(12)
`
`United States Patent
`Bordenkircher et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,514,750 B2
`Feb. 4, 2003
`
`US006514750B2
`
`(54) PCR SAMPLE HANDLING DEVICE
`
`(75) Inventors: Gary L: Bordenklrcher, Lrvermore;
`Gary Llm, San Francisco; Jacob
`Koppel Freudenthal, Alameda, all of
`CA (US)
`
`(73) Assignee: PE Corporation (NY), Foster City, CA
`(Us)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`_
`21 A l.N .. 09897500
`0
`(
`)
`pp
`/
`’
`(22) Filed:
`Jul. 3, 2001
`(65)
`Prior Publication Data
`
`US 2003/0008383 A1 Jan. 9 2003
`7
`
`................................
`435/288.4; 435/288.7; 435/809; 435/810;
`422/50; 422/102; 422/104
`(58) Field of Search ....................... .. 422/50, 68.1, 102,
`422/104, 2862; 435/2872, 2884, 2887,
`809 810
`’
`
`(56)
`
`References Cited
`
`Us PATENT DOCUMENTS
`3 080 759 A
`3/1963 McQuaid
`37933165 A
`1/1976 Budzak et aL
`4,948,564 A
`8/1990 Root et al. ................ .. 422/101
`5,210,015 A
`5/1993 Gelfand et a1,
`____ __ 435/6
`5,456,360 A * 10/1995 Gri?n ......... ..
`206/443
`5,538,848 A
`7/1996 Livak et al
`---- -- 435/5
`57710381 A : 1/1998 Atwood et al
`73/86491
`2
`‘lil‘fnssatertlet a1‘
`9;
`5,928,907 A
`7/1999 Woudenberg et al.
`435/912
`6,015,674 A
`1/2000 Woudenberg et al. ....... .. 435/6
`6,148,878 A * 11/2000 GanZ et al. ............... .. 141/129
`
`,
`
`,
`
`ise e a .
`
`........ ..
`
`.
`
`6,159,368 A 12/2000 Moring et al. ....... .. 210/321.75
`6,272,939 B1 * 8/2001 Frye et al. ............. .. 73/864131
`FOREIGN PATENT DOCUMENTS
`
`DE
`EP
`EP
`EP
`W0
`W0
`W0
`
`3/1999
`197 39 119 A1
`2/1999
`0 895 240 A1
`0 955 097 A1 11/1999
`1 088 590 A1
`4/2001
`WO 91/17239
`11/1991
`WO 97/36681
`10/1997
`WO 01/28684 A2
`4/2001
`OTHER PUBLICATIONS
`
`U. Landegren et al., “A Ligase—Mediated Gene Detection
`Techni ue ” Science 241:1077—80 (Au . 1988).
`‘1
`’
`’
`g
`D. Nickerson et al., “Automated DNA diagnostics using an
`ELISA—based olignucleotide ligation assay,” Proc. Natl.
`Acad. Sci USA, 87:8923—27 (Nov. 1990).
`
`(List Continued on next page‘)
`Primary Examiner—David A. Redding
`(74) Attorney) Agent) Or Firm_Finnegan, Henderson,
`Farabow’ Garrett & Dunner’ L'L'P'
`(57)
`ABSTRACT
`_
`_
`_
`_
`Adevrce for handlmg PCR microcards, each havmg an array
`of sample chambers closed by a transparent material on one
`side thereof, in relation to a PCR instrument, the device
`including a carrier having an apertured region With an array
`of holes corresponding in number and relative location With
`the array of sample chambers in each of the microcards, and
`a provision for retaining a microcard on the carrier so that
`the transparent material faces the apertured region With the
`reagent Sample Chambers aligned, respectively, With the
`holes in the apertured region, and so that the side of the
`microcard opposite the transparent material is unobstructed
`at least throughout the array of sample chambers. The device
`cooperates With the PCR instrument to ensure accurate
`positioning of the carrier and the microcard retained thereon
`for real “me PCR processmg'
`
`-
`
`-
`
`38 Claims, 6 Drawing Sheets
`
`THERMO FISHER EX. 1046
`
`

`

`US 6,514,750 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`P. Grossman et al., “High—density multiplex detection of
`nucleic acid sequences: olignucleotide ligation assay and
`sequence—coded separation,” Nucl. Acids Res., 22:4527—34
`(1994).
`Co—pending Application No. 09/496,408.
`Inventors: Hon Shin et a1.
`Title: Apparatus and method for ejecting sample Well trays.
`Attorney Docket No. 7414.0018—00.
`Co—pending Application No. 09/848,270.
`Inventors: Frye et a1.
`Filed: May 4, 2001.
`Title: System and method for ?lling a substrate With a liquid
`sample.
`
`Attorney Docket No. 7414.0011—01.
`Co—pending Application No. 09/977,225.
`Inventors: Freudenthal et a1.
`Filed: Oct. 16, 2001.
`Title: System for ?lling substrate chambers With liquid.
`Attorney Docket No. 7414.0034—00.
`Co—pending Application No. 09/606,006.
`Inventors: BarZi1ai et a1.
`Filed: Jun. 29, 2000.
`Title: Apparatus and method for transporting sample Well
`trays.
`Attorney Docket No. 7414.0009—00.
`
`* cited by examiner
`
`THERMO FISHER EX. 1046
`
`

`

`US. Patent
`
`Feb. 4, 2003
`
`Sheet 1 0f6
`
`US 6,514,750 132
`
`B
`
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`
`THERMO FISHER EX. 1046
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`

`

`U.S. Patent
`
`Feb. 4, 2003
`
`Sheet 2 0f 6
`
`US 6,514,750 B2
`
`FIG. 2
`
`THERMO FISHER EX. 1046
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`

`

`U.S. Patent
`
`Feb. 4, 2003
`
`Sheet 3 0f 6
`
`US 6,514,750 B2
`
`THERMO FISHER EX. 1046
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`

`

`US. Patent
`
`Feb. 4, 2003
`
`Sheet 4 0f 6
`
`US 6,514,750 132
`
`FIG. 5A
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`
`THERMO FISHER EX. 1046
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`
`
`
`
`
`
`
`

`

`U.S. Patent
`
`Feb. 4, 2003
`
`Sheet 5 0f 6
`
`US 6,514,750 B2
`
`FIG 6A
`
`FIG
`
`f 102
`
`THERMO FISHER EX. 1046
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`

`

`U.S. Patent
`
`Feb. 4, 2003
`
`Sheet 6 6f 6
`
`US 6,514,750 B2
`
`134)
`
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`

`

`US 6,514,750 B2
`
`1
`PCR SAMPLE HANDLING DEVICE
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`The present invention relates to apparatus for handling
`microcracks used for performing polymerase chain reactions
`(PCR), for example, and, more particularly, to a device for
`positioning such microcards in relation to a PCR instrument.
`2. Description of the Related Art
`A substrate for simultaneously testing a large number of
`analytes, Which has a small sample siZe and a large number
`of detection chambers, has been described in published PCT
`International Application, WO97/36681, assigned to the
`assignee of the present application, the disclosure of Which
`is incorporated herein by reference. Also, in commonly
`assigned US. patent application Ser. No. 09/549,382, ?led
`Apr. 13, 2000, now US. Pat. No. 6,272,939, the complete
`disclosure of Which is incorporated by reference, a further
`development of a card-like substrate member having a
`plurality of sample detection chambers is disclosed together
`With a system for ?lling the member With a liquid sample to
`react With reagents located in the sample detection chambers
`during thermal cycling of a PCR process. Such card-like
`substrate members are a spatial variant of the microtiter
`plate and are referred to hereinafter as “microcards.”
`HoWever, the microcards are often referred to in the art as
`“consumables” because they are relatively inexpensive and
`disposable after use, and as such, may be made from a
`variety of different materials and may assume different
`shapes and siZes.
`Microcards typically contain 96, 384, or more, individual
`sample chambers, each having a volume of about 1.0 pL or
`less in a card siZe of 7 cm><11 cm><0.2 cm, for example.
`Although both the number of sample chambers and the
`volume siZe of the individual sample chambers may vary
`Widely, the relatively small siZe of the microcards present
`problems in transporting them into and out of a PCR
`instrument, such as instrument models 7700 or 7900
`HTavailable from Applied Biosystems of Foster City, Calif.,
`and aligning the microcard With a thermal cycling block and
`an optical system in the PCR instrument.
`Handling, including placing and removing microcards
`into and from thermal cyclers of a PCR instrument, storing,
`and transporting of the microcards may be accomplished
`either manually or robotically. Arobot typically functions by
`gripping the sides of the microcard by “?ngers”, or grips.
`Because a microcard may have a relatively thin body, With
`side edges as thin as 0.5 mm or less in thickness, robotic
`handling may become impractical or inconsistent, especially
`When multiple microcards are stacked together.
`Additionally, to accomplish real time PCR processing the
`microcard must be aligned With an optical reading device,
`such as a CCD or laser scanner. To be effective, such
`alignment requires high precision usually greater than tol
`erances provided by the edges of the microcard. There is a
`need for reliable alignment of a microcard With a scanner,
`camera, or luminometer of a PCR instrument.
`In addition to the problems associated With alignment,
`PCR processing requires uniform and complete contact of
`the sample chambers of the microcard With a thermal
`cycling block of a PCR instrument. In some instances, Where
`the microcard is formed by laminated plastic materials, there
`is a tendency for Warpage of the card from an initial planar
`con?guration. Thus, to ensure complete contact of the
`sample chambers of the microcard With the surface of the
`
`2
`thermal cycling block, a ?exing of the microcard is required
`so that is conforms to the typically planar surface of that
`block. In other instances, the microcard may be formed of
`?exible material incapable, in itself, to maintain a shape that
`conforms to the surface of the thermal cycling block. In
`positioning the latter types of microcards relative to the
`thermal cycling block of a PCR instrument, therefore, pro
`vision must be made to conform the microcard to the surface
`of the thermal cycling block.
`Thus, it Will be appreciated that there is a need for
`improvements in apparatus for positioning microcards of the
`types mentioned above in relation to a PCR instrument, and
`to facilitate handling of such microcards in general.
`SUMMARY OF THE INVENTION
`The advantages and purpose of the invention Will be set
`forth in part in the description Which folloWs, and in part Will
`be obvious from the description, or may be learned by
`practice of the invention. The advantages and purpose of the
`invention Will be realiZed and attained by means of the
`elements and combinations particularly pointed out in the
`appended claims.
`To attain the advantages and in accordance With the
`purpose of the invention, as embodied and broadly described
`herein, the invention is directed to a device for handling
`PCR microcards, each having an array of sample chambers
`closed by a transparent material on one side thereof, in
`relation to a PCR instrument. The device includes a carrier
`having an apertured region With an array of holes corre
`sponding in number and relative location With the array of
`sample chambers in each of the microcards, and a structure
`for retaining a microcard on the carrier so that the transpar
`ent material faces the apertured region With the sample
`chambers aligned, respectively, With the holes in the aper
`tured region, and so that the side of the microcard opposite
`the transparent material is unobstructed at least throughout
`the array of sample chambers. Also structure is provided for
`positioning the microcard retained on the carrier in relation
`to the PCR instrument.
`In another aspect, the advantages and purpose of the
`invention are attained by such a device having a carrier plate
`including the apertured region, and a peripherally closed
`retention frame having an opening at least as large as the
`array of sample chambers and being ?tted to the carrier to
`retain the microcard in relation to the carrier plate.
`In yet another aspect, the advantages and purpose of the
`invention are attained by such a device for a microcard that
`has through-holes in marginal portions thereof outside the
`array of sample chambers, a plate member including the
`apertured region, and pins projecting from the plate member
`outside of the apertured region to engage in the through
`holes in the marginal areas of the microcard.
`In a further aspect, the advantages and purpose of the
`invention are attained by a PCR kit including at least one
`handling device, a supply of microcards, and optionally, the
`appropriate thermal block for processing the supplied micro
`card. Other kits might include microcards ?lled With
`reagents of a supplier’s design or custom reagents ordered
`by a customer. The appropriate handling device Would be
`included With the ?lled microcards.
`It is to be understood that both the foregoing general
`description and the folloWing detailed description are exem
`plary and explanatory only and are not restrictive of the
`invention, as claimed.
`
`10
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`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`The accompanying draWings, Which are incorporated in
`and constitute a part of this speci?cation, illustrate several
`
`THERMO FISHER EX. 1046
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`

`

`US 6,514,750 B2
`
`3
`exemplary embodiments of the invention and together With
`the description, serve to explain the principles of the inven
`tion. In the drawings,
`FIG. 1A is a top plan vieW of a laminated plastic micro
`card that may be used With the present invention;
`FIG. 1B is an enlarged fragmentary cross section on line
`B—B of FIG. 1A;
`FIG. 2 is an exploded perspective vieW of an embodiment
`of the invention together With a thermal cycling device of a
`PCR instrument;
`FIG. 3 is an enlarged fragmentary perspective vieW of the
`embodiment shoWn in FIG. 2;
`FIG. 4 is an exploded perspective vieW shoWing the
`bottom of the microcard of FIG. 1 in relation to a carrier
`component of the embodiment of FIG. 2;
`FIG. 5A is a perspective vieW a ?exible laminated foil
`microcard that may be used With the present invention;
`FIG. 5B is an enlarged fragmentary cross section taken on
`line B—B of FIG. 5;
`FIG. 6A is an exploded perspective vieW shoWing an
`alternative embodiment of the present invention for use With
`the microcard shoWn in FIG. 5;
`FIG. 6B is a longitudinal cross section taken through the
`carrier plate of FIG. 6A;
`FIG. 7 is a plan vieW of a thermal cycling block used With
`the embodiment of FIG. 6;
`FIG. 8 is a side vieW of the thermal cycling block of FIG.
`7;
`FIG. 9 is a cross section on line 9—9 of FIG. 7;
`FIG. 10 is an enlarged fragmentary plan vieW of the
`thermal cycling block shoWn in FIG. 7; and
`FIG. 11 is a cross section on line 11—11 in FIG. 10.
`
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`Reference Will noW be made in detail to the exemplary
`embodiments of the invention, examples of Which are illus
`trated in the accompanying draWings. Wherever possible,
`the same reference numbers Will be used throughout the
`draWings to refer to the same or like parts.
`In accordance With the present invention, a device is
`provided for handling PCR microcards, each having an array
`of discreet reagent containing sample chambers closed by a
`transparent material on one side thereof, in relation to a PCR
`instrument. Each sample chamber preferably contains an
`analyte-speci?c reagent that reacts With a selected analyte
`that may be present in the liquid sample. The device is
`designed for retaining a micro-card on a carrier so that a
`transparent side of the microcard faces an apertured region
`of the carrier With the reagent sample chambers aligned,
`respectively, With the holes in the apertured region, and so
`that the opposite side of the microcard is unobstructed at
`least throughout the array of reagent containing sample
`chambers. As disclosed herein and shoWn in FIGS. 1A and
`1B, one embodiment of the apparatus is particularly appli
`cable to a microcard generally designated by the reference
`number 10.
`Although the microcard 10 and a system for ?lling it With
`sample liquid is fully disclosed in the above cited US. patent
`application Ser. No. 09/549,382, ?led Apr. 13, 2000, now
`US. Pat. No. 6,272,939, incorporated herein by reference,
`the features of the microcard 10 that are applicable to the
`apparatus of the present invention Will be described beloW.
`The microcard 10 is formed by a laminated substrate
`shoWn in FIG. 1A as being generally rectangular in shape,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`65
`
`4
`but can be a variety of shapes and siZes, and in the illustrated
`embodiment, by Way of example only, is approximately 7
`cm><11 cm><0.2 cm. A chamfered corner 11 is provided to
`ensure proper orientation of the microcard With a PCR
`instrument. The microcard 10 de?nes a netWork 12 of
`passageWays including a plurality of sample detection cham
`bers 14. Each sample detection chamber can hold a pre
`de?ned volume of liquid sample, such as, for example,
`approximately 1 pl. This volume can be varied depending on
`the speci?c application.
`As embodied herein and shoWn in FIG. 1B, the microcard
`10 is preferably formed as including a top plate 16 and a
`bottom plate 18. The top plate 16 has an upper surface 20
`that contains raised surfaces 22. The raised surfaces 22
`de?ne the top portion of each sample detection chamber 14,
`and are tapered doWnWardly and outWardly in relation to a
`central axis 23 of each sample detection chamber 14.
`Preferably, the raised surfaces are those of truncated spheres,
`but other tapered surfaces, such as those of a cone or
`pyramid could be used.
`The top and bottom plates 16 and 18 can be joined to each
`other by a variety of methods so that the netWork of
`passageWays may be evacuated by a vacuum source, so that
`the liquid sample does not leak from the substrate, and to
`Withstand temperature ?uctuations that can occur during
`thermal cycling. Preferably, the plates 16 and 18 are joined
`using ultrasonic Welding, but other suitable methods include
`the use of adhesives, pressure-sealing, or heat curing.
`As embodied herein and shoWn in FIGS. 1A and 1B, the
`microcard 10 is provided With a sample inlet port 24 for the
`entrance of the liquid sample into the netWork 12 of pas
`sageWays. The sample inlet port 24 is located preferably in
`the center of an attachment/bladder groove 26, in the top
`plate 16 of the microcard 10, and extends through the
`attachment/bladder groove 26. The attachment/bladder
`groove 26 extends across a portion of the Width of the top
`surface of the substrate plate 16 in a region outside of the
`sample detection chambers 14 and has a top surface slightly
`recessed from the upper surface 20 of the top plate 16.
`As described fully in the above-cited US. application Ser.
`No. 09/549,382, now US. Pat. No. 6,272,939, the
`attachment/bladder groove 26 provides an air pocket for the
`liquid sample in the netWork of passageWays so that When
`the ?lled substrate undergoes temperature ?uctuations dur
`ing thermal cycling operations expansion of the liquid
`sample in the netWork 12 of passageWays occurs Without
`signi?cantly increasing the pressure on the substrate. Also,
`the liquid sample may ?oW into the attachment/bladder
`groove 26 through sample port 24 under such conditions.
`The top and bottom plates 16 and 18 may be made out of
`any suitable material that can be manufactured according to
`the required speci?cations, can Withstand any temperature
`?uctuations that may later occur, i.e., during thermal cycling
`or other operations performed on the substrate, and can be
`suitably joined. In addition, for real time optical detection of
`liquid samples during thermal cycling, the top of each
`sample detection chamber 14 must be optically transparent
`for detection of the reaction. For this purpose, silica-based
`glasses, quartZ, polycarbonate, or any optically transparent
`plastic layer, for example, may be used. For use in PCR
`reactions, the material should be PCR compatible, and the
`material should be preferably be substantially ?uorescence
`free. In one embodiment, the material for the top plate is a
`polycarbonate manufactured by “BAYER” TM, referred to as
`FCR 2458-1112 and the material for the bottom plate is a
`0.015 inch thickness polycarbonate manufactured by
`
`THERMO FISHER EX. 1046
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`

`US 6,514,750 B2
`
`5
`“BAYER” TM, referred to as Makrofol DE1-1D. The sub
`strate plates can be formed by a variety of methods known
`in the art. For example, top plate 16 may be injection
`molded, Whereas bottom plate 18 may be die-cut. Any other
`suitable method of manufacturing the plates is also accept
`able.
`Prior to assembly of the top and bottom plates 16 and 18,
`an analyte-speci?c reagent is typically placed in each detec
`tion chamber 14. One or more of the detection chambers
`may be left empty to function as a control. These analyte
`speci?c reagents in the detection chambers may be adapted
`to detect a Wide variety of analyte classes in the liquid
`sample, including polynucleotides, polypeptides,
`polysaccharides, and small molecule analytes, by Way of
`example only. The polynucleotide analytes are detected by
`any suitable method, such as polymerase chain reaction,
`ligase chain reaction, oligonucleotide ligation assay, or
`hybridiZation assay. A preferred method of polynucleotide
`detection is the exonuclease assay referred to as “TAQ
`MAN”TM. Nonpolynucleotide analytes may also be detected
`by any suitable method, such as antibody/antigen binding.
`The above detection methods are Well-knoWn in the art.
`They are described in detail in the folloWing articles and
`patents: US. Pat. No. 5,210,015 of Gelfand et al.; US. Pat.
`No. 5,538,848 of Livak et al.; WO 91/17239 of Barany et al.
`published on Nov. 14, 1991; “A Ligase-Mediated Gene
`Detection Technique” by Landegren et al published in
`Science 241:1077—90 (1988); “High-density multiplex
`detection of nucleic acid sequences: oligonucleotide ligation
`assay and sequence-coded separation” by Grossman et al.,
`published in Nucleic Acid Research 22:4527—34 (1994); and
`“Automated DNA diagnostics using an ELISA-based oligo
`nucleotide ligation assay” by Nickerson et al., published in
`Proc. Natl. Acad. Sci. USA 87:8923—27 (1990).
`In FIG. 2, an embodiment of a handling device for the
`microcard 10 is designated generally by the reference num
`ber 30 and shoWn relative to a thermal cycling device 32 of
`a PCR instrument, such as models 7700 or 7900 HTavailable
`from Applied Biosystems of Foster City, Calif. Such instru
`ments are capable of automated PCR processing and include
`an optical system positioned above the thermal cycling
`device 32 for reading sample ?uorescence in real time While
`the samples are subjected to thermal cycling. The thermal
`cycling device 32 includes a ?at top 34, a depending heat
`sink 36 and a replaceable thermal block 38. Although shoWn
`only partially in FIGS. 2 and 3, the thermal block 38 takes
`the form of a generally rectangular plate having a ?at top and
`a uniform thickness such that the ?at top of the thermal
`block 38 is elevated above the level of the ?at top 34 of the
`thermal cycling device 32. As shoWn most clearly in FIG. 3,
`the thermal block 38 has laterally projecting, bifurcated lugs
`39 on each side thereof for securing it against thermal
`heating/cooling panels (not shoWn), and to the top 34 of the
`thermal cycling device 32 by bolts 40.
`A heated cover plate 42, represented schematically by
`phantom lines in FIG. 2, is supported in the PCR instrument
`for vertical movement toWard and aWay from the thermal
`block 38 and in angular registry thereWith. The function of
`the cover plate is to press the microcard against the thermal
`block 38, While at the same time enabling operation of an
`optical scanning system (not shoWn) to read the samples in
`the respective sample chambers 14 of the microcard.
`In accordance With the present invention, the handling
`device 30 includes a carrier having an apertured region With
`an array of holes corresponding in number and relative
`location With the array of reagent containing sample cham
`bers in each of the micro-cards, means for retaining a
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`micro-card on the carrier so that the transparent material of
`the microcard faces the apertured region With the reagent
`sample chambers aligned, respectively, With the holes in the
`apertured region, and so that the side of the micro-card
`opposite the transparent material is unobstructed at least
`throughout the array of reagent containing sample chambers.
`The handling device 30 additionally includes means for
`positioning the carrier and the micro-card retained thereon in
`relation to the PCR instrument.
`In the illustrated embodiment, the handling device 30
`de?nes a tWo-part carrier for the microcard 10, the tWo parts
`being a peripherally closed frame-like retention frame 44
`and a carrier 46 having an array of holes 48 in a central
`apertured region, the holes corresponding in number and in
`location With the sample chambers 14 in the microcard 10.
`As may be seen in FIGS. 2 and 3, the retention frame 44
`includes a continuous peripheral Wall 49 extending
`upWardly from a ?ared bottom 50 that seats against the ?at
`top 34 of the thermal cycling device 32. A marginal ?ange
`52 of the retention frame 44 extends inWardly from the
`peripheral Wall 49 but elevated slightly above the ?ared
`bottom 50 that seats against the top 34. The marginal ?ange
`52 de?nes a central opening 54 that is shaped to complement
`the peripheral shape of the thermal block 38 a slight periph
`eral clearance betWeen the inner edges of the marginal ?ange
`52 and the outer edges of the thermal block 38. Also, as
`shoWn in FIG. 3, the thickness of the marginal ?ange 52 is
`less than that of the thermal block 38, so that When the ?ared
`bottom of the retention frame 44 is seated on the top 34 of
`the thermal cycling device 32, the top surface of the mar
`ginal ?ange 52 is loWer than the top surface of the thermal
`block 38 even though the marginal ?ange is slightly elevated
`above the seating ?ared bottom 50.
`To retain the microcard 10 by the retention frame 44, both
`ends of the microcard 10 overlie a pair of tabs 56 that project
`from opposite inner edges of the marginal ?ange 52 of the
`retention frame 44. Except for those retained end portions
`that overlie the tabs 56, the entire bottom surface of the
`microcard 10 is exposed through the opening 54 de?ned by
`the inner edges of the marginal ?ange 52.
`The carrier 46 is de?ned in substantial measure by a ?at
`plate 58, in Which the array of holes 48 are formed. A
`peripheral Wall 60, of a depth to project both above and
`beloW the plate 58, extends about three sides of the plate 58,
`as shoWn in FIG. 2. On the fourth side, the Wall 60 is
`continued as a skirt 62 depending from the plate 58. A
`recessed portion 64 on the fourth side of the plate 58,
`together With a complementing recessed portion 66 in the
`Wall 49 of the retention frame 44, provides a WindoW for
`observation of identifying indicia on the microcard 10 When
`the carrier 46 and the retention frame 44 are closed about the
`microcard.
`The peripheral edge surfaces of the carrier 46 are shaped
`and siZed to ?t someWhat loosely into the peripheral Wall 49
`of the retention frame 44. When the carrier 46 and retention
`frame 44 are assembled about a microcard 10 in a manner to
`be described beloW, a pair of clips 68 on each of opposite
`sides of the carrier 46 engage in apertures 70 on opposite
`sides of the retention frame 44 to secure the assembly. The
`clips 68 may be released from the apertures 70 by distorting
`the retention frame of by inserting a tool, such as a small
`screW driver, through the apertures and ?exing the clips to
`permit removal of the microcard 10 from the device 30.
`In FIG. 4, the bottom of the carrier 46 is shoWn to include
`pairs of Wedge-shaped projections 72 on the bottom mar
`ginal regions of the carrier plate 58, outside of the region
`
`THERMO FISHER EX. 1046
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`

`

`US 6,514,750 B2
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`7
`containing the array of holes 48. One such pair of projec
`tions 72 is provided on each side of the carrier 46. Also, a
`single Wedge-shaped projection 72 is located in the corner of
`the carrier 46 that receives the chamfered corner 11 of the
`microcard 10. The Wedge-shaped projections 72 function as
`positioning ramps such that When the carrier 46 is inverted,
`as shoWn in FIG. 4, the microcard 10, also inverted, may be
`placed into the inverted carrier and guided against the
`bottom of the carrier plate 58 so that the raised tapered
`surfaces 22 on the microcard are coarsely aligned With the
`respective holes 48. The retention frame 44 is then inverted
`and pressed against the carrier 46 until the clips 68 on the
`carrier 46 engage in the apertures 70 in the retention frame
`44. The microcard 10 is then secured Within the handling
`device 30, but With freedom of movement Within the device
`30 limited by the carrier plate 58 on the top, by the marginal
`?ange 52 in the retention frame 44 on the bottom, and by the
`positioning ramps on the Wedge-shaped projections 72 on
`the peripheral edges of the microcard
`As shoWn in FIG. 2, the top of the carrier 46 is also
`provided With pairs of Wedge-shaped ramp members 74, one
`such pair on each side of the plate 58. These ramp members
`cooperate With the heated cover plate 42 of the PCR instru
`ment so that When the cover plate 42 is loWered against the
`assembled handling device 30 positioned on the thermal
`block 38, precise ?nal positioning of the handling device and
`of the microcard Will be obtained by cooperation of the
`carrier 46 With the heated cover plate 42, and by cooperation
`of the holes 48 in the carrier 46 With the raised tapered
`surfaces 22 on the microcard 10. In particular, the ?nal
`position of the carrier Will be determined by the camming
`action of the heated cover plate 42 on the ramp members 74
`on the top of the carrier 46, and the ?nal position of the
`microcard 10 Will be determined by the camming action of
`the holes 48 on the raised tapered surfaces 22 of the
`microcard 10.
`As mentioned above With reference to FIG. 3, the thick
`ness of the marginal ?ange 52 is less than that of the thermal
`block 38, so that When the retention frame 44 is seated on the
`top 34 of the thermal cycling device 32, the top surface of
`the marginal ?ange is loWer than the top surface of the
`thermal block 38. This difference in elevation betWeen the
`top of the marginal ?ange 52 and the top surface of the
`thermal block 38 represents the amount of vertical freedom
`of movement that the microcard has in the handling device
`30 When the carrier 46 and retention frame are initially
`closed on each other, and permits the relative vertical
`movement of the carrier 46 and microcard 10 needed to
`effect the cam action ?nal positioning of the microcard.
`Also, movement of the marginal ?ange 52 aWay from the
`bottom of the microcard 10 ensures that only the thermal
`block is in contact With the bottom of the card and that there
`Will be no interference With heat transfer betWeen the
`thermal block 38 and the microcard 10.
`The carrier 46 and retention frame 44 are preferably
`constructed of a polymer that is able to Withstand the heat
`used in a typical thermal cycling process, e.g., about 60° to
`100° C. Thus, the handling device 30 should be able to
`maintain its original shape even after multiple thermal
`cycling processes. The device 30, described herein by Way
`of eXample, is intended to be reusable and able to substan
`tially maintain its shape after 50 or more hours of thermal
`cycling. Ashelf life of about 5 years Would also be expected.
`Materials that may be used for construction of the device 30
`include polymers, plastics, glass, ceramics, metals, or others
`knoWn in the art that are able to Withstand the thermal
`cycling process. Furthermore, the handling device 30 of this
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`invention may be manufactured in a variety of Ways knoWn
`in the art, including injection molding, machining, or metal
`stamping methods.
`In FIGS. 5A and SE, a microcard, representing a variant
`of the microcard 10 of FIGS. 1A and 1B, is designated
`generally by the reference number 80. As shoWn, the micro
`card 80 contains three hundred and eighty-four (384) sample
`chambers 82 connected With a ?ll port 84 via a netWork 86
`of passageWays, but may contain feWer chambers, such as
`ninety-six (96) chambers, for eXample. Also, the illustrat