid, at least in part, in capillary retention of assay 1000 within staging capillary 410.
  • the plurality of microfluidic channels 406 can have any one of a plurality of configurations for carrying assay 1000 to each of the plurality of staging capillaries 410.
  • each of the plurality of staging capillaries 410 can be in fluid communication with only one of the plurality of microfluidic channels 406 (FIGS. 23(a)- (b), 25(a)-(d), and 25(f)) in a series-type configuration.
  • each of the plurality of staging capillaries 410 can be in fluid communication with two or more of the plurality of microfluidic channels 406 (FIGS.
  • base structure 513 comprises at least one alignment feature 517 operably sized to engage a corresponding alignment feature 58 on microplate 20 to, at least in part, facilitate proper alignment of each of the plurality of staging capillaries 410 relative to each of the plurality of wells 26.
  • alignment feature 517 can further engage a corresponding alignment feature 519 formed in at least one of input layer 404, intermediate layer 494, and output layer 408.
  • vent feature 529 can be sized to have a capillary force associated therewith that is lower than a capillary force within microfluidic channels 406 and/or each of the plurality of staging capillaries 410 to reduce the likelihood of assay 1000 flow through or into vent feature 529.
  • vent feature 529 comprises a vent hole 531 extending through input layer 404 (FIGS. 114-118) and in communication with atmosphere.
  • vent hole 531 can be coupled to a chamber or manifold 533 (FIGS. 115 and 116) that can couple two or more vent apertures 535 formed in vent layer 523 to atmosphere.
  • Pressure bores 537 in some embodiments, can be sized to act as an air capacitor trapping a portion of air therein that can contract or expand during filling of assay 1000 into filling apparatus 400 and/or centrifuging assay 1000 into each of the plurality of wells 26, respectively.
  • vent layer 523 can be aligned with input layer 404 and output layer 408 such that vent apertures 535 are positioned above or between each of the plurality of staging capillaries 410.
  • vent apertures 535 can be a circular bore (FIG. 117) or any other shape, such as oblong (FIG. 118), to accommodate for potential misalignment between input layer 404 and vent layer 523 and/or potential misalignment between vent layer 523 and output layer 408.
  • vent layer 523 can be made of any material conducive to joining with input layer 404 and/or output layer 408.
  • vent layer 523 can comprise PDMS, which can aid in joining vent layer 523 to input layer 404 due to the intrinsic tackiness properties of PDMS.
  • vent layer 523 can be made using a double stick adhesive tape. In such embodiments, the double stick adhesive tape can be first applied to input layer 404 and then laser cut to accurately place vent apertures 535 to simplify assembly of input layer 404 and vent layer 523.
  • a predetermined amount of assay 1000 can be placed at each assay input port 402.
  • Such placement can be effected, for example, using an automated pipette system (e.g., a Biomek) or hand-operated single- or multi-channel pipette device (e.g., a Pipetman).
  • Capillary force at least in part, can draw at least a portion of assay 1000 from assay input port 402 into microfluidic channels 406 and further fill at least some of the plurality of staging capillaries 410.
  • filling apparatus 400 can comprise assay input ports 402 positioned within and/or upon output layer 408.
  • assay input ports 402 can be positioned at an end 420 of output layer 408.
  • such assay input ports can be positioned along a short dimension of a major surface (e.g., a top surface) of the output layer, adjacent and parallel to an end thereof.
  • assay input ports 402 can be positioned at a side 422 of output layer 408.
  • such assay input ports can be positioned along a long dimension of a major surface (e.g., a top surface) of the output layer, adjacent and parallel to a side thereof.
  • assay input ports 402 can be positioned at opposing ends 420 or opposing sides 422 (not illustrated) of output layer 408.
  • assay input ports 402 can be positioned at opposing ends 420 or opposing sides 422 (not illustrated) of output layer 408 with a fluid interrupt 409 (e.g. wall or barrier) to fluidly isolate those assay input ports 402 on one end or side from the remaining assay input ports 402 on the other end or side.
  • a fluid interrupt 409 e.g. wall or barrier
  • assay input ports 402 can each comprise a fluid well 424 bound by a plurality of upstanding walls 426.
  • fluid well 424 of each assay input port 402 can be in fluid communication with one or more corresponding microfluidic channels 406 through a throat 430 formed in fluid well 424.
  • a throat can be formed in a lower region of the fluid well, so as to fluidly communicate the fluid well with the microfluidic channels.
  • Throat 430 can comprise a diameter of, for example, 2 mm or less, 1mm or less, 0.5 mm or less, or 0.25 mm or less. In some embodiments, such as illustrated in FIG.
  • throat 430 comprises a reservoir in fluid communication with one or more microfluidic channel 406.
  • surface tension relief post 418 can be disposed in throat 430 to, at least in part, evenly spread assay 1000 throughout the plurality of microfluidic channels 406 and/or engage a meniscus of assay 1000 to encourage fluid flow.
  • Surface tension relief post can, according to some embodiments, comprise a hydrophilic surface in order to further encourage fluid flow into the throat and, thus, the microchannels.
  • microfluidic channels 406 can be in fluid communication with the plurality of staging capillaries 410 extending from microfluidic channel 406, through output layer 408, to a bottom surface 429.
  • bottom surface 429 can be spaced apart from first surface 22 of microplate 20 (FIG. 124) or can be in contact with first surface 22 of microplate 20.
  • each of the plurality of staging capillaries 410 can be generally aligned with a corresponding one of the plurality of wells 26 of microplate 20.
  • a protective covering (not shown) can be disposed over microfluidic channels 406 to provide, at least in part, protection from contamination, reduced evaporation, and the like. It should be understood that such protective covering can be used with any of the various configurations set forth herein.
  • a Zbig valve can be used to achieve fluid isolation between the plurality of staging capillaries 410 and assay input port 402, such as those described in commonly- assigned U.S. Patent Application No. 10/336,274, filed January 3, 2003 and PCT Application No. WO 2004/011147 A1.
  • excess assay 1000 in assay input ports 402 and/or the upstream portion of microfluidic channels 406 relative to stake cut 435 can be removed, if desired. In some embodiments, this can be accomplished by employing a wicking member 441 , as illustrated in FIG. 131.
  • output layer 408 and microplate 20 can be placed into a swing- arm centrifuge.
  • the centripetal force of the swing-arm centrifuge can be sufficient to overcome the surface tension of assay 1000 in each the plurality of staging capillaries 410, thereby forcing a metered volume of assay 1000 into each of the plurality of wells 26 of microplate 20 (FIG. 133).
  • filling apparatus 400 can be configured in any one of a number of configurations as desired. As described above, as illustrated in FIG. 120, assay input ports 402 can be positioned at end 420 of output layer 408. When this configuration is used with a microplate comprising 6,144 wells, filling apparatus 400 can comprise, for example, eight assay input ports 402 that can each be in fluid communication with eight respective microfluidic channels 406. Each of the eight microfluidic channels 406 can be in fluid communication with ninety-six respective staging capillaries 410. In some embodiments, as illustrated in FIG. 121 , assay input ports 402 can be positioned at side 422 of output layer 408.
  • filling apparatus 400 can comprise, for example, eight assay input ports 402 that can each be in fluid communication with twelve respective microfluidic channels 406. Each of the twelve microfluidic channels 406 can be in fluid communication with sixty-four respective staging capillaries 410.
  • This configuration can provide shorter channel lengths, which, in some circumstances, can have more rapid capillary filling times relative to the configuration of FIG. 120.
  • Each of the eight microfluidic channels 406 can be in fluid communication with forty-eight respective staging capillaries 410. These configurations can provide shorter channel lengths, which, in some circumstances, can have more rapid capillary filling times relative to the configurations of FIGS. 120 and 121.
  • the plurality of microfluidic channels 406 can be oriented such that, during centrifugation, they are perpendicular to an axis of revolution of the centrifuge. In some embodiments, this orientation can limit the flow of assay 1000 along the plurality of microfluidic channels 406 during centrifugation.
  • Assay 1000 can continue to flow down the one or more microfluidic channels 406 until each of the plurality of staging capillaries 410 can be at least partially filled with assay 1000 (FIG. 136).
  • fluid overfill reservoir 442 can generally inhibit assay 1000 from flowing into fluid overfill reservoir 442, at least in part because of the single opening therein generally preventing air within fluid overfill reservoir 442 from exiting.
  • fluid overfill reservoir can have a diameter equal to that of staging capillaries 410 and a depth of about 0.05 inch, or less.
  • assay 1000 in each of the plurality of staging capillaries 410 can be held therein by capillary or surface tension forces to aid in the equal metering of assay 1000 to be loaded in each of the plurality of wells 26.
  • a lower-end opeing or open-air outlet 434 of each of the plurality of staging capillaries 410 permit venting of air within each of the plurality of staging capillaries 410 during filling.
  • filling apparatus 400 can be stake cut, generally indicated at 435, via device 436 along a portion of one or more microfluidic channels 406. It should be appreciated that stake-cutting or staking can be carried out, as previously described.
  • At least output layer 408 and microplate 20 can be placed into a swing-arm centrifuge.
  • the centripetal force of the centrifuge can be sufficient to overcome the capillary force and/or surface tension of assay 1000 in each the plurality of staging capillaries 410, thereby forcing a metered volume of assay 1000 into each of the plurality of wells 26 of microplate 20 (FIG. 139).
  • the centripetal force of the centrifuge can be sufficient to force overfill fluid (e.g.
  • overfill reservoir 442 can act as a reservoir for excess assay 1000. As illustrated in FIG. 140, in some embodiments, overfill reservoir 442 can be disposed within output layer 408 and generally aligned with and positioned below at least one assay input port 402 in output layer 408.
  • microfluidic channel 406 can have a single channel portion 446 fluidly coupled to two or more rows of staging capillaries 410.
  • single channel portion 446 comprises a centrally disposed feature 448 to, in part, aid in fluid splitting between adjacent rows of staging capillaries 410.
  • microfluidic channels 406 can be of capillary size, for example, microfluidic channels 406 can be formed with a width of less than about 500 micron, and in some embodiments less than about 125 microns, less than about 100 microns, or less than about 50 microns. In some embodiments, microfluidic channels 406 can be formed, for example, with a depth of less than about 500 micron, and in some embodiments less than about 125 microns, less than about 100 microns, or less than about 20 microns.
  • microfluidic channels 406 can be provided with an interior surface that is hydrophilic, i.e., wettable.
  • the interior surface of microfluidic channels 406 can be formed of a hydrophilic material and/or treated to exhibit hydrophilic characteristics.
  • the interior surface comprises native, bound, or covalently attached charged groups.
  • one suitable surface is a glass surface having an absorbed layer of a polycationic polymer, such as poly-l-lysine.
  • filling apparatus 400 comprises output layer 408, a floating insert 460, a cover 464, port member 467, or any combination thereof for loading assay 1000 into at least some of the plurality of wells 26 in microplate 20.
  • output layer 408 comprises one or more recessed regions or depressions 454 formed in an upper surface 456 of output layer 408.
  • Each depression 454 can be, in some embodiments, sized and/or shaped to receive floating insert 460 therein.
  • at least one wall 458 can be used to separate each depression 454 to define grouping 407 of staging capillaries 410 of any desired quantity and orientation.
  • bottom surface 470 of floating insert 460 and/or top surface 472 of depression 454 can be treated and/or coated to enhance the hydrophilic properties of capillary gap 468.
  • capillary gap 468 can be in fluid communication with an aperture 462 extend through floating insert 460.
  • Aperture 462 can be centrally located relative to floating insert 460 or can be located to one side and/or corner thereof.
  • aperture 462 comprises an assay receiving well 463 (FIG. 145-157). In such embodiments, port member 467 is optional.
  • floating insert 460 comprises a flanged base portion 490 to reduce the potential capillary surface between sidewall 474 of floating insert 460 and wall 458 of depression 454.
  • a hydrophic surface can be employed between floating insert 460 and wall 458 of depression 454 to reduce capillary force therebetween.
  • floating insert 460 can be shaped to, at least in part, achieve any particular capillary and/or flow characteristics.
  • floating insert 460 can comprise a plurality of flow features 478 to, at least in part, extend the capillary surface to facilitate capillary flow.
  • each of the plurality of flow features 478 comprises a post member 480 (FIG. 147) extending orthogonally from bottom surface 470 of floating insert 460.
  • post member 480 comprises a radiused root portion 482 to facilitate capillary flow, if desired.
  • post member 480 can be offset within the corresponding staging capillary 410 and can, if desired, contact a sidewall of staging capillary 410.
  • each of the plurality of flow features 478 comprises a tapered member 484 (FIGS. 148-152) extending from bottom surface 470 of floating insert 460.
  • each of the plurality of staging capillaries 410 comprises a corresponding mating entrance feature 486 (FIG. 148, 150, and 151) to closely conform to each flow feature 478 to define a transition capillary gap 488.
  • Tapered member 484 can be conically shaped (FIGS.
  • floating insert 460 can comprise any material conducive to encourage capillary action along capillary gap 468, such as but not limited to plastic, glass, elastomer, and the like.
  • floating insert 460 can be made of at least two materials, such that an upper portion can be made of a first material and a lower portion can be made of a second material.
  • the second material can provide a desired compliancy, hydrophilicity, or any other desire property for improved fluid flow and/or sealing of staging capillaries 410.
  • the tapered members can include a seal-facilitating film, coating, or gasket thereon.
  • cover 464 can be used, at least in part, to retain floating insert 460 within each depression 454, if desired.
  • cover 464 comprises an aperture 466 generally aligned with an aperture 462 of floating insert 460.
  • cover 464 comprises a pressure sensitive adhesive to, at least in part, retain floating insert 460 within depression 454.
  • port member 467 comprises assay input port 402.
  • port member 467 can comprise a material comprising sufficient weight such that during centrifugation, the centripetal force of port member 467 exerted upon floating insert 460 and output layer 408 can aid in closing off cross-communication of fluid between adjacent staging capillaries 410, as the upper-end openings of staging capillaries 410 can be covered and sealed by the lower surface of floating insert 460.
  • port member 467 can be sized such that its footprint (e.g. the surface area of a bottom surface 476 of port member 467) can be smaller than the opening of depression 454 to aid in the exertion of centripetal force on floating insert 460 during centrifuge.
  • a predetermined amount of assay 1000 can be placed at each assay input port 402 when used with port member 467 or receiving well 463.
  • Capillary gap 468 can be sized to provide sufficient capillary force to draw at least a portion of assay 1000 from assay input port 402 or receiving well 463 into capillary gap 468.
  • the capillary force of capillary gap 468 can be, at least in part, due to the non- rigid connection between floating insert 460 and output layer 408.
  • each of the plurality of staging capillaries 410 in fluid communication with capillary gap 468 can begin to fill, at least in part, by capillary force as described herein.
  • the centripetal force of the centrifuge can also cause floating insert 460 to be forced and, thus, pressed against top surface 472 of depression 454.
  • this additional weight can further apply a force upon floating insert 460 to force floating insert 460 against top surface 472 of depression 454.
  • This force on floating insert 460 against top surface 472 of depression 454 can help to fluidly isolate each staging capillaries 410 from adjacent staging capillaries 410 for improved metering.
  • any component of filling apparatus 400 can comprise a plate, tile, disk, chip, block, wafer, laminate, and any combinations thereof, and the like.
  • filling apparatus 400 does include the plurality of microfluidic channels 406.
  • filling apparatus 400 comprises output layer 408 and a surface wipe assembly 1800 for loading assay 1000 into at least some of the plurality of wells 26 in microplate 20.
  • surface wipe assembly 1800 comprises one or more of a base support 1810, a drive assembly 1812, a funnel assembly 1814, or any combination thereof.
  • base support 1810 can be a generally planar support member operable to support microplate 20 and output layer 408 thereon.
  • base support 1810 comprises an alignment feature 1818 that can engage corresponding alignment feature 58 (refer to previous figures) of microplate 20 and/or alignment feature 519 of output layer 408 to maintain microplate 20 and output layer 408 in a predetermined alignment relative to each other and/or funnel assembly 1814.
  • drive assembly 1812 comprises a drive motor 1816; a guide member 1820, coupled to or formed in base support 1810; a tracking member 1822, coupled to or formed in funnel assembly 1814; and control system 1010.
  • guide member 1820 and tracking member 1822 are sized and/or shaped to slidingly engage with each other to provide guiding support for funnel assembly 1814 as it moves relative to base support 1810.
  • drive motor 1816 can be operably coupled to tracking member 1822 or base support 1810 to move tracking member 1822 relative to guide member 1820 via known drive transmission interfaces, such as mechanical drives, pneumatic drives, hydraulic drives, electromechanical drives, and the like.
  • drive motor 1816 can be controlled in response to control signals from control system 1010 or a separate control system.
  • drive motor 1816 can be operably controlled in response to a switch device controlled by a user.
  • funnel assembly 1814 comprises a spanning portion 1824 generally extending above output layer 408.
  • spanning portion 1824 can be supported on opposing ends by tracking member 1822 of drive assembly 1812 and a foot member 1826.
  • Tracking member 1822 and foot member 1826 can each be coupled to spanning portion 1824 via conventional fasteners in some embodiments.
  • Foot member 1826 can be generally arcuately shaped so as to reduce the contact area between foot member 1826 and base support 1810.
  • foot member 1826 can be made of a reduced friction material, such as Delrin®.
  • spanning portion 1824 of funnel assembly 1814 comprises a slot 1828 formed vertically therethrough that can be sized and/or shaped to receive a funnel member 1830 therein.
  • funnel member 1830 can comprise one or more assay chambers 1832 for receiving one or more different assays therein.
  • drive assembly 1812 and funnel assembly 1814 can be configured to track in a direction perpendicular to that illustrated in the accompanying figures to provide an increased number of assay chambers 1832 and reduced track distances.
  • funnel member 1830 can comprise a flange portion 1834 extending about a top portion thereof.
  • Assay chambers 1832 in some embodiments, can be shaped to provide a predetermined assay capacity for filling all of a predetermined number and/or grouping of the plurality of staging capillaries 410 in output layer 408. In some embodiments, assay chamber 1832 comprises converging sidewalls 1838 that terminate at a tip portion 1840.
  • each assay chamber 1832 comprises a different assay.
  • Assay 1000 is drawn down along sidewalls 1838 to tip portion 1840 to form a fluid bead 1842 extending from tip portion 1840 that can be in contact with upper surface 456 of output layer 408.
  • fluid bead 1842 can be bound by a lip or wiper member 1844 extending downwardly from tip portion 1840 of funnel member 1830.
  • wiper member 1844 can, at least in part, wipe and/or remove excess assay 1000 on upper surface 456 of output layer 408 as funnel member 1830 moves thereabout.
  • drive assembly 1812 can be actuated to advance funnel assembly 1814 across output layer 408 at a predetermined rate, as illustrated in FIG. 161.
  • funnel assembly 1814 can be advanced manually across output layer 408.
  • fluid bead 1842 can contact the upper-end opening or entrance of each of the plurality of staging capillaries 410 and begin to fill, at least in part, by capillary force as described herein.
  • some assay 1000 can be forced off upper surface 456 of output layer 408 at an edge 1846 into at least one overflow channel 1848.
  • at least output layer 408 and microplate 20 can be placed into a centrifuge.
  • the centripetal force of the centrifuge can be sufficient to overcome the capillary force and/or surface tension of assay 1000 in each the plurality of staging capillaries 410, thereby forcing a metered volume of assay 1000 into each of the plurality of wells 26 of microplate 20.
  • the excess assay 1000 in overflow channel 1848 can be contained using one or more reservoir pockets 1850.
  • reservoir pocket 1850 can be in fluid communication with at least one overflow channel 1848.
  • reservoir pocket 1850 can be deeper than overflow channel 1848 to encourage flow of assay 1000 to reservoir pocket 1850. During centrifugation, centripetal force can further encourage assay 1000 to flow to reservoir pocket 1850, thereby reducing the likelihood of any contamination or cross-feed between adjacent staging capillaries 410.
  • an extended wall member 1852 can be positioned about reservoir pocket 1850 to further contain assay 1000.
  • the excess assay 1000 in overflow channel 1848 can be contained using a reservoir trough 1854.
  • an absorbent member 1856 can be disposed in reservoir trough 1854 to absorb excess assay 1000 therein.
  • absorbent member 1856 can be a hydrophilic fiber membrane.
  • reservoir trough 1854 can be sloped toward absorbent member 1856 to facilitate absorption of excess assay 1000.
  • absorbent member 1856 can be removable to permit removal and relocating of the excess assay 1000 prior to centrifugation.
  • funnel member 1830 of funnel assembly 1814 can be any one of a number of configurations sufficient to maintain fluid bead 1842 in contact with upper surface 456 of output layer 408.
  • a predetermined shape of fluid bead 1842 and/or a predetermined flowrate of assay 1000 through tip portion 1840 can be achieved through the particular configuration of funnel member 1830.
  • funnel member 1830 comprises one or more assay chambers 1832 in fluid communication with tip portion 1840.
  • assay chambers 1832 in fluid communication with tip portion 1840.
  • FIG. 168 multiple assays can be used such that a different assay can be disposed in each assay chamber 1832. It should be understood that any number of assay chambers 1832 can be used (e.g., 2, 4, 6, 8, 10, 12, 16, 20, 32, 64, or more).
  • tip portion 1840 can be configured to define a capillary force and/or surface tension sufficient to prevent assay 1000 from exiting assay chamber 1832 prior to fluid bead 1842 engaging upper surface 456 and to permit assay 1000 to be pulled into each of the plurality of staging capillaries 410 during filling of the staging capillaries.
  • tip portion 1840 comprises a restricted orifice 1860 that is sized to increase surface tension to retain assay 1000 with assay chamber 1832.
  • tip portion 1840 can be spaced apart from an underside surface 1862 to, at least in part, inhibit assay 1000 from collecting between funnel member 1830 and output layer 408. In some embodiments, as illustrated in FIG.
  • restricted orifice 1860 can be used with wiper member 1844 to increase surface tension to retain assay 1000 and to wipe and/or remove excess assay 1000 on upper surface 456 of output layer 408.
  • tip portion 1840 can comprise a planar cavity 1864 disposed in fluid communication with restricted orifice 1860.
  • planar cavity 1864 can encourage the formation of wider and/or shallower fluid bead 1842 relative to similar configurations not employing planar cavity 1864.
  • the wider and/or shallower fluid bead 1842 can, at least in part, prolong the time fluid bead 1842 is in contact with each of the plurality of staging capillaries 410.
  • restricted orifice 1860 comprises an elongated slot 1866 (FIG. 174) generally extending from one edge of tip portion 1840 to the opposing edge to define an elongated fluid bead 1842.
  • restricted orifice 1860 comprises one or more apertures 1868.
  • the reduced cross-sectional area of apertures 1868 relative to that of elongated slot 1866 can serve to withstand a fluid head pressure exerted by assay 1000 in assay chamber 1832 that would otherwise overcome the surface tension of fluid bead 1842 exiting elongated slot 1866 and possibly lead to premature discharge of assay 1000.
  • the restricted orifice 1860 can be collinear as well as offset as illustrated in (FIG. 174).
  • funnel member 1830 can be formed with a two- or more-piece construction. As illustrated in FIG. 179, funnel member 1830 can comprise a first section 1880 and a second section 1882. First section 1880 can comprise one or more desired features. For example, as illustrated in FIG. 179, upturned section 1876 of FIG. 178 can be formed in first section 1880. First section 1880 and second section 1882 can then be joined or otherwise mated along a generally vertical joining line 1884 (FIG. 178) to form funnel member 1830. In some embodiments, first section 1880 and second section 1882 can be joined or otherwise mated along a generally horizontal joining line 1886 (FIG. 180).
  • first section 1880 and second section 1882 can be made from different materials to achieve a predetermined performance.
  • second section 1882 can be made of an elastomer to provide enhance flexibility to accommodate for variations in output layer 408 and enhanced wiping performance of wiper member 1844.
  • surface energy can be improved, for example, when using a polymer material in the manufacture of filling apparatus 400, through surface modification of the polymer material via Michael addition of acrylamide or PEO- acrylate onto laminated surface; surface grafting of acrylamide or PEO-acrylate via atom transfer radical polymerization (ARTP); surface grafting of acrylamide via Ce(IV) mediated free radical polymerization; surface initiated living radical polymerization on chloromethylated surface; coating of negatively charged polyelectrolytes; plasma CVD of acrylic acid, acrylamide, and other hydrophilic monomers; or surface adsorption of an ionic or non-ionic surfactant.
  • surfactants such as those set forth in Tables 2 and 3, can be used.
  • filling apparatus 400 can comprise polyolefins; poly(cyclic olefins); polyethylene terephthalate; poly(alkyl (meth)acrylates); polystyrene; poly(dimethyl siloxane); polycarbonate; structural polymers, for example, poly(ether sulfone), poly(ether ketone), poly(ether ether ketone), and liquid crystalline polymers; polyacetal; polyamides; polyimides; poly(phenylene sulfide); polysulfones; polyvinyl chloride); polyvinyl fluoride); poly(vinylidene fluoride); copolymers thereof; and mixtures thereof.
  • a co-agent can be employed to enhance the hydrophilicity and/or improve the shelf life of filling apparatus 400.
  • Co-agents can be, for example, a water-soluble or slightly water-soluble homopolymer or copolymers prepared by monomers comprising, for example, (meth)acrylamide; N-methyl (methyl)acrylamide, N,N-dimethyl (methyl)acrylamide, N-ethyl (meth)acrylamide, N-n- propyl (meth)acrylamide, N-iso-propyl (meth)acrylamide, N-ethyl-N-methyl (meth)acrylamide, N,N-diethyl (meth)acrylamide, N-hydroxymethyl (meth)acrylamide, N- (3-hydroxypropyl) (meth)acrylamide, N-vinylformamide, N-vinylacetamide, N-methyl-N- vinylacetamide, vinyl acetate that can be hydrolyzed to give vinylalcohol after polymer
  • (meth)acrylamide N-amido(meth)acrylamide, N-acetamido (meth)acrylamide, N- tris(hydroxymethyl)methyl (meth)acrylamide, N-
  • sealing cover 80 can be generally disposed across microplate 20 to seal assay 1000 within each of the plurality of wells 26 of microplate 20 along a sealing interface 92 (see FIGS. 4, 5, 26, and 27). That is, sealing cover 80 can seal (isoloate) each of the plurality of wells 26 and its contents (i.e. assay 1000) from adjacent wells 26, thus maintaining sample integrity between each of the plurality of wells 26 and reducing the likelihood of cross contamination between wells.
  • sealing cover 80 can be positioned within an optional depression 94 (FIG. 30) formed in main body 28 of microplate 20 to promote proper positioning of sealing cover 80 relative to the plurality of wells 26.
  • sealing cover 80 can be made of any material conducive to the particular processing to be done.
  • sealing cover 80 can comprise a durable, generally optically transparent material, such as an optically clear film exhibiting abrasion resistance and low fluorescence when exposed to an excitation light.
  • sealing cover 80 can comprise glass, silicon, quartz, nylon, polystyrene, polyethylene, polycarbonate, copolymer cyclic olefin, polycyclic olefin, cellulose acetate, polypropylene, polytetrafluoroethylene, metal, and combinations thereof.
  • sealing cover 80 comprises an optical element, such as a lens, lenslet, and/or a holographic feature.
  • sealing cover 80 comprises features or textures operable to interact with (e.g., by interlocking engagement) circular rim portion 32 or square-shaped rim portion 38 of the plurality of wells 26.
  • sealing cover 80 can provide resistance to distortion, cracking, and/or stretching during installation.
  • sealing cover 80 can comprise water impermeable-moisture vapor transmission values below 0.5 (cc-mm)/(m2-24hr-atm).
  • sealing cover 80 can maintain its physical properties in a temperature range of 4°C to 99 0 C and can be generally free of inclusions (e.g. light blocking specks) greater than 50 ⁇ m, scratches, and/or striations.
  • sealing cover 80 can comprise a liquid such as, for example, oil (e.g., mineral oil).
  • the sealing material can provide sufficient adhesion between sealing cover 80 and microplate 20 to withstand about 2.0 lbf per inch or at least about 0.9 lbf per inch at 95°C. In some embodiments, the sealing material can provide sufficient adhesion at room temperature to contain assay 1000 within each of the plurality of wells 26. This adhesion can inhibit sample vapor from escaping each of the plurality of wells 26 by either direct evaporation or permeation of water and/or assay 1000 through sealing cover 80. In some embodiments, the sealing material maintains adhesion between sealing cover 80 and microplate 20 in cold storage at 2 ° C to 8 ° C range (non-freezing conditions) for 48 hours.
  • compliant layer 86 can be a soft silicone elastomer or other material known in the art that is deformable to allow pressure sensitive adhesive 88 to conform to irregular surfaces of microplate 20, increase bond area, and resist delamination of sealing cover 80.
  • pressure sensitive adhesive 88 and compliant layer 86 can be a single layer, if the pressure sensitive adhesive exhibit sufficient compliancy. Release liner 90 is removed prior to coupling pressure sensitive adhesive 88 to microplate 20.
  • adhesive 88 can selected so as to be compatible with assay 1000.
  • adhesive 88 is free of nucleases, DNA, RNA and other assay components, as discussed below.
  • sealing cover 80 comprises one or more materials that are selected so as to be compatible with detection probes in assay 1000.
  • adhesive layer 88 is selected for compatibility with detection probes.
  • Methods of matching a detection probe with a compatible sealing cover 80 include, in some embodiments, varying compositions of sealing cover 80 by different weight percents of components such as polymers, crosslinkers, adhesives, resins and the like. These sealing covers 80 can then be tested as a function of their corresponding fluorescent intensity level for different dyes. In such embodiments, comparison can be analyzed at room temperature as well as at elevated temperatures typically employed with PCR. Comparisons can be analyzed over a period of time and in some embodiments, the time period can be, for example, up to 24 hours. Data can be collected for each of the varying compositions of sealing cover 80 and plotted such that fluorescence intensity of the dye is on the X-axis and time is on the Y-axis.
  • Some embodiments of the present teachings include a method of testing compatibility of the detection probe comprising an oligonucleotide and a fluorophore to a composition of a sealing cover.
  • the method includes depositing a quantity of the fluorophore into a plurality of containers, providing a plurality of sealing covers that have different compositions and sealing the containers with the sealing covers.
  • Methods also include exciting the fluorophore in each of the containers and then measuring an emission intensity from the fluorophore in each of the containers.
  • the method can also include an evaluation of the emission intensity from the fluorophore of each of the containers and then a determination of which sealing cover composition is compatible with the fluorophore.
  • kits comprising, for example, a sealing cover 80 and one or more compatible detection probes that are compatible (e.g., emission intensity does not degrade when in contact) with sealing cover 80.
  • a kit can comprise one or more detection probes that are compatible (e.g., do not degrade over time when in contact) with adhesive 88 of sealing cover 80.
  • Kits may comprise a group of detection probes that are compatible with sealing cover 80 comprising adhesive 88 and microplate 20.
  • the present teachings include methods for matching a group of detection probes that are compatible with sealing cover 80 and spotting into at least some of plurality of wells 26 of microplate 20.
  • sealing cover 80 can be configured as a roll 512.
  • the use of sealing cover roll 512 can provide, in some embodiments, and circumstances, improved ease in storage and application of sealing cover 80 on microplate 20 when used in conjunction with a manual or automated sealing cover application device, as discussed herein.
  • sealing cover roll 512 can be manufactured using a laminate comprising a protective liner 514, a base stock 516, an adhesive 518, and/or a carrier liner 520. During manufacturing, protective liner 514 can be removed and discarded.
  • Base stock 516 and adhesive 518 can then be kiss-cut, such that base stock 516 and adhesive 518 are cut to a desired shape of sealing cover 80, yet carrier liner 520 is not cut. Excess portions of base stock 516 and adhesive 518 can then be removed and discarded.
  • base stock 516 can be a scuff resistant and water impermeable layer with low to no fluorescence.
  • carrier liner 520 can then be punched or otherwise cut to a desired shape and finally the combination of carrier liner 520, base stock 516, and adhesive 518 can be rolled about a roll core 522 (see FIG. 182).
  • Roll core 522 can be sized so as not to exceed the elastic limitations of base stock 516, adhesive 518, and/or carrier liner 520.
  • adhesive 518 is sufficient to retain base stock 516 to carrier liner 520, yet permit base stock 516 and adhesive 518 to be released from carrier liner 520 when desired.
  • base stock 516, adhesive 518, and carrier liner 520 are rolled upon roll core 522 such that base stock 516 and adhesive 518 face toward roll core 522 to protect base stock 516 and adhesive 518 from contamination and reduce the possibility of premature release.
  • such a desired shape of carrier liner 520 can comprise a plurality of drive notches 524 formed along and slightly inboard of at least one of the elongated edges 526.
  • the plurality of drive notches 524 can be shaped, sized, and spaced to permit cooperative engagement with a drive member to positively drive sealing cover roll 512 and aid in the proper positioning of sealing cover 80 relative to microplate 20.
  • the desired shape of carrier liner 520 can further comprise a plurality of staging notches 528 to be used to permit reliable positioning of sealing cover 80.
  • the plurality of staging notches 528 can be formed along at least one elongated edge 526.
  • the plurality of staging notches 528 can be shaped and sized to permit detection by a detector, such as an optical detector, mechanical detector, or the like.
  • An end/start of roll notch or other feature 530 can further be used in some embodiments to provide notification of a first and/or last sealing cover 80 on sealing cover roll 512. Similar to the plurality of staging notches 528, end/start of roll notch 530 can be shaped and sized to permit detection by a detector, such as an optical detector, mechanical detector, or the like. It should be appreciated that the foregoing notches and features can have other shapes than those set forth herein or illustrated in the attached figures. It should also be appreciated that other features, such as magnetic markers, non-destructive markers (e.g.
  • sealing cover 80 can be laminated onto microplate 20 using a hot roller apparatus 540, as illustrated in FIG. 29.
  • hot roller apparatus 540 comprises a heated top roller 542 heated by a heating element 544 and an unheated bottom roller 546.
  • a first plate guide 548 can be provided for guiding microplate 20 into hot roller apparatus 540, while similarly a second plate guide 550 can be provided for guiding microplate 20 out of hot roller apparatus 540.
  • hot roller apparatus 540 can variably control he amount of heat applied to sealing cover 80. In this regard, sufficient heat can be supplied to provide adhesive flow or softening of the adhesive of sealing cover 80 without damaging assay 1000.
  • hot roller apparatus 540 can /ariably control a drive speed of heated top roller 542 and unheated bottom roller 546.
  • hot roller apparatus 540 can variably control a clamping force between heated top roller 542 and unheated bottom roller 546. By varying these parameters, optimal sealing of sealing cover 80 to microplate 20 can be achieved with minimal negative effects to assay 1000.
  • alignment feature 560 can comprise a comer chamfer, a pin, a slot, a cut corner, an indentation, a graphic, a nub, a protrusion, and/or other unique feature that can be capable of interfacing with alignment feature 58 or other feature of microplate 20.
  • fixture 554 can comprise one or more recesses 562 formed in generally planar substrate 556 to permit, among other things, improved grasping of microplate 20 for ease of insertion and withdrawal of microplate 20 from fixture 554.
  • one or more recesses 562 can be positioned along opposing ends of microplate 20.
  • plane assembly 588 comprises a plate member 618 and a plane roller 620 rotatably coupled to plate member 618 along axis 622.
  • plane roller 620 can be a generally cylindrical member comprising a pair of pins 624 disposed on opposing ends thereof along axis 622.
  • the pair of pins 624 can engage apertures formed in plate member 618 to permit rotating movement of plane roller 620 relative thereto.
  • plane roller 620 can be made of, at least in part, a compliant material to permit plane roller 620 accommodate variations in fixture 554 and/or microplate 20.
  • plane roller 620 can carry carrier liner 520 of sealing cover roll 512.
  • plane roller 620 can be sized to apply a force on a backside of carrier liner 520 and, consequently, on sealing cover 80 to adhere sealing cover 80 to microplate 20 during application.
  • carrier liner 520 can then travel along plate member 618 to intermediate roller 590.
  • plane roller 620 can comprise posts (not illustrated) formed thereon to engage the plurality of drive notches 524 formed on some embodiments of carrier liner 520 to aid in alignment.
  • intermediate roller 590 can be sized to apply a force on a backside of carrier liner 520 and, consequently, on sealing cover 80 to adhere sealing cover 80 to microplate 20 during application.
  • a pair of knob portions 630 can be used and disposed on opposing ends of drive roller 632 to permit both left-handed and right-handed operation.
  • Knob portion 630 can be manually manipulated by a user to manually advance carrier liner 520 of sealing cover roll 512.
  • drive roller 632 can be comprised of, at least in part, a compliant material to permit drive roller 632 to accommodate variations in fixture 554 and/or microplate 20.
  • drive roller 632 can be sized to apply a force on a backside of carrier liner 520 and, consequently, on sealing cover 80 to adhere sealing cover 80 to microplate 20 during application.
  • drive roller 632 can be sized to operably engage pressure roller 594 to receive carrier liner 520 of sealing cover roll 512 therebetween (see FIG. 185).
  • pressure roller 594 can be a generally cylindrical member comprising a pair of pins 638 disposed on opposing ends thereof along axis 640.
  • the pair of pins 638 can engage apertures formed in a support bracket 642 to permit rotating movement of pressure roller 594 relative thereto.
  • support bracket 642 can be fixedly mounted to or integrally formed with at least one cover section 568.
  • pressure roller 594 can be biased to apply a force against drive roller 632 to, at least in part, positively grab, and/or advance carrier liner 520.
  • carrier liner 520 of sealing cover roll 512 can be fed from a lower portion of sealing cover roll 512 forward along a top side of plate member 618. Carrier liner 520 can then be fed around plane roller 620, along an bottom side of plate member 618, around intermediate roller 590, between pressure roller 594 and drive roller 632, and finally out of waste gate 596.
  • sealing cover roll 512 can be loaded in manual sealing cover applicator 552 by positioning latch member 580 in the unlocked position (FIG. 187) and pivoting at least one cover section 568 upward. Sealing cover roll 512 can then be place on roll hub 584. Carrier liner 520 can then be routed through manual sealing cover applicator 552 as described above.). In some embodiments, closing of the at least one cover section 568 causes pressure roller 594 to apply a force on carrier liner 520. In some embodiments, drive roller 632 and/or knob section 630 can be ratcheted to maintain carrier liner 520 under tension.
  • sealing cover 80 can be laminated onto microplate 20 using an automated sealing cover applicator 1100.
  • automated sealing cover applicator 1100 comprises a housing 1102 sized to receive sealing cover roll 512 therein.
  • housing 1102 can comprise a base section 1104 and cover section 1106 connectable therewith.
  • cover section 1106 can comprise an opening 1108 for receiving a sealing cover cassette 1110 therein.
  • base section 1104 comprises at least one of a microplate tray assembly 1112, a tray drive system 1114, a sealing cover drive system 1116 for at least in part alignment control of sealing cover roll 512, a heated roller assembly 1118, and an applicator control system 1120.
  • alignment feature 1126 can a corner chamfer, a pin, a slot, a cut comer, an indentation, a graphic, a nub, a protrusion, or other unique feature that can be capable of interfacing with alignment feature 58 or other feature of microplate 20.
  • microplate tray assembly 1112 comprises one or more recesses 1128 formed in generally planar tray member 1122 to permit, among other things, improved grasping of microplate 20 for ease of insertion and withdrawal of microplate 20 from microplate tray assembly 1112.
  • one or more recesses 1128 can be positioned along opposing ends of microplate 20.
  • generally planar tray member 1122 comprises a uniquely sized and/or shaped insert 1130 that can be fastened within recessed portion 1124 to accommodate varying sizes of microplates or other devices.
  • microplate tray assembly 1112 can be moved between the extended position and the retracted position via tray drive system 1114.
  • tray drive system 1114 comprises at least one of a drive motor 1132 and a drive track member 1134.
  • drive track member 1134 can be a threaded member, such as but not limited to a worm gear, threadedly engaging a receiver 1136 fixedly coupled to microplate tray assembly 1112.
  • Drive motor 1132 can be actuated by a control switch and/or applicator control system 1120 to rotatably turn drive track member 1134.
  • microplate tray assembly 1112 can travel relative to drive track member 1134 between the extended and retracted positions.
  • sealing cover cassette 1110 comprises at least one of a support structure 1138, a cover member 1140, a roll hub 1142, a plane roller 1144, at least one feed roller 1146, a sprocket drive member 1148, and a waste gate 1150.
  • roll hub 1142 can be fixedly coupled to support structure 1138 to support sealing cover roll 512 thereon and permit relative rotation therebetween.
  • roll hub 1142 comprises pair of friction legs 598 extending outwardly from tangential sections 600 of central portion 602 as discussed herein.
  • roll hub 1142 can comprise a cylindrical support member 1152.
  • the at least one feed roller 1146 can comprise a pair of cylindrical members rotatably supported by support structure 1138 to permit rotating movement of feed roller 1146 relative thereto.
  • feed rollers 1146 can be made of a material to, at least in part, positively grab and/or advance carrier liner 520.
  • Feed roller 1146 can also be configured to impart a drag force on carrier liner 520 opposing a driving force by sprocket drive member 1148 to ensure carrier liner 520 and sealing cover 80 disposed thereon are generally flat between feed roller 1146 and sprocket drive member 1148.
  • sprocket drive member 1148 can be a generally cylindrical member comprising at least one sprocket portion 1154 disposed on at least one end of a support rod 1156 (FIG. 189) rotatable about an axis 1157.
  • a pair of sprocket portions 1154 can be provided such that each of the pair of sprocket portions 1154 can be disposed on opposing ends of support rod 1156.
  • support rod 1156 can be rotatably coupled to support structure 1138.
  • the pair of sprocket portions 1154 can each comprise a plurality of engaging portions 1158 that are each sized and spaced to enmesh with each of the plurality of drive notched 524 formed on carrier liner 520 of sealing cover roll 512.
  • sealing cover drive system 1116 can comprise a drive motor 1160 (FIG. 189) enmeshingly engaging a drive gear 1162 (FIG. 191) fixed coupled at an end of support rod 1156 of sprocket drive member 1148 (FIG. 191).
  • drive motor 1160 can be actuated by a control switch and/or applicator control system 1120 to rotatably turn sprocket drive member 1148 and drive carrier liner 520 of sealing cover roll 512.
  • drive motor 1160 can be fixedly mounted within base section 1104.
  • a vibration isolation member 1164 can be disposed between drive motor 1160 and a support structure 1166 within base section 1104.
  • carrier liner 520 of sealing cover roll 512 can be fed from sealing cover roll 512 downward between feed roller 1146 and around sprocket drive members 1148 and out waste gate 1150.
  • a guide wall 1168 can be provided to direct an end of carrier liner 520 toward waste gate 1150.
  • sealing cover cassette 1110 can further comprise a latch system 1170 for operably coupling sealing cover cassette 1110 to cover section 1106.
  • latch system 1170 comprises a lip member 1172 disposed on one end of cover member 1140 and at least one biasing members 1174. As best seen in FIG. 192, lip member 1172 can engage an underside of cover section 1106.
  • at least one biasing member 1174 can be generally U-shaped and have a retaining feature 1177 that can be sized to engage an underside of cover section 1106.
  • At least one biasing member 1174 can impart a locking force such that retaining feature 1177 remains engaged with the underside of cover section 1106 until a user overcomes the biasing force to disengage retaining feature 1177 from cover section 1106.
  • cover section 1106 To install sealing cover cassette 1110 into cover section 1106, one can simply insert lip member 1172 under cover section 1106 and pivot a front end of sealing cover cassette 1110 downward until the at least one biasing member 1174 engages cover section 1106. This motion can further engage drive gear 1162 with drive motor 1160.
  • heated roller assembly 1118 can be used to apply at least one of heat and pressure to sealing cover 80 and/or microplate 20 as tray generally planar tray member 1122 passed therebelow.
  • heat and/or pressure can be used to activate adhesive 518 on sealing cover 80 to effect sealing interface 112.
  • heated roller assembly 1118 comprises a heated roller 1178 rotatably supported within a removable housing 1180.
  • heated roller 1178 can be heated internally via a heating member 1182 and/or heated externally via a heating device 1184.
  • heating member 1182 and/or heating device 1184 can be controlled by applicator control system 1120. It should be appreciated that heated roller assembly 1118 can be manufactured as a sub-assembly to permit easy retrofitting of existing
  • heating device 1184 can serve as a convective and/or indirect heater of sealing cover 80 as microplate 20 passes therebelow. In such embodiments, heated roller 1178 can be eliminated.
  • applicator control system 1120 can be operable to control tray drive system 1114 and/or sealing cover drive system 1116 to apply sealing cover 80 to microplate 20.
  • Applicator control system 1120 comprises an electrical circuit operable to output various control signals to drive motor 1132 and/or drive motor 1160 in response to a program mode of operation and/or data input.
  • applicator control system 1120 can receive data input from at least one sensor disposed in automated sealing cover applicator 1100, such as, but not limited to, a tray drive sensor for detecting encumbered operation of microplate tray assembly 1112, a sealing cover drive sensor for detecting encumbered operation of sealing cover cassette 1110, a sealing cover position sensor for detecting one of the plurality of staging notches 528 formed in carrier liner 520, an end/start of roll sensor for detecting end/start of roll notch 530, a temperature sensor for detecting a temperature of heated roller 1178, or any other sensor for detecting a desired operating parameter of automated sealing cover applicator 1100.
  • a tray drive sensor for detecting encumbered operation of microplate tray assembly 1112
  • sealing cover drive sensor for detecting encumbered operation of sealing cover cassette 1110
  • sealing cover position sensor for detecting one of the plurality of staging notches 528 formed in carrier liner 520
  • an end/start of roll sensor for detecting end/start of roll
  • applicator control system 1120 can be response to at least one of a power switch 1186, a tray activation button 1188, and/or a seal application button 1190 (FIG. 188). Still further, in some embodiments, applicator control system 1120 can output a control status indicia 1192 that can include, but is not limited to, a TEMP alert indicia, a SEAL EMPTY alert indicia, a TRAY JAM alert indicia, a SEAL JAM alert indicia, a POWER alert indicia, a READY alert indicia, or the like. In some embodiments, the TEMP alert indicia can be used to indicate when a desired temperature has been reached.
  • the SEAL EMPTY alert indicia can be used to indicate when sealing cover roll 512 is at or near empty of sealing covers 80. In some embodiments, the TRAY JAM alert indicia can be used to indicate when microplate tray assembly 1112 is encumbered. In some embodiments, the SEAL JAM alert indicia can be used to indicate when at least one sealing cover 80 is encumbered.
  • automated sealing cover applicator 1100 comprises a single sheet applicator assembly 1194.
  • single sheet applicator assembly 1194 comprises at least one of a plate member 1196, a cartridge receiving assembly 1198, a sealing cover cartridge 1200, and a planer drive system 1202.
  • sealing cover cartridge 1200 comprises at least one of a top cover 1204, a bottom cover 1206, a separator 1208, at least one wheel member 1210, and a sealing cover carrier assembly 1212.
  • sealing cover carrier assembly 1212 comprises a carrier liner 1214 and a sealing cover 80 disposed on carrier liner 1214.
  • carrier liner 1214 can be sized larger than sealing cover 80 to define a flap 1216 along a leading edge of carrier liner 1214.
  • carrier liner 1214 can be similar in material to carrier liner 520.
  • top cover 1204 can be generally planar in construction and comprises a pair of feed slots 1218 formed along a leading edge 1220 thereof.
  • the pair of feed slots 1218 can be sized to reveal a portion of sealing cover carrier assembly 1212, specifically flap 1216, for later use in dispensing sealing cover 80.
  • bottom cover 1206 can be generally planar in construction and can comprise a pair of feed slots 1222 formed along a leading edge 1224 thereof.
  • the pair of feed slots 1222 can be sized to generally align with the pair of feed slots 1218 of top cover 1204 to reveal a portion of sealing cover carrier assembly 1212, specifically flap 1216, for later use in dispensing sealing cover 80.
  • separator 1208 can be generally planar in construction and can be sized to be generally received within top cover 1204 and bottom cover 1206.
  • separator 1208 can comprise at least one rib 1226 extending about a periphery of separator 1208 and/or traversing thereabout to support sealing cover carrier assembly 1212 thereon.
  • Separator 1208 can further comprise at least one coupling member 1228 for retaining at least one wheel member 1210.
  • the at least one coupling member 1228 can be a C- shaped members sized to engage and retain a reduced cross-section portion 1230 of at least one wheel member 1210.
  • the outer diameter of the at least one coupling member 1228 can be less than the outer diameter the at least one wheel member 1210 to reduce interference between the at least one coupling member 1228 and sealing cover carrier assembly 1212.
  • top cover 1204, separator 1208, and bottom cover 1206 can be coupled together to encapsulate sealing cover carrier assembly 1212 and sealing cover 80 therein, as illustrated in FIG. 196.
  • Bottom cover 1206 can comprise at least one mounting stud 1232 formed on an interior side thereof.
  • Top cover 1204 and separator 1208 can comprise at least one aperture 1234 generally aligned with the at least one mounting stud 1232 to receive a threaded fastener therethrough.
  • planer drive system 1202 comprises a generally triangular mounting block 1252 and at least one drive roller 1254 mounted thereto that can be sized and generally aligned with at least one feed slot 1218, 1222 to operably engage flap 1216 of carrier liner 1214 to drive sealing cover carrier assembly 1212 and urge sealing cover 80 out of slot 1236.
  • at least one drive roller 1254 can be operably driven via a drive motor, such as drive motor 1160, through a gear assembly 1256 (FIG. 194).
  • planer drive system 1202 can further comprise a plane roller 1258.
  • plane roller 1258 can be a generally cylindrical member rotatably supported by support structure 1166 to permit rotating movement of plane roller 1258 relative thereto.
  • plane roller 1258 can be made of, at least in part, a compliant material to permit plane roller 1258 to accommodate variations in microplate tray assembly 1112 and/or microplate 20.
  • plane roller 1258 can be sized and/or positioned to engage microplate tray assembly 1112 and/or microplate 20 to apply a compressing force upon sealing cover 80 and microplate 20 to impart at least an initial sealing engagement.
  • plane roller 1258 can be heated.
  • sealing cover carrier assembly 1212 carrying a single sealing cover 80, can be preloaded or loaded by a user into sealing cover cartridge 1200 such that flap 1216 of carrier liner 1214 can be exposed through at least one feed slot 1218, 1222.
  • This arrangement can provide reduced contamination of sealing cover 80 and microplate 20.
  • sealing cover cartridge 1200 can then be loaded into removable cartridge support 1238 and inserted into opening 1240 of cover section 1242 until urging member 1250 engages removable cartridge support 1238 such that flap 1216 can be urged against at least one drive roller 1254 of planer drive system 1202.
  • Microplate 20 can be loaded into microplate tray assembly 1112. As illustrated in FIG.
  • thermocycler system 100 comprises at least one thermocycler block 102.
  • Thermocycler system 100 provides heat transfer between thermocycler block 102 and microplate 20 during analysis to vary the temperature of a sample to be processed.
  • thermocycler block 102 can also provide thermal uniformity across microplate 20 to facilitate accurate and precise quantification of an amplification reaction.
  • a control system 1010 (FIGS. 30, 41 , and 42) can be operably coupled to thermocycler block 102 to output a control signal to regulate a desired thermal output of thermocycler block 102.
  • the control signal of control system 1010 can be varied in response to an input from a temperature sensor (not illustrated).
  • thermocycler block 102 continuously cycles the temperature of microplate 20. In some embodiments, thermocycler block 102 cycles and then holds the temperature for a predetermined amount of time. In some embodiments, thermocycler block 102 maintains a generally constant temperature for performing isothermal reactions upon or within microplate 20.
  • thermocycler blocks 102 can be employed to thermally cycle a plurality of microplates 20 to permit higher throughput of microplates 20 through high-density sequence detection system 10.
  • each of the plurality of thermocycler blocks 102 can thermally cycle a separate microplate 20 to increase the overall duty cycle of detection system 300 and, in turn, high-density sequence detection system 10.
  • temperature cycles are used, at least in part, to denature (at a high temperature, e.g, about 95 ° C) and then extend (at a low temperature, e.g., about 60°C) a DNA target.
  • Conventional detection systems can then measure a resultant emission while at the low temperature. However, as can be appreciated, during these temperature cycles, conventional detection systems are idle until the next low temperature portion of the cycle. For instance, in cases where about 40 temperature cycles are completed over a 2-hour period, the conventional detection system is active to measure the resultant emission about 40 times. The remaining time the conventional detection system is idle. Therefore, it should be appreciated that conventional thermocycler systems limit the duty cycle of conventional excitation systems and/or conventional detection systems.
  • the plurality of thermocycler blocks 102 can be synchronized to provide offset temperature cycles. In some embodiments, the plurality of thermocycler blocks 102 can be synchronized to maximize or provide at or near 100% usage of detection system 300. The exact number of thermocycler blocks 102 to be used is, at least in part, dependent on the time required to measure all the samples on a single thermocycler and the degree of time offset between the cycling profiles of each thermocycler system.
  • detection system 300 can comprise a driving device to position detection system 300 and, in some embodiments, excitation system 200 above one of the plurality of thermocycler blocks 102 to measure a resultant emission from the corresponding microplate 20.
  • detection system 300 can comprise a movable mirror to permit measurement of the resultant emission of multiple microplates 20 from a fixed position.
  • each of the plurality of thermocycler blocks 102 can be positioned on a carousel or track system for movement relative to detection system 300. It should be appreciated that any system, in addition to those described herein, can be used to permit detection of resultant emission from one or more microplates 20 positioned on the plurality of thermocycler blocks 102 by a single detection system 300 to increase the duty cycle thereof.
  • thermal compliant pad 140 can be disposed between thermocycler block 102 and any adjacent component, such as microplate 20 or a sealing cover 80. It should be understood that thermal compliant pad 140 is optional. Thermal compliant pad 140 can better distribute heating or cooling through a contact interface between thermocycler block 102 and the adjacent component. This arrangement can reduce localized hot spots and compensate for surface variations in thermocycler block 102, thereby providing improved thermal distribution across microplate 20.
  • pressure clamp system 110 can apply a clamping force upon sealing cover 80, microplate 20, and thermocycler block 102 to, at least in part, operably seal assay 1000 within the plurality of wells 26 during thermocycling and further improve thermal communication between microplate 20 and thermocycler block 102.
  • Pressure clamp system 110 can be configured in any one of a number of orientations, such as described herein. Additionally, pressure clamp system 110 can comprise any one of a number of components depending upon the specific orientation used. Therefore, it should be understood that variations exist that are still regarded as being within the scope of the present teachings.
  • pressure clamp system 110 can comprise an inflatable transparent bag 116 positioned between and in engaging contact with a transparent window 112 and sealing cover 80.
  • transparent window 112 and thermocycler block 102 are fixed in position against relative movement.
  • Inflatable transparent bag 116 comprises an inflation/deflation port 118 that can be fluidly coupled to a pressure source 122, such as an air cylinder, which can be controllable in response to a control input from a user or control system 1010.
  • a pressure source 122 such as an air cylinder
  • inflatable transparent bag 116 can comprise a plurality of inflation/deflation ports to facilitate inflation/deflation thereof.
  • pressurized fluid such as air
  • inflatable transparent bag 116 Upon actuation of pressure source 122, pressurized fluid, such as air, can be introduced into inflatable transparent bag 116, thereby inflating transparent bag 116 in order to exert a generally uniform force upon transparent window 112 and upon sealing cover 80 and microplate 20.
  • such generally uniform force can serve to provide a reliable and consistent sealing engagement between sealing cover 80 and microplate 20. This sealing engagement can substantially prevent water evaporation or contamination of assay 1000 during thermocycling.
  • inflatable transparent bag 116 can be part of the transparent window 112, thereby forming a bladder.
  • transparent window 112, inflatable transparent bag 116, and sealing cover 80 permit free transmission therethrough of an excitation light 202 generated by an excitation system 200 and the resultant fluorescence emission.
  • Transparent window 112, inflatable transparent bag 116, and sealing cover 80 can be made of a material that is non-fluorescent or of low fluorescence.
  • transparent window 112 can be comprised of Vycor®, fused silica, quartz, high purity glass, or combination thereof.
  • window 112 can be comprised of Schott Q2 quartz glass.
  • window 112 can be from about ⁇ A to about Vz inch thick; e.g., in some embodiments, about 3/8 inch thick.
  • a broadband anti-reflective coating can be applied to one or both sides of window 112 to reduce glare and reflections.
  • the transparent window 112 can comprise optical elements such as a lens, lenslets, and/or a holographic feature.
  • transparent window 112 can be movable to exert a generally uniform force upon transparent bag 116 and, additionally, upon sealing cover 80 and microplate 20.
  • transparent bag 116 can comprise a fixed internal amount of fluid, such as air.
  • Transparent window 112 can be movable using any moving mechanism (not illustrated), such as an electric drive, mechanical drive, hydraulic drive, or the like.
  • pressure clamp system 110 can further employ a pressure chamber 150 in place of transparent bag 116.
  • Pressure chamber 150 can be a pressurizable volume generally defined by transparent window 112, a frame 152 that can be coupled to transparent window 112, and a circumferential chamber seal 154 disposed along an edge of frame 152.
  • Circumferential chamber seal 154 can be adapted to engage a surface to define the pressurizable, airtight, or at least low leakage, pressure chamber 150.
  • Transparent window 112, frame 152, circumferential chamber seal 154, and the engaged surface bound the actual volume of pressure chamber 150.
  • Circumferential chamber seal 154 can engage one of a number of surfaces that will be further discussed herein.
  • a port 120 in fluid communication with pressure chamber 150 and pressure source 122, can provide fluid to pressure chamber 150.
  • circumferential chamber seal 154 can be positioned such that it engages a portion of sealing cover 80.
  • a downward force from transparent window 112 can be exerted upon microplate 20 to maintain a proper thermal engagement between microplate 20 and thermocycler block 102. Additionally, such downward force can further facilitate sealing engagement of sealing cover 80 and microplate 20.
  • pressure chamber 150 can then be pressurized to exert a generally uniform force upon sealing cover 80 and sealing interface 92. Such generally uniform force can provide a reliable and consistent sealing engagement between sealing cover 80 and microplate 20. This sealing engagement can reduce water evaporation or contamination of assay 1000 during thermocycling.
  • circumferential chamber seal 154 of pressure chamber 150 can be positioned to engage thermocycler block 102, rather than microplate 20.
  • Microplate 20 can be positioned within pressure chamber 150. As pressure chamber 150 is pressurized, force is exerted upon sealing cover 80, thereby providing a sealing engagement between sealing cover 80 and microplate 20.
  • optional posts 156 can be employed.
  • Optional posts 156 can be adapted to be coupled with transparent window 112 and downwardly extend therefrom.
  • Optional posts 156 can then engage at least one of microplate 20 or sealing cover 80 to ensure proper contact between microplate 20 and thermocycler block 102 during thermocycling.
  • microplate 20 can be inverted such that each of the plurality of wells 26 is generally inverted, such that the opening of each of the plurality of wells 26 is directed downwardly.
  • this arrangement can provide improved fluorescence detection. As illustrated in FIG. 27, this inverted arrangement causes assay 1000 to collect adjacent sealing cover 80 and, thus, addresses the occurrence of condensation effecting fluorescence detection and improves optical efficiency, because assay 1000 is now disposed adjacent to the opening of each of the plurality of wells 26.
  • thermocycler block 102 remains stationary and is positioned above microplate 20 and transparent window 112 is positioned below microplate 20.
  • Inflatable transparent bag 116 can then be positioned in engaging contact between transparent window 112 and sealing cover 80.
  • transparent window 112, inflatable transparent bag 116, and sealing cover 80 can permit free transmission therethrough of excitation light 202 generated by excitation system 200 positioned below transparent window 112 and the resultant fluorescence therefrom.
  • detection system 300 can be positioned below microplate 20 to detect such fluorescence generated in response to excitation light 202 of excitation system 200.
  • microplate 20 can be positioned in an inverted orientation, similar to that described in connection with FIG. 32, and further employ pressure chamber 150.
  • Circumferential chamber seal 154 can then be positioned such that it engages a portion of sealing cover 80.
  • a force from transparent window 112 can be exerted upon microplate 20 to maintain a proper thermal engagement between microplate 20 and thermocycler block 102 and sealing engagement between sealing cover 80 and microplate 20.
  • Pressure chamber 150 can then be pressurized to exert a generally uniform force across sealing cover 80.
  • vacuum assist system 170 can be formed in transparent window 112.
  • transparent window 112 can comprise a heating device 160.
  • Heating device 160 can be operable to heat transparent window 112, which in turn heats each of the plurality of wells 26 to reduce the formation of condensation within each of the plurality of wells 26.
  • condensation can reduce optical performance and, thus, reduce the efficiency and/or stability of fluorescence detection.
  • a chamber body 1402 has a first side 1404 and a second side 1406.
  • chamber body 1402 can be formed from aluminum or other materials such as steel, stainless steel, standard plastic, or fiber- reinforced plastic compound, such as a resin or polymer, and mixtures thereof.
  • An opening 1408 extends through first side 1404 and second side 1406.
  • a chamber cover 1410 has an opening 1412 surrounded by circumferential chamber seal 154.
  • Circumferential chamber seal 154 can have a peripheral lip that 1413 that defines a sealing plane abutting sealing cover 80 of microplate 20.
  • peripheral lip 1413 can be positioned radially inward of a periphery of opening 1412.
  • a reactive surface 1415 can span between opening 1412 and peripheral lip 1413. Reactive surface 1415 can react to fluid pressure in pressure chamber 150 by increasingly urging peripheral lip 1413 against sealing cover 80 as the fluid pressure increases from zero to about 25 pounds per square inch (PSI).
  • chamber cover 1410 is formed from stainless steel.
  • a gasket 1414 (FIG.
  • thin film heater 1418 can have a power dissipation of at least 50 watts.
  • circumferential chamber seal 154 can be molded from a silicone material. In some embodiments, circumferential chamber seal 154 can be insert-molded with chamber cover 1410.
  • An alignment ring 1426 can be fastened to chamber body 1402 through chamber cover 1410, and secure chamber cover 1410 to second side 1406.
  • Microplate 20 can fit within an inner periphery of alignment ring 1426. Alignment ring 1426 can locate microplate 20 with respect to thermocycler system 100.
  • an alignment feature 1428 can interface with alignment feature 58 of microplate 20.
  • recesses 1430 can be formed in the inner periphery of alignment ring 1426. Recesses 1430 reduce a contact area between alignment ring 1426 and microplate 20 and can thereby reduce heat flow between microplate 20 and alignment ring 1426.
  • a flange 1432 can protrude radially inward from the periphery of opening 1408 and support a window seal 1434. In some embodiments, flange 1432 can be about %" wide. A surface of transparent window 112 can abut window seal 1434. In some embodiments, for example when window seal 1434 is a non-adhesive type seal, a window-retaining ring 1436 can be secured to chamber body 1402 and clamp transparent window 112 against window seal 1434. A connector 1438 can provide a connection to port 120 (FIGS. 34-37, 39-40) that is in fluid communication with the internal volume of pressure chamber 150.
  • At least one catch 1440 can be positioned on frame 152.
  • a pair of catches 1440 can be positioned on opposing sides of a perimeter of frame 152.
  • Each of the pair of catches 1440 can have a centering feature 1442.
  • clamp mechanism 1400 can allow pressure chamber 150 to be moved to an undamped position away from thermocycler system 100.
  • the undamped position can provide a gap of 3/8 inch between thermocycler block 102 (FIG. 203) and microplate 20.
  • clamp mechanism 1400 can be actuated manually.
  • clamp mechanism 1400 can be actuated by pneumatics, hydraulics, electric machines and/or motors, electromagnetics, or any other suitable means.
  • a ball joint 1472 can pivotally connect telescoping end 1468 to input end 1466.
  • a mounting end 1474 of pneumatic cylinder 1470 can pivotally connect to support structure 1444. In various other embodiments, mounting end 1474 of pneumatic cylinder 1470 can pivotally connect to clamp frame 1446.
  • Bellcrank 1452 can have a clamp end 1476.
  • a clamp pin 1478 can project from clamp end 1476 and engage centering feature 1442 when clamp mechanism 1400 is in the locked condition. It should be appreciated that the clamp mechanism 1400 on one side of thermocycler system 100 has been described.
  • a second clamp mechanism 1401 can be positioned on the other side of thermocycler system 100 (FIG. 206). Second clamp mechanism 1401 can be symmetrical with the side just described and operate similarly.
  • a transverse member 1479 can connect lever arm 1456 to the lever arm of the other side.
  • Pneumatic cylinder 1470 can be movable between an extended condition (FIG. 205) and a contracted condition (FIGS. 204 and 206). As pneumatic cylinder 1470 moves to the contracted condition, it can cause lever arm 1456 to pivot as indicated by a curved arrow A. Lever arm 1456 can in turn cause bellcrank 1452 to pivot as indicated by a curved arrow B, thereby moving clamp pin 1478 towards centering feature 1442. Clamp pin 1478 can then become centered in centering feature 1442.
  • bellcrank 1452 As bellcrank 1452 completes rotating in the direction of arrow B, it can cause clamp pin 1478 to move chamber 150 from an undamped position towards the clamped position against thermocycler assembly 100. This can cause circumferential chamber seal 154 to press against microplate 20 (best seen in FIG. 203). A clamping pressure between chamber seal 154 and microplate 20 can be adjusted by varying the pivot location of first end 1450 of over-center link 1448.
  • an adjustment mechanism 1477 such as, by way of non-limiting example, a screw, can be used to vary the pivot location as indicated by arrows A (FIG. 205).
  • optical sensor 1491 can read marking indicia 94 (FIG. 16) on microplate 20 as it is moved to the thermocycler position. Optical sensor 1491 can provide a marking data signal indicative of marking indicia 94 to control system 1010.
  • rails 1480 can be telescoping rails. Rails 1480 can be moved manually or can be motorized. In some motorized embodiments, a rack gear 1482 can be positioned on at least one of rails 1480. A rotating actuator 1484 can be adapted with a pinion gear 1486 that engages rack gear 1482. Rotating actuator 1484 can rotate in response to control signals from control system 1010. In some embodiments, rotating actuator 1484 can be an electric motor, such as a stepper motor. For example, actuator 1484 can be a Vexta PK245-02AA stepper motor available from Oriental Motor U.S.A. Corp. In other embodiments, rotating actuator 1484 can be pneumatic or hydraulic. Pressure chamber 150 can be attached between rails 1480.
  • a lost motion mechanism 1488 can be positioned between rails 1480 and pressure chamber 150. Lost motion mechanism 1488 can allow pressure chamber 150 limited perpendicular movement with respect to rails 1480. The limited perpendicular movement facilitates moving pressure chamber 150 between the clamped and undamped positions as clamp assembly 1400 moves between the locked and unlocked conditions, respectively.
  • Pneumatic system 1500 can provide pneumatic control for various pneumatic devices used in sequence detection system 10.
  • the pneumatic devices can include, alone or in any combination, pressure chamber 150, pneumatic cylinders 1470, and vacuum source 172.
  • Particle filter 1506 and condensation separator 1508 can provide a conditioned fluid supply 1510 to a remainder of pneumatic system 1500.
  • a first pressure regulator 1512 can be in fluid communication with conditioned fluid supply 1510.
  • First pressure regulator 1512 can provide a first fluid supply 1516 to a chamber pressurization subsystem 1518 and/or to other subsystems.
  • a check valve 1520 can be connected in series with first pressure regulator 1512.
  • Check valve 1520 can reduce a risk of depressurization of the internal volume of pressure chamber 150 in the event conditioned fluid supply 1510 is interrupted.
  • a ballast tank 1522 can be in fluid communication with the first fluid supply 1516 and increase a fluid volume of chamber pressurization subsystem 1518. The increased volume can reduce pressure variations of the first fluid supply 1516.
  • Ballast tank 1522 can also provide a fluid reserve to help maintain pressure in the event first fluid supply 1516 is interrupted.
  • One side of a charge valve 1524 can be in fluid communication with the first fluid supply 1516. The other side of charge valve 1524 can be in fluid communication with the internal volume of pressure chamber 150.
  • a flexible fluid line can connect chamber pressurization subsystem 1518 to connector 1438 of chamber 150.
  • Charge valve 1524 can be controlled by control system 1010 in accordance with a method described later herein.
  • charge valve 1524 can be a part number MKH0NBG49A available from .
  • a pressure sensor 1526 can be in fluid communication with the internal volume of pressure chamber 150 and can provide a chamber pressure signal 1527 to control system 1010.
  • pressure sensor 1526 can be a part number MPS-P6N-AG available from Parker-Hannifin Corp.
  • a chamber pressure relief valve 1528 can be in fluid communication with the internal volume of pressure chamber 150 and establish a maximum pressure that can be applied thereto. In some embodiments, the maximum pressure of 1528 chamber pressure relief valve can be less than, or equal to, 30 PSI.
  • Pressurization subsystem 1518 can also comprise a release valve 1530 in fluid communication with the internal volume of pressure chamber 150.
  • the other side of release valve 1530 can be vented to atmosphere.
  • Release valve 1530 can be controlled by control system 1010 in accordance with a method described later herein.
  • release valve 1530 can be a part number
  • the charge and release valves 1524, 1530 can maintain chamber pressure at about 18 PSI while the microplate temperature is greater than 40 degrees Celsius. This combination of pressure and temperature conditions can help reduce a possibility of pressure within wells 26 overcoming the chamber pressure and causing wells 26 to leak between sealing cover 80.
  • a first silencer 1532 can be in fluid communication with the other side of release valve 1530 to reduce noise as fluid is vented.
  • a second pressure regulator 1534 can be in fluid communication with conditioned fluid supply 1510.
  • Second pressure regulator 1534 can provide a second fluid supply 1536 to a cylinder control subsystem 1538.
  • Second pressure regulator 1540 can also provide second fluid supply 1536 to a vacuum control subsystem 1540.
  • a pressure transducer 1542 can be in fluid communication with second fluid supply 1536 and provide a pressure signal 1544 to control system 1010.
  • pressure transducer 1542 can comprise a part number MPS-P6N-AG available from Parker-Hannifin Corp.
  • second fluid supply 1536 is greater than, or equal to, 50 PSI.
  • a cylinder valve 1546 can have a pressure port 1548, an exhaust port 1550, a first port 1552, and a second port 1554.
  • Cylinder valve 1546 can be referred to as a 3-position, 2-port valve, commonly referred to as a 3/2 valve.
  • cylinder valve 1546 can comprise a part number P2MISGEE2CV2DF7 available from or a part number B360BA549C available from .
  • Pressure port 1548 can be in fluid communication with second fluid supply 1536.
  • Exhaust port 1550 can be vented to atmosphere.
  • Cylinder silencer 1556 can be in fluid communication with exhaust port 1550 to reduce noise when fluid is vented from pneumatic cylinder 1470.
  • Cylinder valve 1546 can have three positions that route fluid between ports 1548-1554.
  • a first position can route pressure port 1548 to first port 1552 and route second port 1554 to exhaust port 1550.
  • a second position can block pressure port 1548 and route first and second ports 1552, 1554 to exhaust port 1550.
  • a third position can route pressure port 1548 to second port 1554 and route first port 1552 to exhaust port 1550.
  • the first, second, and third positions of cylinder valve 1546 can be referred to as the lock, release, and unlock positions, respectively.
  • fluid routing through cylinder valve 1546 can cause pneumatic cylinder 1470 to move to the contracted condition, thereby moving clamp mechanism 1400 to the locked condition (FIG. 204).
  • a first limit switch 1560 can sense, either directly or indirectly, when pneumatic cylinder 1470 is in the extended condition and provide a corresponding signal 1562 to control system 1010.
  • a second limit switch 1564 can be used to sense, either directly or indirectly, when pneumatic cylinder 1470 is in the contracted condition and provide a corresponding signal 1566 to control system 1010.
  • first and second limits switches 1560, 1564 can be integral to pneumatic cylinder 1470.
  • pneumatic cylinder 1470 can be a Parker-Hannifin Corp. SRM Series pneumatic cylinder with piston sensing capability.
  • pneumatic cylinder 1470 can be a part number L06DP- SRMBSY400 from Parker-Hannifin Corp.
  • vacuum control system 1540 selectively actuates vacuum source 172. Vacuum generated by vacuum source 172 can be provided to thermocycler system 100 or other systems. Vacuum control system 1572 can comprise a vacuum control valve 1568. In some embodiments, vacuum control valve 1568 can comprise a part number P2MISDEE2CV2BF7 available from
  • Vacuum control valve 1568 can have a pressure port 1570, an exhaust port 1572, a first port 1574, and a second port 1576.
  • Vacuum control valve 1568 can be referred to as a 3-position, 2-port valve, commonly referred to as a 3/2 valve.
  • Pressure port 1570 can be in fluid communication with second fluid supply 1536.
  • exhaust port 1572 can be blocked.
  • exhaust port 1572 can be vented to atmosphere.
  • First port 1574 can be in fluid communication with vacuum source 172.
  • Second port 1576 can be blocked in some embodiments having exhaust port 1572 vented to atmosphere. In other embodiments, second port 1576 can be vented to atmosphere.
  • Vacuum control valve 1568 can be manually controlled. In some embodiments, vacuum control valve 1568 is a servovalve controlled by control system 1010 in accordance with a method described later herein.
  • Vacuum control valve 1568 can have three positions that route fluid between ports 1570-1576.
  • a first position can route pressure port 1570 to first port 1574, and can block exhaust port 1572 and second port 1576.
  • a second position can block pressure port 1570, and route first and second ports 1574, 1576 through exhaust port 1572.
  • a third position can route pressure port 1570 to second port 1576, and block first port 1574 and exhaust port 1572.
  • the first, second, and third positions of vacuum control valve 1568 can also be referred to as the vacuum on, vacuum off, and vent positions, respectively.
  • vacuum control valve 1568 When vacuum control valve 1568 is in the vacuum on position, the fluid routing through vacuum control valve 1568 can flow through vacuum source 172. Vacuum source 172 generates a vacuum in response thereto that can be fluidly coupled to the thermocycler system 100 or other systems. When vacuum control valve 1568 is in the vacuum off position, second fluid supply 1536 is disconnected from vacuum source 172 and vacuum source 172 can be routed to atmosphere through exhaust port 1572 and/or second port 1576. When vacuum control valve 1568 is in the vent position, second fluid supply 1536 can be purged to atmosphere through second port 1576.
  • Method 1580 for clamping pressure chamber 150 to thermocycler system 100.
  • Method 1580 can be executed by control system 1010 when pressure chamber 150 is placed in proximity to thermocycler block 102.
  • Method 1580 can begin in step 1582 and can proceed to decision step 1584 to determine whether pressure chamber 150 is properly located within clamp mechanism 1400.
  • Position signal 1489 FIG. 204 can be used to make the determination.
  • method 1580 can proceed to step 1586 and move cylinder valve 1546 to the lock position.
  • Method 1580 can then proceed to decision step 1588 and determine whether pneumatic cylinder 1470 has moved to the contracted condition, thereby placing clamp mechanism 1400 in the locked condition.
  • Decision step 1588 can make the determination by using signal 1566 (FIG. 207) from second limit switch 1570.
  • Method 1580 can execute decision step 1588 until pneumatic cylinder 1470 moves to the contracted condition.
  • Method 1580 can then proceed to step 1590 and can perform a leak test 1590 as described later herein.
  • Method 1580 can then proceed to decision step 1592 and determine, from results of leak test 1590, whether leak test 1590 passed. If leak test 1590 passed, then method 1580 can proceed to step 1594 and exit. If leak test 1590 failed, then method 1580 can proceed to step 1610 and release chamber 150 according to a method described later herein.
  • step 1596 method 1580 can indicate that chamber 150 is improperly located within clamp mechanism 1400. Method 1580 can then proceed to method 1610 and assure clamp mechanism 1400 is in the unlocked condition. Method 1580 can indicate the improper location of chamber 150 though, by way of example, a buzzer, lamp, writing to a computer memory in control system 1010, or any other suitable means.
  • the chamber leak rate can be expressed in units of PSI/minute.
  • method 1590 can compare the chamber leak rate to a predetermined leak rate. If the chamber leak rate is less than the predetermined leak rate, method 1590 can proceed to step 1598, indicating that the leak test has passed. Method 1590 can then proceed to step 1600 and open charge valve 1524 to connect ballast tank 1536 to the internal volume of pressure chamber 150. In step 1600, method 1590 can also provide an indication to control system 1010 that thermocycling can begin.
  • Method 1610 can also be executed after thermocycling is completed. Method 1610 can begin in step 1612 and then can proceed to step 1614. In step 1614, method 1610 can move cylinder valve 1546 to the unlock position, which can cause pneumatic cylinder 1470 to begin moving to the extended condition and changing clamp mechanism to the unlocked condition. Method 1610 can then proceed to decision step 1616 and determine whether pneumatic cylinder 1470 has moved to the extended condition. Decision step 1616 can make the determination by using signal 1562 (FIG 207) from first limit switch 1560. Method 1610 can execute decision step 1616 until pneumatic cylinder 1470 moves to the extended condition. Method 1610 can then proceed to step 1618 and exit.
  • step 1614 method 1610 can move cylinder valve 1546 to the unlock position, which can cause pneumatic cylinder 1470 to begin moving to the extended condition and changing clamp mechanism to the unlocked condition. Method 1610 can then proceed to decision step 1616 and determine whether pneumatic cylinder 1470 has moved to the extended condition. Decision step 1616 can make the determination by using signal 1562 (FIG
  • excitation system 200 generally comprises a plurality of excitation lamps 210 generating excitation light 202 in response to control signals from control system 1010. Excitation system 200 can direct excitation light 202 to each of the plurality of wells 26 or across the plurality of wells 26. In some embodiments, excitation light 202 can be a radiant energy comprising a wavelength that permits detection of photo-emitting detection probes in assay 1000 disposed in at least some of the plurality of wells 26 of microplate 20 by detection system 300.
  • the quantitative analysis of assay 1000 can involve measurement of the resultant fluorescence intensity or other emission intensity.
  • fluorescence from the plurality of wells 26 on microplate 20 can be measured simultaneously using a CCD camera.
  • CCD camera In an idealized optical system, if all of the plurality of wells 26 have the same concentration of dye, each of the plurality of wells 26 would produce an identical fluorescence signal.
  • wells near the center of the microplate may appear significantly brighter (i.e. output more signal) than those wells near the edge of the microplate, despite the fact that all of the wells may be outputting the same amount of fluorescence. There are several reasons for this condition in some current designs — vignetting, shadowing, and the particular illumination/irradiance profile.
  • the excitation light is brighter at the center, then the fluorescence signal from a well near the edge of the irradiance zone would be less than an identical well near the center. Shadowing can occur due to the depth of the wells. Unless the excitation light is perpendicular to the microplate, some part of the well may not be properly illuminated. In other words, the geometry of the well may block some of the light from reaching the bottom of the well. In addition, the amount of fluorescence emitted, which can be collected, may vary from center to edge. As should be appreciated by one skilled in the art, noise sources are often constant across the field of view of the camera.
  • a graph illustrates the relative intensity or light transmission versus well location on a plate. As can be seen from the graph, the effects of vignetting and shadowing causes the light intensity to drop off along the edges of the field of view of the plate.
  • the present teachings address these effects so that identical wells output generally identical fluorescence irrespective of their location on microplate 20.
  • the optimum irradiance profile can be calculated.
  • a corresponding irradiance profile represented by a dashed line, can provide a higher irradiance along the edges. This irradiance profile, when coupled with the effects of vignetting and shadowing, creates generally uniform signal strength across all of the plurality of wells 26 of microplate 20.
  • the plurality of excitation lamps 210 of excitation system 200 can be fixedly mounted to a support structure 212.
  • the plurality of excitation lamps 210 can be removably mounted to support structure 212 to permit convenient interchange, exchange, replacement, substitution, or the like.
  • support structure 212 can be generally planar in construction and can be adapted to be mounted within housing 1008 (FIG. 1).
  • the plurality of excitation lamps 210 can be arranged in a generally circular configuration and directed toward microplate 20 to promote uniform excitation of assay 1000 in each of the plurality of wells 26.
  • the present teachings permit a generally uniform excitation that is substantially free of shadowing.
  • the plurality of excitation lamps 210 can be arranged in a generally circular configuration about an aperture 214 formed in support structure 212. Aperture 214 permits the free transmission of fluorescence therethrough for detection by detection system 300, as described herein.
  • each of the plurality of excitation lamps 210 can be configured to achieve the desired irradiance profile.
  • each of the plurality of excitation lamps 210 can comprise a lens 216.
  • Lens 216 can be shaped to provide a desired irradiance profile (see FIG. 51). The exact shape of lens 216 can depend, at least in part, upon one or more of the desired irradiance profile at microplate 20, the illumination/irradiance profile at each of the plurality of excitation lamps 210, and the size and position of microplate 20 relative to the plurality of excitation lamps 210.
  • the shape of lens 216 can be calculated in response to the particular application using commercially available software, such as ZEMAX and/or ASAP.
  • each of the plurality of excitation lamps 210 can comprise a mirror 218.
  • Mirror 218 can be shaped to provide a desired irradiance profile (see FIG. 51).
  • the exact shape of mirror 218 can be dependent, at least in part, upon the desired irradiance profile at microplate 20, the illumination/irradiance profile at each of the plurality of excitation lamps 210, and the size and position of microplate 20 relative to the plurality of excitation lamps 210.
  • the shape of mirror 218 can be calculated in response to the particular application using commercially available software, such as ZEMAX and/or ASAP.
  • each of the plurality of excitation lamps 210 can comprise a combination of lens 216 and mirror 218 to achieve the desired irradiance profile. Again, lens 216 and mirror 218 can be calculated in response to the particular application using commercially available software, such as ZEMAX and/or ASAP. [0581] Turning now to FIG. 55, in some embodiments, each of the plurality of excitation lamps 210 can be aligned such that their optical centers converge on a single point 220. Additionally, in some embodiments, a desired irradiance profile (see FIG.
  • each of the plurality of excitation lamps 210 can comprise lens 216 and/or mirror 218 and can further be aligned as illustrated in FIG. 56 to achieve more complex irradiance profiles.
  • employing any of the above techniques described herein can provide improved irradiance across microplate 20, thereby improving the resultant signal to noise ratio of the plurality of wells 26 along the edge of microplate 20.
  • the plurality of excitation lamps 210 can be any one of a number of sources.
  • the plurality of excitation lamps 210 can be a laser source having a wavelength of about 488 nm, an Argon ion laser, an LED, a halogen bulb, or any other known source.
  • the LED can be a MR16 from Opto Technologies (Wheeling IL; http://www.optotech.com/MR16.htm).
  • the LED can be provided by LumiLEDS.
  • the halogen bulb can be a 75 W, 21 V DC lamp or a 50 W, 12 V DC lamp.
  • each of the plurality of excitation sources 210 can be removably coupled to support structure 212 to permit convenient interchange, exchange, replacement, substitution, or the like thereof.
  • the particular excitation source(s) employed can be selected by one skilled in the art to exhibit desired characteristics, such as increased power, better efficiency, improved uniformity, multi-colors, or having any other desired performance criteria.
  • additional detection probes and/or dyes can be used to, in some circumstances, increase throughput of high-density sequence detection system 10 by including multiple assays in each of the plurality of wells 26.
  • the temperature of the plurality of excitation lamps 210 can be controlled to decrease the likelihood of intensity and spectral shifts.
  • the temperature control can be, for example, a cooling device.
  • the temperature control can maintain each of the plurality of excitation lamps 210 at an essentially constant temperature.
  • the intensity can be controlled via a photodiode feedback system, utilizing pulse width modulation (PWM) control to modulate the power of the plurality of excitation lamps 210.
  • PWM pulse width modulation
  • shutters can be used to control each of the plurality of excitation lamps 210. It should be appreciated that any of the excitation assemblies 200 illustrated in FIGS. 42-49 and described above can be interchanged with each other.
  • detection system 300 can be used to detect and/or gather fluorescence emitted from assay 1000 during analysis.
  • detection system 300 can comprise a collection mirror 310, a filter assembly 312, and a collection camera 314. After excitation light 202 passes into each of the plurality of wells 26 of microplate 20, assay 1000 in each of the plurality of wells 26 can be illuminated, thereby exciting a detection probe disposed therein and generating an emission (i.e. fluorescence) that can be detected by detection system 300.
  • collection mirror 310 can collect the emission and/or direct the emission from each of the plurality of wells 26 towards collection camera 314.
  • collection mirror 310 can be a 120 mm-diameter mirror having 1/4 or 1/2 wave flatness and 40/20 scratch dig surface.
  • filter assembly 312 comprises a plurality of filters 318. During analysis, microplate 20 can be scanned numerous times — each time with a different filter 318.
  • collection camera 314 comprises a multielement photo detector 324, such as, but not limited to, charge coupled devices (CCDs), diode arrays, photomultiplier tube arrays, charge injection devices (CIDs), CMOS detectors, and avalanche photodiodes.
  • CCDs charge coupled devices
  • CIDs charge injection devices
  • CMOS detectors charge injection devices
  • avalanche photodiodes the emission from each of the plurality of wells 26 can be focused on collection camera 314 by a lens 316.
  • collection camera 314 is an ORCA-ER cooled CCD type available from Hamamatsu Photonics.
  • lens 316 can have a focal length of 50 mm and an aperture of 2.0.
  • collection camera 314 can be mounted to, and prealigned with, lens 316.
  • detection system 300 can comprise a light separating element, such as a light dispersing element.
  • Light dispersing element can comprise elements that separate light into its spectral components, such as transmission gratings, reflective gratings, prisms, beam splitters, dichroic filters, and combinations thereof that are can be used to analyze a single bandpass wavelength without spectrally dispersing the incoming light.
  • a detection system can be limited to analyzing a single bandpass wavelength. Therefore, one or more light detectors, each comprising a single bandpass wavelength light dispersing element, can be provided.
  • an alignment mount 320 can mate collection camera 314 and lens 316.
  • Alignment mount 320 can provide a mechanism to adjust an axial alignment and a distance between an optic assembly 322 and multi-element photo detector 324.
  • Lens 316 can receive optic assembly 322 and can mount to a mounting face 326 of a base plate 328.
  • Base plate 328 can have an aperture 330 formed therein that can allow light to pass from optic assembly 322 to multi-element photo detector 324.
  • base plate 328 can be formed from a metal, such as steel, stainless steel, or aluminum.
  • Collection camera 314 can contain multi-element photo detector 324 and can mount to a camera mounting plate 332.
  • Mounting plate 332 can have an aperture 334 that can align with aperture 330.
  • Mounting plate 332 can have a face 336 generally parallel to a mating face 338 of base plate 328.
  • mounting plate 332 can be formed from a metal, such as steel, stainless steel, or aluminum.
  • At least one resilient member 340 can attach to mounting plate 332 and to base plate 328.
  • Resilient member 340 can be formed, by non-limiting example, from a spring and/or other elastic structure. Resilient member 340 can provide a bias force that urges face 336 towards mating face 338.
  • a planarity adjustment feature such as, by way of non-limiting example, at least one setscrew 342, can be positioned between face 336 and mating face 338. At least one setscrew 342 can apply a force opposite the bias force provided by resilient member 340 and maintain face 336 in a spaced relationship from mating face 338.
  • At least one set screw 342 can have a thread pitch between 80 and 100 threads per inch (TPI), inclusive.
  • at least one setscrew 342 can be a ball-end type.
  • three setscrews 342 can be radially spaced around mounting plate 332.
  • the planarity adjustment feature can comprise cams, motorized screws, fluid-containing bags, or inclined planes.
  • the space between face 336 and mating face 338 can be less than 1/8 inch.
  • a light blocking gasket 344 can be positioned in the space between face 336 and mating face 338.
  • light blocking gasket 344 can be formed from closed cell foam.
  • Light blocking gasket 344 can have apertures formed therein that align with apertures 330 and 334, and with the planarity adjustment feature.
  • at least one of collection camera 314 and lens 316 can have a mount comprising a threaded mount or a bayonet mount.
  • the threaded mount can comprise, for example, a C-mount or a CS-mount.
  • the bayonet mount can comprise, for example, an F-mount or a K-mount.
  • collection camera 314 can be mounted to mounting plate 332 using a mounting ring 346 and a retaining ring 348.
  • mounting plate 332 can be formed from a metal, such as steel, stainless steel, or aluminum.
  • Collection camera 314 can be secured to mounting ring 346.
  • Mounting ring 346 can fit into a groove 350 formed around a periphery of aperture 334.
  • Retaining ring 348 can fasten to mounting plate 332 and can cover at least a portion of groove 350 and a portion of mounting ring 346, thereby retaining mounting ring 346 within groove 350.
  • retaining ring 348 can be formed from a metal, such as steel, stainless steel, or aluminum.
  • a concentricity adjustment feature such as at least one set screw 352, can protrude radially into groove 350 and can press against an outer periphery 354 of mounting ring 346.
  • the concentricity adjustment feature can locate mounting ring 350 in an x-y plane of groove 350.
  • the x-y plane can be illustrated by a coordinate system 356.
  • at least one setscrew 352 can have a thread pitch between 80 TPI and 100 TPI, inclusive.
  • at least one setscrew 352 can be a ball-end type.
  • the concentricity adjustment feature in other embodiments can include cams, motorized screws, fluid-containing bags, and/ or inclined planes.
  • a line segment 358 can represent an image plane of optic assembly 322.
  • An arrow 360 can be centered on optic assembly 322 and normal to its image plane 358.
  • a line segment 362 can represent an image plane of multi-element photo detector 324.
  • An arrow 364 can be centered on multi-element photo detector 324 and normal to its image plane 362.
  • the planarity adjustment feature such as at least one set screw 342, can be used to tilt mounting plate 332 such that image plane 362 can become parallel with image plane 322.
  • the planarity adjustment feature can also used to adjust the distance between optic assembly 322 and multi-element photo detector 324.
  • the concentricity adjustment feature such as at least one setscrew 352, can translate mounting ring 346 in the x-y plane. Translating mounting ring 346 can adjust arrow 364 concentrically with arrow 360.
  • alignment features 368 can align base plate 328 with support structure 212. Locations of alignment features 368 and dimensions of alignment mount 320 can be selected to place the arrow 360 concentric with a center of microplate 20. Locations of alignment features 356 and dimensions of alignment mount 320 can be selected to place image plane 358 in parallel with an image plane of microplate 20. In some embodiments having collection mirror 310 (of FIGS. 42 and 43), locations of alignment features 356 and dimensions of alignment mount 320 can be selected to place image plane 358 perpendicular with the image plane of microplate 20. In some embodiments, base plate 328 can include a foot plate 366. By way of non- limiting example, alignment features 368 can comprise any combination of dowels and keys.
  • control system 1010 can archive data within a database, database retrieval, database analysis and manipulation, and bioinformatics.
  • control system 1010 can be operable to analyze raw data and among other actions, control operation of high-density sequence detection system 10.
  • Such analysis of raw data can comprise compensating for point spread (PSF), background or base emissions, a unique intensity profile, optical crosstalk, detector and/or optical path variability and noise, misalignment, or movement during operation. This can be accomplished, in some embodiments, by utilizing internal controls in several of the plurality of wells 26, as well as calibrating high-density sequence detection system 10.
  • data analysis can comprise difference imaging, such as comparing an image from one point in time to an image at a different point in time, or image subtracting.
  • data analysis can comprise curve fitting based on a specific gene or a gene set.
  • data analysis can comprise using no template control (NTC) background or baseline correction.
  • NTC no template control
  • data analysis can comprise error estimation using confidence values derived in terms of CT. See U.S. Patent Application No. 60/517,506 filed November 4, 2003 and U.S. Patent Application No. 60/519,077 (Attorney Docket No. AB 5043) filed November 10, 2003.
  • the present teachings can provide a method of characterizing signal noise associated with operation of a charge-coupled device (CCD) adapted for analysis of biological samples, wherein the signal noise comprises a dark current contribution, readout offset contribution, and spurious change contribution.
  • CCD charge-coupled device
  • the method can comprise providing a plurality of first data points associated with first outputs provided from the CCD under a substantially dark condition during a first exposure duration, providing a plurality of second data points associated with second outputs provided from the CCD under the substantially dark condition during a second exposure duration wherein the second duration is different from the first duration, providing a plurality of third data points associated with third outputs provided from a cleared output register of the CCD without comprising charge transferred thereto, determining the dark current contribution per unit exposure time by comparing the first data points and the second data points, determining the readout offset contribution from the third data points, and determining the spurious charge contribution based on the dark current contribution and the readout offset contribution.
  • a high-density sequence detection system or components thereof are used for the amplification of polynucleic acids, such as by PCR.
  • PCR can be used to amplify a sample of target Deoxyribose Nucleic Acid (DNA) for analysis.
  • DNA Deoxyribose Nucleic Acid
  • the PCR reaction involves copying the strands of the target DNA and then using the copies to generate additional copies in subsequent cycles. Each cycle doubles the amount of the target DNA present, thereby resulting in a geometric progression in the number of copies of the target DNA.
  • the temperature of a double-stranded target DNA is elevated to denature the DNA, and the temperature is then reduced to anneal at least one primer to each strand of the denatured target DNA.
  • the target DNA can be a cDNA.
  • primers are used as a pair— a forward primer and a reverse primer-and can be referred to as a primer pair or primer set.
  • the primer set comprises a 5' upstream primer that can bind with the 5' end of one strand of the denatured target DNA and a 3' downstream primer that can bind with the 3' end of the other strand of the denatured target DNA.
  • the primer can be extended by the action of a polymerase.
  • the polymerase can be a thermostable DNA polymerase, for example, a Taq polymerase.
  • the product of this extension which sometimes may be referred to as an amplicon, can then be denatured from the resultant strands and the process can be repeated. Temperatures suitable for carrying out the reactions are well known in the art. Certain basic principles of PCR are set forth in U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188, each issued to Mullis et al.
  • PCR can be conducted under conditions allowing for quantitative and/or qualitative analysis of one or more target DNA.
  • detection probes can be used for detecting the presence of the target DNA in an assay.
  • the detection probes can comprise physical (e.g., fluorescent) or chemical properties that change upon binding of the detection probe to the target DNA.
  • Some embodiments of the present teaching can provide real time fluorescence-based detection and analysis of amplicons as described, for example, in PCT Publication No. WO 95/30139 and U.S. Patent Application No. 08/235,411.
  • assay 1000 can be a homogenous polynucleotide amplification assay, for coupled amplification and detection, wherein the process of amplification generates a detectable signal and the need for subsequent sample handling and manipulation to detect the amplified product is minimized or eliminated.
  • Homogeneous assays can provide for amplification that is detectable without opening a sealed well or further processing steps once amplification is initiated.
  • Such homogeneous assays 1000 can be suitable for use in conjunction with detection probes.
  • the use of an oligonucleotide detection probe, specific for detecting a particular target DNA can be included in an amplification reaction in addition to a DNA binding agent of the present teachings. Homogenous assays among those useful herein are described, for example, in commonly assigned U.S. Patent No. 6,814,934.
  • methods for detecting a plurality of targets. Such methods include those comprising forming an initial mixture comprising an analyte sample suspected of comprising the plurality of targets, a polymerase, and a plurality of primer sets.
  • each primer set comprises a forward primer and a reverse primer and at least one detection probe unique for one of the plurality of primer sets.
  • the initial mixture can be formed under conditions in which one primer elongates if hybridized to a target.
  • the location of a fluorescent signal on a solid support, such as microplate 20 can be indicative of the identity of a target comprised by the analyte sample.
  • a plurality of detection probes are distributed to identify loci of at least some of the plurality of wells 26 of microplate 20.
  • a signal deriving from a detection probe such as, for example, an increase in fluorescence intensity of a fluorophore at a particular locus can be detected if an amplification product binds to a detection probe and is then amplified.
  • the location of the locus can indicate the identity of the target, and the intensity of the fluorescence can indicate the quantity of the target.
  • reagents comprising a master mix comprising at least one of catalysts, initiators, promoters, cofactors, enzymes, salts, buffering agents, chelating agents, and combinations thereof.
  • reagents can include water, a magnesium catalyst (such as MgCI2), polymerase, a buffer, and/or dNTP.
  • specific master mixes can comprise AmpliTaq® Gold PCR Master Mix, TaqMan® Universal Master Mix, TaqMan® Universal Master Mix No AmpErase® UNG, Assays-by-DesignSM, Pre-Developed Assay Reagents (PDAR) for gene expression, PDAR for allelic discrimination and Assays-On- Demand®, (all of which are marketed by Applied Biosystems).
  • PDAR Pre-Developed Assay Reagents
  • PDAR Pre-Developed Assay Reagents
  • allelic discrimination and Assays-On- Demand® all of which are marketed by Applied Biosystems.
  • the present teachings should not be regarded as being limited to the particular chemistries and/or detection methodologies recited herein, but may employ Taqman®; Invader®; Taqman Gold®; protein, peptide, and immuno assays; receptor binding; enzyme detection; and other screening and analytical methodologies.
  • high-density sequence detection system 10 is operable for analysis of materials (e.g., polynucleotides) comprising or derived from genetic materials from organisms.
  • materials e.g., polynucleotides
  • such materials comprise or are derived from substantially the entire genome of an organism.
  • organisms include, for example, humans, mammals, mice, Arabidopsis or any other plant, bacteria, fungi, or animal species.
  • assay 1000 comprises at least one of a homogenous solution of a DNA sample, at least one primer set for detection of a polynucleotide comprising or derived from such genetic materials, at least one detection probe, a polymerase, and a buffer.
  • assay 1000 comprises at least one of a plurality of different detection probes and/or primer sets to perform multiplex PCR, which can be particularly useful when analyzing a whole genome having, for example, about 30,000 different genes.
  • analysis of substantially the entire genome of an organism is conducted on a single microplate 20, or on multiple microplates (e.g., two, three, four or more) each comprising subparts of such materials comprising or derived from the genetic materials of the organism.
  • a plurality of plates contain a plurality of assay 1000 having essentially identical materials and a plurality of assay 1000 having different materials.
  • a plurality of plates do not contain assay 1000 having essentially identical materials.
  • microplate 20 comprises a fixed subset of a genome. It should also be recognized that the present teachings can be used in connection with genotyping, gene expression, or other analysis.
  • the microplate can be covered with a sealing liquid prior to performance of analysis or reaction of assay 1000.
  • a sealing liquid is applied to the surface of a microplate comprising reaction spots comprising an assay 1000 for amplification of polynucleotides.
  • a sealing liquid can be a material which substantially covers the material retention regions (e.g., reaction spots) on the microplate so as to contain materials present in the material retention regions, and substantially prevent movement of material from one reaction region to another reaction region on the substrate.
  • the sealing liquid can be any material which is not reactive with assay 1000 under normal storage or usage conditions.
  • the sealing liquid can be substantially immiscible with assay 1000.
  • the sealing liquid can be transparent, have a refractive index similar to glass, have low or no fluorescence, have a low viscosity, and/or be curable.
  • the sealing liquid can comprise a flowable, curable fluid such as a curable adhesive selected from the group consisting of. ultra-violet-curable and other light-curable adhesives; heat, two-part, or moisture activated adhesives; and cyanoacrylate adhesives.
  • the sealing liquid can be selected from the group consisting of mineral oil, silicone oil, fluorinated oils, and other fluids which are substantially non-miscible with water.
  • the sealing liquid can be a fluid when it is applied to the surface of the microplate and in some embodiments, the sealing liquid can remain fluid throughout an analytical or chemical reaction using the microplate. In some embodiments, the sealing liquid can become a solid or semi-solid after it is applied to the surface of the microplate.
  • the present teachings can find utility in a wide variety of amplification methods, such as PCR, Reverse Transcription PCR (RT-PCR), Ligation Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), self-sustained sequence replication (3SR), strand displacement activation (SDA), Q (3replicase) system, isothermal amplification methods, and other known amplification method or combinations thereof.
  • amplification methods such as PCR, Reverse Transcription PCR (RT-PCR), Ligation Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), self-sustained sequence replication (3SR), strand displacement activation (SDA), Q (3replicase) system, isothermal amplification methods, and other known amplification method or combinations thereof.
  • amplification methods such as PCR, Reverse Transcription PCR (RT-PCR), Ligation Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), self-sustained sequence replication (3SR), strand displacement activation
  • the present teachings can be used in connection with such amplification methods and analytical techniques using not only spectrometeric measurements, such as absorption, fluorescence, luminescence, transmission, chemiluminescence, and phosphorescence, but also colorimetric or scintillation measurements or other known detection methods. It should also be appreciated that the present teachings may be used in connection with microcards and other principles, such as set forth in U.S. Patent Nos. 6,126,899 and 6,124,138.
  • the two oligonucleotides can be joined by ligation, e.g., by treatment with ligase.
  • ligation e.g., by treatment with ligase.
  • microplate 20 is heated to dissociate unligated detection probes, and the presence of ligated, target-bound detection probe is detected by reaction with an intercalating dye or by other means.
  • the oligonucleotides for OLA can also be designed to bring together a fluorescer-quencher pair, as discussed above, leading to a decrease in a fluorescence signal when the analyte sequence is present.
  • the concentration of a target region from an analyte polynucleotide can be increased, if desired, by amplification with repeated hybridization and ligation steps.
  • Simple additive amplification can be achieved using the analyte polynucleotide as a target and repeating denaturation, annealing, and ligation steps until a desired concentration of the ligated product is achieved.
  • the ligated product formed by hybridization and ligation can be amplified by ligase chain reaction (LCR).
  • LCR ligase chain reaction
  • two complementary sets of sequence-specific oligonucleotide detection probes are employed for each target DNA.
  • One of the two sets of sequence-specific oligonucleotide detection probes comprises first and second oligonucleotides designed for sequence-specific binding to adjacent, contiguous regions of a first strand of target DNA.
  • the second of the two sets of sequence-specific oligonucleotide detection probes comprises first and second oligonucleotides designed for sequence-specific binding to adjacent, contiguous regions of a second strand of target DNA.
  • the target DNA is amplified exponentially, allowing small amounts of target DNA to be detected and/or amplified.
  • the oligonucleotides for OLA or LCR assay bind to adjacent regions in a target that are separated by one or more intervening bases, and ligation is effected by reaction with (i) a DNA polymerase, to fill in the intervening single stranded region with complementary nucleotides, and (ii) a ligase enzyme to covalently link the resultant bound oligonucleotides.
  • a detection probe comprises a moiety that facilitates detection of a nucleic acid sequence, and in some embodiments, quantifiably.
  • a detection probe can comprise, for example, a fluorophore such as a fluorescent dye, a hapten such as a biotin or a digoxygenin, a radioisotope, an enzyme, or an electrophoretic mobility modifier.
  • the level of amplification can be determined using a fluorescently labeled oligonucleotide.
  • a detection probe can comprise a fluorophore further comprising a fluorescence quencher.
  • a detection probe can comprise a fluorophore and can be, for example, a 5'-exonuclease assay probe such as a TaqMan® probe (marketed by Applied Biosystems), a stem-loop Molecular Beacon (see, e.g., U.S. Patent Nos. 6,103,476 and 5,925,517, Nature Biotechnology 74:303-308 (1996); Vet et al., Proc Natl Acad Sci U S A. 96:6394-6399 (1999)), a stemless or linear molecular beacon (see., e.g., PCT Patent Publication No.
  • a 5'-exonuclease assay probe such as a TaqMan® probe (marketed by Applied Biosystems)
  • a stem-loop Molecular Beacon see, e.g., U.S. Patent Nos. 6,103,476 and 5,925,517, Nature Biotechnology 74:303-308 (1996); Vet e
  • a peptide nucleic acid (PNA) light-up probe a self-assembled nanoparticle probe, or a ferrocene-modified probe described, for example, in U.S. Patent No. 6,485,901; Mhlanga et al., Methods 25:463-471 (2001); Whitcombe et al., Nature Biotechnology 77:804-807 (1999); lsacsson et al., Molecular Cell Probes 74:321-328 (2000); Svanvik et al., Anal. Biochem.
  • PNA peptide nucleic acid
  • a detection probe can comprise a sulfonate derivative of a fluorescent dye, a phosphoramidite form of fluorescein, or a phosphoramidite forms of CY5. Detection probes among those useful herein are also disclosed, for example, in U.S. Patent Nos.
  • a detection probe can comprise a fluorescent dye.
  • the fluorescent dye can comprise at least one of rhodamine green (R110), 5-carboxyrhodamine, 6-carboxyrhodamine, N,N'-diethyl-2',7'-dimethyl-5- carboxy-rhodamine (5-R6G), N,N'-diethyl-2',7'-dimethyl-6-carboxyrhodamine (6-R6G), 5-carboxy-2',4',5',7',-4,7-hexachlorofluorescein, 6-carboxy-2',4',5',7',-4,7-hexachloro- fluorescein, 5-carboxy-2',7'-dicarboxy-4',5'-dichlorofluorescein, 6-carboxy-2',7'- dicarboxy-4',5'-dichlorofluorescein, 5-carboxy-2',7'- dicarboxy
  • amplified sequences can be detected in double-stranded form by a detection probe comprising an intercalating or a crosslinking dye, such as ethidium bromide, acridine orange, or an oxazole derivative, for example, SYBR Green® (marketed by Molecular Probes, Inc.), which exhibits a fluorescence increase or decrease upon binding to double-stranded nucleic acids.
  • a detection probe comprises SYBR Green® or Pico Green® (marketed by Molecular Probes, Inc.).
  • a detection probe can comprise an enzyme that can be detected using an enzyme activity assay.
  • An enzyme activity assay can utilize a chromogenic substrate, a fluorogenic substrate, or a chemiluminescent substrate.
  • the enzyme can be an alkaline phosphatase
  • the chemiluminescent substrate can be (4-methoxyspiro [1 ,2-dioxetane-3,2'(5'-chloro)- tricyclo [3.3.1.13, 7]decan]-4-yl) phenylphosphate.
  • a chemiluminescent alkaline phosphatase substrate can be CDP-Star® chemiluminescent substrate or CSPD® chemiluminescent substrate (marketed by Applied Biosystems).
  • the present teachings can employ any of a variety of universal detection approaches involving real-time PCR and related approaches.
  • a first reaction vessel such as for example, an eppendorf tube
  • a plurality of decoding reactions are then performed in microplate 20 described herein.
  • a multiplexed oligonucleotide ligation reaction OLA
  • OLA multiplexed oligonucleotide ligation reaction
  • a given encoded target DNA can be amplified by that distinct primer pair in a given well of plurality of wells 26.
  • a universal detection probe such as, for example, a nuclease cleavable TaqMan® probe
  • Such approaches can result in a universal microplate 20, with its attendant benefits including, among other things, one or more of economies of scale, manufacturing, and/or ease-of-use.
  • the nature of the multiplexed encoding reaction can comprise any of a variety of techniques, including a multiplexed encoding PCR pre-amplification or a multiplexed encoding OLA. Further, various approaches for encoding a first sample with a first universal detection probe, and a second sample with a second universal detection probe, thereby allowing for two sample comparisons in a single microplate 20 , can also be performed according to the present teachings. Illustrative embodiments of such encoding and decoding methods can be found for example in PCT Publication No. WO2003US0029693 to Aydin et al., PCT Publication No. WO2003US0029967 to Andersen et al., U.S. Provisional Application Nos.
  • SNP Single Nucleotide Polymorphism
  • the detection probes can be suitable for detecting single nucleotide polymorphisms (SNPs).
  • SNPs single nucleotide polymorphisms
  • a specific example of such detection probes comprises a set of four detection probes that are identical in sequence but for one nucleotide position. Each of the four detection probes comprises a different nucleotide (A, G, C, and T/U) at this position.
  • the detection probes can be labeled with probe labels capable of producing different detectable signals that are distinguishable from one another, such as different fluorophores capable of emitting light at different, spectrally resolvable wavelengths (e.g., 4-differently colored fluorophores).
  • two colors can be used for two known variants.
  • At least one of the forward primer and the reverse primer can further comprise a detection probe.
  • a detection probe (or its complement) can be situated within the forward primer between the first primer sequence and the sequence complementary to the target DNA, or within the reverse primer between the second primer sequence and the sequence complementary to the target DNA.
  • a detection probe can comprise at least about 10 nucleotides up to about 70 nucleotides and, more particularly, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 50 nucleotides, or about 60 nucleotides.
  • a detection probe (or its complement) can further comprise a Zip-CodeTM sequence (marketed by Applied Biosystems).
  • a detection probe can comprise an electrophoretic mobility modifier, such as a nucleobase polymer sequence that can increase the size of a detection probe, or in some embodiments, a non- nucleobase moiety that increases the frictional coefficient of the detection probe, such as those mobility modifier described in commonly-owned U.S. Patent Nos. 5,514,543, 5,580,732, 5,624,800, and 5,470,705 to Grossman.
  • a detection probe comprising a mobility modifier can exhibit a relative mobility in an electrophoretic or chromatographic separation medium that allows a user to identify and distinguish the detection probe from other molecules comprised by the sample.
  • a detection probe comprising a sequence complementary to a detection probe and an electrophoretic mobility modifier can be, for example, a ZipChuteTM detection probe (marketed by Applied Biosystems).
  • a detection probe with an amplicon, followed by electrophoretic analysis can be used to determine the identity and quantity of the target DNA.
  • the present teaching provide methods and apparatus for Reverse Transcriptase PCR (RT-PCR), which include the amplification of a Ribonucleic Acid (RNA) target.
  • assay 1000 can comprise a single-stranded RNA target, which comprises the sequence to be amplified (e.g., an mRNA), and can be incubated in the presence of a reverse transcriptase, two primers, a DNA polymerase, and a mixture of dNTPs suitable for DNA synthesis. During this process, one of the primers anneals to the RNA target and can be extended by the action of the reverse transcriptase, yielding an RNA/cDNA doubled-stranded hybrid.
  • thermostable reverse transcriptases can comprise, but are not limited to, reverse transcriptases such as AMV reverse transcriptase, MuLV, and Tth reverse transcriptase.
  • assay 1000 can be an assay for the detection of RNA, including small RNA. Detection of RNA molecules can be, in various circumstances, very important to molecular biology, in research, industrial, agricultural, and clinical settings. Among the types of RNA that are of interest in some embodiments are, for example, naturally occurring and synthetic regulatory RNAs such as small RNA molecules (Lee, et al., Science 294: 862-864, 2001; Ruvkun, Science 294: 797-799; Pfeffer et al., 304: Science 734-736, 2004; Ambros, Cell 107: 823-826, 2001; Ambros et al., RNA 9: 277-279, 2003; Carrington and Ambros, Science 301: 336-338, 2003; Reinhart et al., Genes Dev.
  • small RNA molecules Lee, et al., Science 294: 862-864, 2001; Ruvkun, Science 294: 797-799; Pfeffer
  • Small RNA molecules such as, for example, micro RNAs (miRNA), short interfering RNAs (siRNA), small temporal RNAs (stRNA) and short nuclear RNAs (snRNA), can be, typically, less than about 40 nucleotides in length and can be of low abundance in a cell.
  • miRNA micro RNAs
  • siRNA short interfering RNAs
  • stRNA small temporal RNAs
  • snRNA short nuclear RNAs
  • high-density sequence detection system 10 can detect miRNA expression found in, for instance, cell samples taken at different stages of development.
  • coexpression patterns can be analyzed across microplate 20 with TaqMan sensitivity, specificity, and dynamic range.
  • high-density sequence detection system 10 can be used to validate that siRNA molecules have successfully, post-translationally regulated the gene expression patterns of interest.
  • such methods may be useful during the manipulation of gene expression patterns using siRNAs in order to elucidate gene function and/or interrelationships amongst genes.
  • the methods of the present teachings can include forming a detection mixture comprising a detection probe set ligation sequence, and a primer set.
  • any detection probe set ligation sequence comprised by the detection mixture can be amplified using PCR on high-density sequence detection system 10 and thereby form an amplification product.
  • detection of amplification of any detection probe ligation sequence of an analyte can comprise detection of binding of a detection probe to a detection probe hybridization sequence comprised by a probe set ligation sequence or an amplification product thereof.
  • detecting can comprise contacting a PCR amplification product such as an amplified probe set ligation sequence with a detection probe comprising a label under hybridizing conditions.
  • assay 1000 can comprise a preamplification product, wherein one or more polynucleotides in an analyte has been amplified prior to being deposited in at least one of the plurality of wells 26.
  • these methods can further comprise forming a plurality of preamplification products by subjecting an initial analyte comprising a plurality of polynucleotides to at least one cycle of PCR to form a detection mixture comprising a plurality of preamplification products.
  • the detection mixture of preamplification products can be then used for further amplification using microplate 20 and high-density sequence detection system 10.
  • preamplification comprises the use of isothermal methods.
  • the methods involve the incorporation of an RNA polymerase promoter into selected cDNA molecule by priming cDNA synthesis with a primer complex comprising a synthetic oligonucleotide containing the promoter.
  • a polymerase generally specific for the promoter can be added, and anti-sense RNA can be transcribed from the cDNA template.
  • the progressive synthesis of multiple RNA molecules from a single cDNA template results in amplified, anti-sense RNA (aRNA) that serves as starting material for cloning procedures by using random primers.
  • the amplification which will typically be at least about 20-40, typically to 50 to 100 or 250-fold, but can be 500 to 1000-fold or more, can be achieved from nanogram quantities or less of cDNA.
  • a two stage preamplification method can be used to preamplify assay 1000 in one vessel by IVT and, for example, this preamplification stage can be 100 X sample.
  • the preamplified product can be divided into aliquots and preamplified by PCR and, for example, this preamplification stage can be 16,000 X sample or more.
  • this preamplification stage can be 16,000 X sample or more.
  • the preamplification can be a multiplex preamplification, wherein the analyte sample can be divided into a plurality of aliquots. Each aliquot can then be subjected to preamplification using a plurality of primer sets for DNA targets. In some embodiments, the primer sets in at least some of the plurality of aliquots differ from the primer sets in the remaining aliquots. Each resulting preamplification product detection mixture can then be dispersed into at least some of the plurality of wells 26 of microplate 20 comprising an assay 1000 having corresponding primer sets and detection probes for further amplification and detection according to the methods described herein.
  • the primer sets of assay 1000 in each of the plurality of wells 26 can correspond to the primer sets used in making the preamplification product detection mixture.
  • the resulting assay 1000 in each of the plurality of wells 26 thus can comprise a preamplification product and primer sets and detection probes for amplification for DNA targets, which, if present in the analyte sample, have been preamplified.
  • the multiplex preamplification can be used in a variety of contexts to effectively increase the concentration or quantity of a sample available for downstream analysis and/or assays.
  • significantly more analyses can be performed with multiplex amplified samples than can be performed with the original sample.
  • multiplex amplification further permits the ability to perform analyses that require more sample or a higher concentration of sample than was originally available. In such embodiments, multiplex amplification enables downstream analysis for assays that could not have been possible with the original sample due to its limited quantity.
  • assay 1000 comprises a first universal primer that binds to a complement of a first target, a second universal primer that binds to a complement of a second target, a first detection probe comprising a sequence that binds to the sequence comprised by the first target, and a second detection probe comprising a sequence that binds to a sequence comprised by the second target.
  • at least some of the plurality of wells 26 of microplate 20 comprise a solution operable to perform multiplex PCR.
  • the first and second detection probes can comprise different labels, for example, different fluorophores such as, in non-limiting example, VIC and FAM.
  • Sequences of the first and second detection probes can differ by as little as one nucleotide, two nucleotides, three nucleotides, four nucleotides, or greater, provided that hybridization occurs under conditions that allow each detection probe to hybridize specifically to its corresponding detection probe.
  • the kit can comprise at least one primer and at least one detection probe disposed in at least some of the plurality of wells 26.
  • the kit can comprise a forward primer, a reverse primer, and at least one FAM labeled MGB quenched PCR detection probe disposed in at least some of the plurality of wells 26.
  • the kit can comprise at least one detection probe, at least one primer, and a polymerase.
  • a kit comprises a container containing assay reagents and a separate data storage medium that contains data about the assay reagents.
  • the assay reagents can be adapted to perform an allelic discrimination or expression analysis reaction when mixed with at least one target polynucleotide.
  • the other reagents can be, for example, components conventionally used for PCR and can comprise non-reactive components.
  • the assay reagents container can have a machine-readable label that provides information about the contents of the container.
  • the data stored on the data storage medium can comprise computer-readable code that can be used to adjust, calibrate, direct, set, run, or otherwise control an apparatus, for example, high-density sequence detection system 10.
  • the data stored on the date storage medium can be used to control high-density sequence detection system 10 to automatically perform PCR or RT-PCR of assay 1000. See, for example, U.S. Patent Application Publication No. 2004/0072195.
  • a plurality of microplates 20 having assay 1000 filled thereon can be analyzed as described herein with high-density sequence detection system 10 to generate data.
  • this data can be stored in a gene expression analysis system database 736. Software can then be used to generate gene expression analysis information 738.
  • a gene expression analysis system can utilize computer software that organizes analysis sessions into studies and stores them in database 738.
  • An analysis session can comprise the results of running microplate 20 in high-density sequence detection system 10.
  • To analyze session data one can load an existing study that contains analysis session data or create a new study and attach analysis session data to it. Studies can be opened and reexamined an unlimited number of times to reanalyze the analysis session data or to add other analysis sessions to the analysis.
  • gene expression analysis system database 736 stores the analyzed data for each microplate 20 run on high-density sequence detection system 10 as an analysis session in database 736.
  • the software can identify each analysis session by marking indicia 64 of the associated microplate 20 and the date on which it was created. Once analysis sessions have been assigned to a study, various functions can be performed. These functions comprise, but are not limited to, designating replicates, removing outliers, filtering data out of a particular view or report, correction of preamplification values via stored values, and computation of gene expression values.
  • real time PCR is adapted to perform quantitative real time PCR (qRT-PCR).
  • two different methods of analyzing data from qRT-PCR experiments can be used: absolute quantification and relative quantification.
  • absolute quantification can determine an input copy number of the target DNA of interest This can be accomplished, for example, by relating a signal from a detection probe to a standard curve.
  • relative quantification can describe the change in expression of the target DNA relative to a reference or a group of references such as, for an example, an untreated control, an endogenous control, a passive internal reference, an universal reference RNA, or a sample at time zero in a time course study.
  • a gene expression analysis system can determine the amount of target DNA, normalized to a reference and relative to a calibrator, by determining:
  • ⁇ C T can be less than ⁇ 1.
  • the above calculations can be adapted for use in multiplex PCR (See, for example, Livak et al. Applied Biosystems User Bulletin #2, updated October 2001 and Livak and Schmittgen, Methods (25) 402- 408 (2001).
  • assay 1000 can be preamplified, as discussed herein, in order to increase the amount of target DNA prior to distribution into the plurality of wells 26 of microplate 20.
  • assay 1000 can be collected, for example, via a needle biopsy that typically yields a small amount of sample. Distributing this sample across a large number of wells can result in variances in sample distribution that can affect the veracity of subsequent gene expression computations.
  • assay 1000 can be preamplified using, for example, a pooled primer set to increase the number of copies of all target DNA simultaneously.
  • a minor proportion of all target DNA can have an observed preamplification efficiency of less than 100%.
  • the amplification bias is reproducible and consistent from one input sample to another, then the ability to accurately compute comparative relative quantitation between any two samples containing different relative amounts of target can be maintained.
  • target DNA X is initially present in sample A at 100 target molecules, then after 10 cycles of PCR amplification (50% of 1000-fold), 50,000 target molecules should be present.
  • target X is initially present in sample B at 500 target molecules, then after 10 cycles of PCR amplification (50% of 1000-fold), 250,000 target molecules should be present.
  • the ratio of template X in samples A/B remains constant before and after the amplification procedure and is the same ratio as the 100% efficiency scenario.
  • an unbiased amplification of each target DNA can be determined by calculating the difference in C ⁇ value of the target DNA (x,y,z, etc.) from the C ⁇ value of a selected endogenous reference, and such calculation is referred to as the ⁇ C ⁇ value for each given target DNA, as described above.
  • a reference for a bias calculation can be non- preamplified, amplified target DNA and an experimental sample can be a preamplified amplified target DNA.
  • the standard sample and experimental sample can originate from the same sample, for example, same tissue, same individual, and/or same species.
  • comparison of ⁇ C T values between the non-preamplified amplified target DNA and preamplified amplified target DNA can provide a measure for the bias of the preamplification process between the endogenous reference and the target DNA (x, y, z, etc.).
  • the difference between the two ⁇ C T values can be zero and as such there is no bias from preamplification. This is illustrated below with reference to FIG. 213.
  • the gene expression analysis system can be calibrated for potential differences in preamplification efficiency that can arise from a variety of sources, such as the effects of multiple primer sets in the same reaction.
  • calibration can be performed by computing a reference number that reflects preamplification bias. Reference number similarity for a given target DNA across different samples is indicative that the preamplification reaction ⁇ C ⁇ s can be used to achieve reliable gene expression computations.
  • a gene expression analysis system can compute these reference numbers by collecting a sample (designated as Sample A and S A ) and processing it with one or more protocols.
  • a first protocol comprises running individual PCR gene expression reactions for each target DNA (T x ) relative to an endogenous reference (endo), such as, for example, 18s or GAPDH. These reactions can yield cycle threshold values for each target DNA relative to the endogenous control; as computed by:
  • a second protocol can comprise running a single PCR preamplification step on assay 1000 with, for example, a pooled primer set.
  • the pooled primer set can contain primers for each target DNA.
  • the preamplified product can be distributed among plurality of wells 26 of microplate 20.
  • PCR gene-expression reactions can be run for each preamplified target DNA (Tx) relative to an endogenous reference (endo). These reactions can yield cycle threshold values for each preamplified target DNA relative to the endogenous control, as computed by:
  • a difference between these ⁇ C T not preamplified T X S A and ⁇ C T preamplified T X S A can be computed by:
  • ⁇ C ⁇ T x S A ⁇ Cy not preampiified T X S A " ⁇ C ⁇ preamplified T X S A
  • an amplification efficiency can be less than 100% for a particular target DNA, therefore ⁇ C T is less than zero for the particular target DNA.
  • An example can be an evaluation of ⁇ C T values for a group of target DNA from a 1536-plex for the multiplex preamplification process including four different human sample input sources: liver, lung, brain and an universal reference tissue composite. In this example, most ⁇ C T values are near zero, however, some of the target DNA have a negative ⁇ C T value but these negative values are reproducible from one sample input source to another.
  • a gene expression analysis system can determine if a bias exists for target DNA analyzed for different sample inputs.
  • a gene expression analysis system can use ⁇ C T values computed for the same target DNA but in different samples (Sample A (S A ) and Sample B (S B )) in order to determine the accuracy of subsequent relative expression computations. This results in the equation,
  • ⁇ C T T X ⁇ C T T X S A - ⁇ C T T X S B
  • a value for ⁇ C T T X can be zero or reasonably close to zero which can indicate that the preamplified ⁇ C T values for T x ( ⁇ C T preampiified T X S A and ⁇ C T preamplified T x S B ) can be used for relative gene expression computation between different samples via a standard relative gene expression calculation.
  • a standard relative gene expression calculation can determine the amount of the target DNA. In some embodiments, a standard relative gene expression calculation employs a comparative C ⁇ . In some embodiments, the above methods can be practiced during experimental design and once the conditions have been optimized so that the ⁇ C T T x is reasonably close to zero, subsequent experiments only require the computation of the ⁇ C T value for the preampiified reactions. In some embodiments, ⁇ C T T X S A values can be stored in a database or other storage medium. In such embodiments, these values can then be used to convert ⁇ C Tpreampiified T x S A values to ⁇ C T not preampii f iedT x S A values.
  • the ⁇ C T preamplified T x S y values can be mapped back to a common domain.
  • a not preampiified domain can be calculated using other gene expression instrument platforms such as, for example, a microarray.
  • the ⁇ C T T X S A values need not be stored for all different sample source inputs (S A ) if it can be illustrated that the ⁇ C T preamplified T x is reasonably consistent over different sample source inputs.
  • real-time PCR data can be directly compared to data from other platforms.
  • a ⁇ C T calculation can be a validation tool to confirm that relative quantitation data can be compared from one amplification/detection process to another.
  • ⁇ C ⁇ calculation can be a validation tool to confirm that relative quantitation data can be compared from one sample input source to another sample input source, for example, comparing a sample from liver to a sample from brain in the same individual.
  • ⁇ C T calculation can be a validation tool to confirm that relative quantitation data can be compared from one high- density sequence detector system 10 to another high-density sequence detection system 10.
  • ⁇ C T calculation can be a validation tool to confirm that relative quantitation data can be compared from one platform to another, for example, data from real time PCR to data from a hybridization array is especially valuable for cross-platform validation.
  • real time PCR and hybridization array data can be directly compared.
  • the TaqMan ⁇ C T can be compared to a microarray output converted to the ⁇ C T format. In such embodiments, the resultant ⁇ C T , if within +/- 1 C ⁇ of zero, can determine a high- degree of confidence that the actual fold difference observed within each of the two platforms is correlative.
  • high-density sequence detection system 10 measures the relative quantities of target DNA using the C ⁇ value from a PCR growth curve, as described herein.
  • the measured C ⁇ value for target DNA for a given assay may vary depending on the system and/or microplate 20 in which the assay 1000 is measured. That is, such variation may arise from manufacturing differences in high- density sequence detection system 10 and/or thermal non-uniformity from variances in production of microplate 20.
  • normalization may be the adjusting of a set of raw measurements.
  • quantities may be represented in copy numbers, according to some transformation function in order to make such data compatible between different samples.
  • adjusting copy numbers for a target DNA quantity will produce measurements normalized against a quantity of total RNA and therefore such data can be expressed in specific meaningful and/or compatible units.
  • raw measurements may not carry information that is easily interpretable.
  • the control comprises a template.
  • the template can be, for example, a synthetic oligonucleotide or plasmid, genomic DNA, or other natural DNA or RNA.
  • the template can contain analogs of naturally occurring nucleotides with modifications to the base, sugar, or phosphate backbone, such as PNAs.
  • exogenous templates can be used as controls and such templates can be introduced into assay 1000 in one of the following ways:
  • the template at a known concentration can be introduced into a reverse transcription reaction along with the sample;
  • the template at a known concentration can be introduced into a preamplification reaction along with the sample;
  • the template at a known concentration can be spotted onto at least one of a plurality of wells 26.
  • the exogenous template can be spotted and dried into at least some of the plurality of wells 26 at a known and defined concentration and the C ⁇ value measured from those of the plurality of wells 26 comprising the control.
  • This Cj value can be used to correct for high-density sequence detection system 10, microplate 20, and sample filling/pipetting variations.
  • assay 1000 can be used to fill at least some of the plurality of wells 26, but assay 1000 would not contain any exogenous template that would be amplified.
  • the plurality of wells 26 used for controls on microplate 20 can be allocated to contain at least one fluorescent dye that can be spotted and dried down into microplate 20 and hydrolyzed at the time of sample filling. Such plurality of wells 26 can be used to improve calibration of detection system 300 for optical aberrations.
  • a dye can be used at known concentration and the signals therefrom can be used to optimize the detection sensitivity of high- density sequence detection system 10 (such as the exposure time of the CCD in a detection system 300).
  • the plurality of wells 26 comprising a series dilution of control wells can be used for such calibrations and optimizations.
  • some of the plurality of wells 26 can be used as controls for identification of the position of the plurality of wells 26.
  • at least some of the plurality of wells 26 on microplate 20 can comprise a passive internal reference dye (PIR), such as for example, ROX.
  • PIR passive internal reference dye
  • the signal from the PIR can be used to locate the plurality of wells 26 by detection system 300.
  • background signals from quenching dyes can be used to determine the locations of the plurality of wells 26 by detection system 300.
  • controls can be used to determine filling errors.
  • signals from the PIR can be used to determine if sample filling errors have occurred by looking for an absent or an abnormally high or low signal in the PIR detection image or channel. These signals can indicate an empty well, or an overfilled or under filled well, respectively.
  • controls can be used to determine spotting errors.
  • the background signals from quenching dyes can be used to determine if spotting errors occurred by looking for an absent or an abnormally high or low signal in the quenching detection image or channel.
  • the plurality of wells 26 without detection probes or primers and/or the plurality of wells 26 that are completely empty or filled with buffer or other solution not containing dye can be used for background correction.
  • the plurality of wells 26 comprising controls without templates can also be used for background correction and/or for confirming lack of contamination of the plurality of wells 26 by other samples.
  • the plurality of wells 26 comprising controls without assay 1000 can be used to confirm lack of contamination during spotting.
  • the plurality of wells 26 containing varying amounts of a single or multiple dyes can be used to determine if high-density sequence detection system 10 is capable of detecting signals within the expected dynamic range independent of assay performance.
  • the plurality of wells 26 containing varying amounts of a single or multiple dyes can be used to correct for optical crosstalk or other means of signal correction or normalization. Examples include serial dilutions, multiple titration points, dye ladders, as well as replicates and combinations thereof.
  • pin hole arrays are used for optical calibration. The controls described above, individually or in combinations thereof, can be incorporated into a single microplate 20 to be used to verify high-density sequence detection system 10 performance in the field at the time of installation or during manufacture.
  • a procedure for calibration of spectral sensitivity can employ a reference standard to apply a correction to a spectrum such that each of the plurality of wells 26 signal for each filter is normalized to a specific value.
  • the reference standard can comprise serial dilutions, multiple titration points or dye ladders, as well as replicates and combinations thereof.
  • the reference comprises multiple dyes (e.g., two, three, four, five, or more) in some of the plurality of wells 26 of microplate 20.
  • the value should be identical across all instruments and time periods in order to preserve the calibration.
  • a reference can be fluorescent reference standard.
  • the reference can be used in normalizing a single high-density sequence system 10. In some embodiments, the reference can be used to normalize a group of high-density sequence systems 10. In some embodiments, the procedure normalizes thresholds and baselines over a group of high- density sequence detector systems 10 so that C ⁇ values are similar across the group for the same assay 1000. In some embodiments, the controls are templates.
  • the templates are introduced into a mixture comprising a sample prior to reverse transcription and the resulting C ⁇ values generated from the templates are used to correct for variations in the efficiency of the reverse transcription reaction relative to the expected CT value.
  • templates are introduced into a mixture comprising a sample prior to preamplification and the resulting C ⁇ values generated from the templates are used to correct for variations in efficiency of the preamplification reaction.
  • the templates are introduced into a mixture comprising the sample prior to amplification and the resulting C ⁇ values generated from the templates are used to correct for variations in efficiency of amplification.
  • different templates are introduced into the mixture comprising a sample at the three different steps (i) reverse transcription, (ii) preamplification and (iii) amplification and the resulting CT values generated from the templates are calculated for each of the three steps.
  • the resulting C ⁇ value generated from the templates can be used to determine which of the three steps can be responsible for large deviations of C ⁇ measurements from the expected values.
  • Multiple exogenous templates with varying relative concentrations can be added to a sample mixture in any of the three steps or all of the steps.
  • a standard plot for absolute quantitation of a sample run on microplate 20 can be calculated. The standard plot can be used to normalize data attained from different microplates 20 or from different samples on the same microplate 20.
  • a control can comprise an endogenous template or a set of endogenous templates within a sample that can be used in a wide range of tissues.
  • the endogenous template can be selected so that the average signal produced during amplification is consistent from sample to sample.
  • the appropriately selected endogenous template can be used to normalize for variations in sample quantity in the plurality of wells 26.
  • results from endogenous controls can be compared from results from exogenous control to distinguish variations in sample quantity and variations in assay performance.
  • a dataset can be normalized by using a function of multiple endogenous templates as controls. For example, a regression of the mean expression values from multiple endogenous controls and can be chosen to be expressed across the entire expression range.
  • normalization using a function examples include functions of the mean signal across microplate 20, median normalization, quantile normalization, and lowness normalization.
  • the endogenous controls are relatively invariantly expressed across standard experimental conditions or biological conditions, for example, a tumor, or non-tumor tissue.
  • the endogenous controls are relatively, invariantly expressed across different tissue types, for example, brain and lung.
  • a single endogenous control can be used for normalization.
  • multiple endogenous controls are used for normalization.
  • microplate 20 comprising a calibrated dilution series of DNA targets and single exon assays can be run on high-density sequence detection system 10 and the data collected can be used to calibrate for absolute quantity or copy number estimations or as in comparison to other array platforms.
  • microplate 20 can comprise a combination of replicated bacterial DNA and human DNA.
  • microplate 20 can be spotted with 96 different primer sets and 64 replications of the ten-fold primer sets.
  • the human sample can be split and then spiked with bacterial targets to make a set of four ten-fold dilutions.
  • Microplate 20 comprising 96 primer sets with 64 replications can be filled with the set of four ten-fold dilutions and run in high-density sequence detection system 10 producing data for 16 replications of each dilution of the set.
  • the data collected can be used for calculation of high-level performance parameters such as tabulating bad data, calibrating random error model, estimating systematic errors, and estimating starting copy number.
  • controls can be used for spatial normalization that compensates between at least two channels of signal that is being collected by detections system 300.
  • the channels for which a signal can be being collected and imaged can be different band passes and the optical performance can change with wavelength and detection probe.
  • spatial normalization can be accomplished by calibration images of each of the at least two channels collected from a mixture of a pure detection probe spotting to the channel.
  • a control comprising a mixture of dyes can be spotted onto microplate. In such embodiments, the control comprising a mixture of dyes produces a high signal to noise ratio when detected in detection system 300 of high-density sequence detection system 10.
  • spatial normalization correction can be calculated by the use of spatial trends of the measurements of the controls.
  • the controls comprising a mixed dye can be placed in the grid throughout microplate 20.
  • a coarse image can be collected and normalized to a 1, 2D median smoothed inner plated under every feature collected is then divided into the image of the extracted normalized intensities.
  • spatial normalization allows for platform comparisons of data, removes specific instrument effects, or improves cross instrument and cross platform comparisons.
  • any of the controls discussed above can be adapted for genotyping applications.
  • a method for supplying a user with assays useful in obtaining structural genomic information, such as the presence or absence of one or more SNPs, and functional genomic information, such as the expression or amount of expression of one or more genes.
  • the assays can be configured to detect the presence or expression of genetic material in the sample.
  • a method of compiling a library of polynucleotide data sets can be provided.
  • the data sets can correspond to polynucleotides that each function as a primer for producing a nucleic acid sequence that can be complementary to at least one target SNP, as a detection probe for rendering detectable the at least one target SNP, or as both.
  • the method can comprise selecting for the library polynucleotide data sets that each correspond to a respective polynucleotide that contains a sequence that is complementary to a respective first allele in each of the at least one target, if, under a set of reaction conditions a number of parameters are met by each polynucleotide corresponding to the data sets in the library.
  • the method can comprise determining a background signal value by calculating a first normalized ratio of a fluorescence intensity of a respective polynucleotide that contains a sequence that is complementary to a first allele comprised in the at least one target nucleic acid sequence, reacted with first assay reactants in the absence of the target nucleic acid sequence, and under first conditions of fluorescence excitation, to a dye fluorescence intensity of a passive-reference dye under the first conditions.
  • the method can comprise comparing a difference between a second normalized ratio of the fluorescence intensity of the respective polynucleotide reacted with the first assay reactants in the presence of the target nucleic acid sequence, to the dye fluorescence intensity, and the background signal value.
  • the method can comprise comparing a difference between a third normalized ratio of the fluorescence intensity of the respective polynucleotide reacted with second assay reactants that contain a second allele comprised in the at least one target nucleic acid sequence to the dye fluorescence intensity, wherein the second allele differs from the first allele, and the background signal value.
  • the method can comprise determining whether at least one individual from a population of individuals has a genotype identifiable under the first conditions that result from reacting the respective polynucleotide with the first assay reactants and in the presence of the target nucleic acid sequence, wherein the population comprises at least one individual that has the identifiable genotype and at least one individual that does not have the identifiable genotype.
  • the method can comprise determining whether at least one individual from the population has an identifiable minor allele of the identifiable genotype, under the first conditions that result from reacting the respective polynucleotide with the first assay reactants in the presence of the target nucleic acid sequence. See U.S. Patent Application Publication No. 2003/0190652 to De La Vega et al.
  • high-density sequence detection system 10 can be used for a variety of biological applications, or assays, other than PCR.
  • high-density sequence detection system 10 comprising optical illumination and detection system 300 can be used in imaging microplates that fit a SBS standard footprint from low density microplates, for example, 96, 384, or 1536 well microplates to high-density microplates, for example, 6144 or 31104 well microplate.
  • using lower density microplates high-density sequence detection system 10 can detect multiple, discrete events within a well, for example, for imaging fluorescently tagged antibodies binding to receptors on the surface of a cell for high- throughput cell-based screening.
  • high-density sequence detection system 10 is not limited to imaging only microplate 20 but can be used in the imaging of gels, blots, nitrocellulose membranes, and the like with features at high- density.
  • high-density sequence detection system 10 can image microplates, nitrocellulose membranes, gels, films, blots, and the like. Detection can be, in some embodiments, for isotopic changes, chemiluminescent emissions, chemifluorescent emissions, fluorescent emissions, calorimetric changes, and time-lapse studies of any of the above detection methods. In some embodiments, high-density sequence detection system 10 can be used as a spectrophotometer or spectrofluorometer for samples contained in microplate 20.
  • high-density sequence detection system can be used for methods for the measurement and/or analysis of absorbance (UV-Vis-NIR) ' by adding a detector to opposite side from excitation side of microplate 20; for methods for the measurement and/or analysis of fluorescence intensity; for methods for the measurement and/or analysis of fluorescence polarization by adding at least one polarizing filter to detection system 300; or for methods for the measurement and/or analysis of time resolved fluorescence.
  • high-density sequence detection system 10 can be modified to increase read out speed of CCD pixels.
  • high-density sequence detection system 10 can be used for methods for the measurement and/or analysis of luminescence; .
  • high-density sequence detection system 10 can be used for time-limited chemiluminescent reactions and in such embodiments, high- density sequence detection system 10 can be modified to manipulate reagents in microplate 20 to begin the reactions.
  • high-density sequence detection system 10 can be used to perform various isothermal procedures in, for example, the areas of molecular diagnostics, genotyping, gene expression monitoring, and drug screening.
  • Such isothermal procedures can include, for example, those useful in genetic, biochemical, and bioanalytic processes, such as processes for detecting a target DNA, processes for detecting a mutation, processes for detecting a polymorphism, processes for detecting a single base insertion or deletion, and for processes for identifying SNPs.
  • the high-density sequence detection system 10 can be used to perform isothermal amplification according to U.S. Patent No. 6,692,917.
  • processes for identifying SNPs can include, for example, assays for single-base discrimination and/or quantitative detection of DNA or RNA sequences, for example, SNPs and mutations (single base changes, insertions or deletions in DNA and RNA molecules), from samples containing genomic DNA, total RNA, cell lysates, purified DNA, purified RNA, or nucleic acid amplification products, for example, PCR or RT-PCR products.
  • Other assays that can be carried out using high- density sequence detection system 10 of the present teachings include the processes and methods taught in U.S. Patent No. 6,692,917.
  • high-density sequence detection system 10 can be used to detect the binding activity of primary antibody reagents as direct labeled conjugates or indirect conjugate forms, for example, conjugate enzymes or conjugate Quantum Dots (Qdots).
  • Cells from a variety of sources can be used including in vitro tissue culture and peripheral blood leukocytes.
  • binding events can be detected or imaged from microplate 20, or alternatively, on nitrocellulose membranes with high-density separation channels and/or bands, for example, using a Western blot technique.
  • one protein in a mixture of any number of proteins can be detected while also providing information about the size of the protein and such information can indicate how much protein has accumulated in cells.
  • first proteins are separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates the proteins by size.
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • Nitrocellulose membrane is placed on the gel and the protein bands are electrokinetically transported onto the nitrocellulose membrane. This results in a nitrocellulose membrane imprinted with the same protein bands as the gel.
  • the nitrocellulose membrane is then incubated with a primary antibody made by inoculating a rabbit and diluting the antisera (from blood).
  • the primary antibody sticks to the protein and forms an antibody-protein complex with the protein of interest.
  • the nitrocellulose membrane is then incubated with a secondary antibody, an antibody enzyme conjugate.
  • the secondary antibody is an antibody against the primary antibody and has the ability to stick to the primary antibody.
  • the conjugate enzyme can comprise a molecular flare stuck onto the antibodies so they can be visualized.
  • the enzyme is incubated in its specific reaction mix resulting in bands wherever there is a protein-primary antibody- secondary antibody-enzyme complex such as wherever the protein of interest is located.
  • high-density sequence detection system 10 can be used to detect a flash of light that is given off by the enzyme and, in some embodiments, detection system 300 of high-density sequence detection system 10 can be customized for the particular conjugated labels.
  • Green Fluorescent Protein is extracted from Aequorea Victoria.
  • GFP is a small protein (about 27 Kd) and the DNA sequences coding for GFP can be manipulated by recombinant DNA technology to create gene fusions between GFP and any protein of interest. Such DNA constructs can then be introduced into living cells to express the GFP fluorescent tags on the protein of interest.
  • the GFP fluorescent tag can be used to localize a protein of interest to a specific cell type and/or subcellular localization in living cells and organisms.
  • high-density sequence detection system 10 optics can be modified to enable 2-40 X magnification of individual wells or a small number of wells, adding an x-y stage and adding z-axis autofocus.
  • high-density sequence detection system 10 can be used to perform GFP-based protein localization assays using microplate 20.
  • the GFP DNA coding sequence can be placed behind a promoter and/or regulatory DNA sequence of interest, and introduced into cells and this can be used to perform promoter studies in living organisms.
  • fluorescence resonance energy transfer (FRET) assays can be used to determine the exact time and place of colocalization. Energy transfers from the excited fluorophore to the nearby acceptor fluorophore.
  • donor and acceptor molecules are less than 10 nm apart and the emission spectra of the donor fluorophore overlap the excitation spectra of the acceptor fluorophore. The farther apart the molecules are, the weaker the transfer energy. Extremely low light levels require, in some embodiments, a highly sensitive cooled CCD with high quantum efficiency and fast readout rates. FRET images can be taken at different wavelengths.
  • high-density sequence detection system 10 can be modified to perform FRET assays in microplate 20.
  • High-density sequence detection system 10 optics can be modified to enable magnification (e.g., 2 - 40 X) of individual wells or a small number of wells, adding an x-y stage, and adding z-axis autofocus.
  • high-density sequence detection system 10 can be used to perform FRET assays using microplate 20.
  • high-density sequence detection system 10 can produce a series of time lapse images for FRET. Assays Using QDots As Labels
  • QDots are fluorescent nanoparticles made of inorganic molecules, for example, CdSe and an emission wavelength of a QDot is determined by its physical size.
  • QDots have large stokes shifts, with excitation wavelengths on the order of 408 nm and emission wavelengths starting at around 520 nm and
  • Qdots can have greater photostability, greater spectral separation, and brighter emission relative to organic fluorescent dyes. It is possible to label, or conjugate QDots to molecules of interest for molecular biology assays, such as antibodies. Further, mixtures of QDots can be employed to provide multiplexing capability.
  • Some embodiments include the use of beads coated with different QDot nanocrystals to detect gene expression levels. For example, 9 ⁇ m paramagnetic beads can be coated with mixtures of QDot nanocrystals. Unique spectral codes can be created using four different fluorescent colors of QDot nanocrystals coated onto the beads at defined ratios. Then an outer protective coat can be applied and cross-linked.
  • gene-specific oligonucleotide probes are conjugated to the bead surface and each gene-specific bead can be identified by its unique QDot nanocrystal spectral code.
  • Gene-specific beads can be combined to form custom gene panels. In some embodiments, many beads of each different type are added to each well 26 with the different bead types having been coated with the spectral code corresponding to the different target DNA.
  • RNA is isolated from cells or tissue and the sample can then be labeled with biotin. Unbound biotin can be separated from the biotynilated-sample complex by washing, size exclusion, or any of a number of other well-known processes. The cleanly separated biotin labeled sample can then be added to the bead mixtures in microplate 20 and allowed to hybridize to the beads. A reporter can be created by attaching streptavidin to a fifth QDot nanocrystal label. Unattached streptavidin can be separated from the QDot labeled streptavidin in a manner similar to that used for separating the unbound biotin, as before.
  • QDots have been linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus.
  • each bead can be identified by reading its spectral code and can quantify the amount of target hybridized to each coded bead.
  • high-density sequence detection system 10 can be optimized for the excitations and emissions of QDots.
  • spectral codes with the multiplexing capabilities afforded by spectral codes, a whole genome gene expression analysis can be completed on a microplate 20.
  • microplate 20 can be modified using coatings, activations, and the like to make it more amenable to a particular assay. For example, for growing and staining adherent cells, for example, high protein binding (affinity to molecules for hydrophobic and hydrophilic domains - high binding of antibodies), and for low binding capacity (affinity to molecules of hydrophobic domains).
  • adherent cells for example, high protein binding (affinity to molecules for hydrophobic and hydrophilic domains - high binding of antibodies), and for low binding capacity (affinity to molecules of hydrophobic domains).
  • high-density sequence detection system 10 comprising microplate 20 can be used to analyze cell differentiation such as identifying morphological changes following membrane dye incorporation; analyze cell cycle employing the detection of G1 , S and G2/M phases of a cell cycle; determine mitotic index by detection using antibodies to identify M-phase specific marker; identify cell adhesion by detecting attachment and morphology; or monitor colony formation by detecting the enumeration of one or more colonies.
  • high-density sequence detection system 10 comprising microplate 20 can be used to study slow ion channels by employing, for example, detection of ion flux fluorescent DiBAC4(3) reporter.
  • high-density sequence detection system 10 comprising microplate 20 can be used to study protein kinase by using standard antibody methods; study translocation by identifying movement of proteins between plasma membrane, cytoplasm, and the nucleus; study fluorescent proteins such as EGFP and Reef Coral Fluorescent Protein in multiplex assays; identify quantum dots using limited spectral overlap from distinct conjugates; or to study cell based screening such as data lactamase, adipogenesis, hybridoma, expression cloning and/or lectin binding.
  • high-density sequence detection system 10 comprising microplate 20 can be used to study G-protein coupled receptors.
  • Such a system can be used to measure changes in the dielectric properties of the samples contained in the plurality of wells 26 of microplate 20.
  • Examples of events that cause changes in dielectric properties include monitoring cell growth and/or death, detecting DNA hybridization, detecting protein-protein and protein-small molecule interactions, detecting protein conformational changes, detecting ion channel flux in cells, and monitoring bulk properties such as pH, and salt concentration.
  • this interaction can generate electron charge density waves called plasmons and can cause a reduction in the intensity of the reflected light.
  • High-density sequence detection system 10 can be modified to illuminate microplate 20 with incident polarized light covering a range of incident angles. In some embodiments with further modifications, high-density sequence detection system 10 can measure reflected light at different angles of transmission from microplate 20.
  • the resonance angle at which the intensity minimum occurs can be a function of the refractive index of the solution close to the gold layer, for example, a biological sample flowing over the gold layer in the plurality of the wells 26 of microplate 20.
  • modified high-density sequence detection system 10 can be used to detect SPR analysis such as protein interactions, small molecule (drug candidates) interactions with their targets, membrane-bound receptor interactions, DNA and RNA hybridization, interactions between whole cells and viruses, recognition of cell surface carbohydrates and molecular interactions, such as binding and dissociation.
  • SPR analysis such as protein interactions, small molecule (drug candidates) interactions with their targets, membrane-bound receptor interactions, DNA and RNA hybridization, interactions between whole cells and viruses, recognition of cell surface carbohydrates and molecular interactions, such as binding and dissociation.
  • An electrochemical signal can be generated when the amplicon hybridizes to the capture probe and the ferrocene-labeled signal probe, thereby bringing a reporter molecule, ferrocene, into contact with the monolayer on the gold electrode.
  • an AC voltammogram is obtained when the specific target DNA is detected in a sample, but no electronic signal is registered when the specific target DNA is absent from the sample.
  • microplate 20 can comprise a high-density array of planar waveguides to selectively excite only fluorophores located at or near the surface of the waveguide.
  • the waveguide can be constructed by depositing a high refractive index material onto a low refractive index material.
  • a parallel laser light beam is coupled into the waveguiding film by a diffractive grating which is etched into the substrate material of microplate 20.
  • the light propagates within the waveguiding film and creates a strong evanescent field perpendicular to the direction of propagation into the adjacent medium, for example, one of plurality of wells 26 in microplate 20.
  • the field strength of the evanescent wave can decay exponentially with distance, so only fluorophores at or near the surface are excited.
  • selective detection of DNA hybridization, immunoaffinity reactions, and membrane receptor based assays can be analyzed using microplate 20 comprising a high-density array of planar waveguides.
  • microplate 20 can comprise heat generating electronics and such electronics can be associated with, or in proximity to, one or more of plurality of wells 26 in microplate 20.
  • temperatures in a plurality of wells 26 or subsets thereof can be controlled to create a gradient thermocycler.
  • microplate 20 comprising heat generating electronics can be used, for example, to determine optimum assay parameters such as oligo melting point temperatures and/or can be used to improve synchronization of thermal cycling with detection system 300 in high-density sequence detection system 10.
  • thermal cycling reactions can be started or stopped selectively by use of microplate 20 comprising heat generating electronics to correspond with optical detection.
  • a web-based user interface can be provided that comprises a web-based gene exploration system operable to provide information to assist a user in selecting one or both of a stock assay and a custom assay.
  • the web-based gene exploration system can comprise a search function operable to identify genetic material based on a portion of known data. The search function can provide one or more parameters identifying gene structure or function for selection by the user.
  • systems comprising a web- based user interface configured for ordering stock assays and/or requesting custom designed assays. Such assays can then be delivered to the user.
  • such assays are configured to detect presence or expression of genetic material.
  • Assays that detect the presence or expression of genetic material can comprise assays for detecting SNPs or for detecting expressed genes.
  • the web-based user interface can be configured to receive criteria related to the SNP or to the expressed transcript for which an assay is ordered.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Clinical Laboratory Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Optical Measuring Cells (AREA)
  • Devices For Use In Laboratory Experiments (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne une microplaque comprenant une partie corps principal présentant une première surface et une deuxième surface opposée, ainsi qu'une gorge disposée à proximité de la première surface. Cette gorge divise la partie corps principal en une section intérieure et une section extérieure. Une pluralité de puits sont formés dans la section intérieure de la première surface, chaque puits étant dimensionné pour recevoir une dose.
PCT/US2006/010373 2005-03-22 2006-03-21 Plaque haute densite creusee d'une gorge WO2006102396A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US11/087,101 US20050225751A1 (en) 2003-09-19 2005-03-22 Two-piece high density plate
US11/087,101 2005-03-22
US11/086,262 US20050280811A1 (en) 2003-09-19 2005-03-22 Grooved high density plate
US11/086,262 2005-03-22

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WO2006102396A2 true WO2006102396A2 (fr) 2006-09-28
WO2006102396A3 WO2006102396A3 (fr) 2007-03-29

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EP4019131A1 (fr) * 2020-12-22 2022-06-29 Albert-Ludwigs-Universität Freiburg Dispositif microfluidique et procédé pour isoler des objets
US11686208B2 (en) 2020-02-06 2023-06-27 Rolls-Royce Corporation Abrasive coating for high-temperature mechanical systems

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* Cited by examiner, † Cited by third party
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US11686208B2 (en) 2020-02-06 2023-06-27 Rolls-Royce Corporation Abrasive coating for high-temperature mechanical systems
EP4019131A1 (fr) * 2020-12-22 2022-06-29 Albert-Ludwigs-Universität Freiburg Dispositif microfluidique et procédé pour isoler des objets
WO2022136170A1 (fr) * 2020-12-22 2022-06-30 Albert-Ludwigs-Universität Freiburg Dispositif microfluidique et procédé pour isoler des objets

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