I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111
`US008835118B2
`
`c12) United States Patent
`Kordunsky et al.
`
`(IO) Patent No.:
`(45) Date of Patent:
`
`US 8,835,118 B2
`*Sep.16,2014
`
`(54) SYSTEMS AND METHODS FOR
`FLUORESCENCE DETECTION WITH A
`MOVABLE DETECTION MODULE
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`(75)
`
`Inventors: Igor Kordunsky, Newton, MA (US);
`Jeffrey A. Goldman, Acton, MA (US);
`Michael J. Finney, San Francisco, CA
`(US)
`
`(73) Assignee: Bio-Rad Laboratories, Inc., Hercules,
`CA (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`CN
`DE
`
`4,626,684 A
`5,091,652 A
`5,184,020 A
`5,210,015 A
`5,315,375 A
`
`12/1986 Landa
`2/ 1992 Mathies et al.
`2/ 1993 Hearst et al.
`5/ 1993 Gelfand et al.
`5/1994 Allen
`(Continued)
`
`FOREIGN PATENT DOCUMENTS
`
`11/2002
`1379236
`5/1986
`3441179
`(Continued)
`
`OTHER PUBLICATIONS
`
`This patent is subject to a terminal dis(cid:173)
`claimer.
`
`(21) Appl. No.: 13/542,587
`
`(22) Filed:
`
`Jul. 5, 2012
`
`(65)
`
`Prior Publication Data
`
`US 2013/0143308 Al
`
`Jun. 6, 2013
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 12/827,521, filed on
`Jun. 30, 2010, now Pat. No. 8,236,504, which is a
`continuation of application No. 11/555,642, filed on
`Nov. 1, 2006, now Pat. No. 7,749,736, which is a
`continuation of application No. 10/431,708, filed on
`May 8, 2003, now Pat. No. 7,148,043.
`
`(51)
`
`(2006.01)
`(2006.01)
`
`Int. Cl.
`C12Q 1/68
`C12P 19/34
`(52) U.S. Cl.
`USPC ........................... 435/6.12; 435/6.1; 435/6.11
`( 58) Field of Classification Search
`None
`See application file for complete search history.
`
`PCT International Preliminary Report on Patentability for PCT/
`US04/14566, (2005).
`
`(Continued)
`
`Primary Examiner - Young J Kim
`(74) Attorney, Agent, or Firm - Kilpatrick Townsend &
`Stockton LLP
`
`ABSTRACT
`(57)
`A fluorescence detection apparatus for analyzing samples
`located in a plurality of wells in a thermal cycler and methods
`of use are provided. In one embodiment, the apparatus
`includes a support structure attachable to the thermal cycler
`and a detection module movably mountable on the support
`structure. The detection module includes one or more chan(cid:173)
`nels, each having an excitation light generator and an emis(cid:173)
`sion light detector both disposed within the detection module.
`When the support structure is attached to the thermal cycler
`and the detection module is mounted on the support structure,
`the detection module is movable so as to be positioned in
`optical communication with different ones of the plurality of
`wells. The detection module is removable from the support
`structure to allow easy replacement.
`
`16 Claims, 7 Drawing Sheets
`
`Agilent Exhibit 1203
`Page 1 of 17
`
`

`

`US 8,835,118 B2
`Page 2
`
`(56)
`
`References Cited
`
`FOREIGN PATENT DOCUMENTS
`
`U.S. PATENT DOCUMENTS
`
`5/1995 Zaun et al.
`5,415,839 A
`5,473,437 A
`12/1995 Blumenfeld et al.
`5,578,818 A
`11/1996 Kain et al.
`5,585,242 A
`12/1996 Bouma et al.
`5,595,708 A
`1/1997 Berndt
`5,736,333 A
`4/1998 Livak et al.
`5,784,152 A
`7/1998 Heffelfinger
`5,928,907 A
`7/1999 Woudenberg et al.
`5,972,716 A
`10/1999 Ragusa et al.
`5,994,056 A
`11/1999 Higuchi
`1/2000 Woudenberg et al.
`6,015,674 A
`6,024,920 A
`2/2000 Cunanan
`6,043,880 A
`3/2000 Andrews et al.
`10/2000 Wittwer et al.
`6,140,054 A
`11/2000 Mitoma
`6,144,448 A
`6,174,670 Bl
`1/2001 Wittwer
`3/2001 Griffith et al.
`6,197,575 Bl
`6,211,989 Bl
`4/2001 Wulf et al.
`1/2002 Chu et al.
`6,337,435 Bl
`3/2002 Hayashi et al.
`6,359,284 Bl
`4/2002 Christel et al.
`6,369,893 Bl
`6/2002 Maher et al.
`6,399,952 Bl
`6,569,631 Bl
`5/2003 Pantoliano et al.
`11/2004 Gambini et al.
`6,818,437 Bl
`7,148,043 B2 * 12/2006 Kordunsky et al. .......... 435/91.2
`7,749,736 B2 * 7/2010 Kordunsky et al. .......... 435/91.2
`8,236,504 B2 * 8/2012 Kordunsky et al. .......... 435/6.12
`11/2001 French et al.
`2001/0046673 Al
`2002/0024026 Al
`2/2002 Kaushikkar
`2002/0060791 Al
`5/2002 Stumbo et al.
`2002/0064780 Al
`5/2002 Gold et al.
`2003/0015668 Al
`1/2003 Montagu
`2004/0014202 Al
`1/2004 King eta!.
`
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`WO
`WO
`WO
`WO
`WO
`WO
`
`H07-506433
`H09-508536
`11-271227
`2000-121559
`2000-511629
`2000-321206
`2001-108684
`2001-509272
`2001-255272
`WO 95/30139
`WO 97/46707
`WO 98/53301
`WO 99/12008
`WO 00/31518
`WO 01/13096
`
`7 /1995
`9/1997
`10/1999
`4/2000
`9/2000
`11/2000
`4/2001
`7/2001
`9/2001
`11/1995
`12/1997
`11/1998
`3/1999
`6/2000
`2/2001
`
`OTHER PUBLICATIONS
`
`Wittwer et al., The LightCycler.TM.: A Microvolume Multisample
`Fluorometer with Rapid Temperature Control, BioTechniques (Jan.
`1997) vol. 22, No. 1, pp. 176-181.
`European Patent Office Notice of Opposition dated Nov. 21, 2007 for
`EP Patent Application No. 04751790.9, 14 pages.
`European Patent Office Preliminary Opinion dated May 19, 2011, 2
`pages.
`Patentee side written arguments with auxiliary requests dated Sep.
`19, 2011, 89 pages.
`Opponent's written argument dated Sep. 19, 2011, 4 pages.
`Opponent's letter in reply dated Oct. 13, 2011, 7 pages.
`
`* cited by examiner
`
`Agilent Exhibit 1203
`Page 2 of 17
`
`

`

`U.S. Patent
`
`Sep.16,2014
`
`Sheet 1 of 7
`
`US 8,835,118 B2
`
`122
`
`112
`
`121
`
`FIG. I
`
`Agilent Exhibit 1203
`Page 3 of 17
`
`

`

`Sep.16,2014
`
`Sheet 2 of '7
`
`U.S. \latent
`
`US S,835,118 B2
`
`0 0 0 0 0 0 0 0 0 0 0 0
`0 0 0 0 0 0 0 0 0 0 0 0
`0 0 0 0 0 0 0 0 0 0 0 0
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`0 0 0 0 ooooooo j
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`2.10
`
`FIG. 2
`
`Agilent Exhibit 1203
`Page 4 of 17
`
`

`

`U.S. Patent
`
`Sep.16,2014
`
`Sheet 3 of 7
`
`US 8,835,118 B2
`
`0
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`Agilent Exhibit 1203
`Page 5 of 17
`
`

`

`U.S. Patent
`
`Sep.16,2014
`
`Sheet 4 of 7
`
`US 8,835,118 B2
`
`420
`
`420
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`
`Agilent Exhibit 1203
`Page 6 of 17
`
`

`

`U.S. Patent
`
`Sep. 16,2014
`
`Sheet 5 of 7
`
`US 8,835,118 B2
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`
`Agilent Exhibit 1203
`Page 7 of 17
`
`

`

`U.S. Patent
`
`Sep.16,2014
`
`Sheet 6 of 7
`
`US 8,835,118 B2
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`Agilent Exhibit 1203
`Page 8 of 17
`
`

`

`U.S. Patent
`
`Sep.16,2014
`
`Sheet 7 of 7
`
`US 8,835,118 B2
`
`;-800
`802
`
`r
`
`PREPARE REACTION VESSELS
`WITH SAMPLES TO BE ANALYZED
`
`+
`
`MOUNT DETECTION MODULE
`ON SHUTTLE
`
`t
`
`PLACE REACTION VESSELS IN
`SAMPLE WELLS
`
`+
`
`CLOSE LID AND PLACE UNIT
`IN THERMAL CYCLER BASE
`
`+
`
`CALIBRATE DETECTION MODULE
`
`+
`
`PERFORM PCR CYCLE
`
`806
`,.C..
`
`r
`
`808
`
`r
`
`810
`
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`
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`
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`
`__________ j __________ cs16_
`
`SCAN AND INTERROGATE REACTION VESSELS
`816a
`~
`
`POSITION DETECTION MODULE IN
`OPTICAL COMMUNICATION WITH WELLS
`
`t
`
`ACTIVATE LED
`
`t
`
`~ 816b
`
`v- 816c
`
`DETECT FLUORESCENT RESPONSE
`
`L----------------------------
`F/G. 8
`
`I
`I
`I
`
`Agilent Exhibit 1203
`Page 9 of 17
`
`

`

`US 8,835,118 B2
`
`1
`SYSTEMS AND METHODS FOR
`FLUORESCENCE DETECTION WITH A
`MOVABLE DETECTION MODULE
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`This application is a continuation of application Ser. No.
`12/827,521, filed Jun. 30, 2010, entitled "Systems and Meth(cid:173)
`ods For Fluorescence Detection With A Movable Detection
`Module," which is a continuation of application Ser. No.
`11/555,642, filed Nov. 1, 2006, entitled "Systems and Meth(cid:173)
`ods For Fluorescence Detection With A Movable Detection
`Module," which is a continuation of application Ser. No.
`10/431,708, filed May 8, 2003, entitled "Systems and Meth(cid:173)
`ods for Fluorescence Detection with a Movable Detection
`Module," now U.S. Pat. No. 7,148,043. The respective dis(cid:173)
`closures of all applications are incorporated herein by refer(cid:173)
`ence.
`
`BACKGROUND OF THE INVENTION
`
`The present invention relates in general to fluorescence
`detection systems and in particular to a fluorescence detection
`system having a movable excitation/detection module for use
`with a thermal cycler.
`Thermal cyders are known in the art. Such devices are used
`in a variety of processes for creation and detection of various
`molecules of interest, e.g., nucleic acid sequences, in
`research, medical, and industrial fields. Processes that can be
`performed with conventional thermal cyders include but are
`not limited to amplification of nucleic acids using procedures
`such as the polymerase chain reaction (PCR). Such amplifi(cid:173)
`cation processes are used to increase the amount of a target
`sequence present in a nucleic acid sample.
`Numerous techniques for detecting the presence and/or
`concentration of a target molecule in a sample processed by a
`thermal cycler are also known. For instance, fluorescent
`labeling may be used. A fluorescent label ( or fluorescent
`probe) is generally a substance which, when stimulated by an
`appropriate electromagnetic signal or radiation, absorbs the
`radiation and emits a signal (usually radiation that is distin(cid:173)
`guishable, e.g., by wavelength, from the stimulating radia(cid:173)
`tion) that persists while the stimulating radiation is continued,
`i.e. it fluoresces. Some types of fluorescent probes are gener(cid:173)
`ally designed to be active only in the presence of a target
`molecule ( e.g., a specific nucleic acid sequence), so that a
`fluorescent response from a sample signifies the presence of
`the target molecule. Other types of fluorescent probes
`increase their fluorescence in proportion to the quantity of
`double-stranded DNA present in the reaction. These types of
`probes are typically used where the amplification reaction is
`designed to operate only on the target molecule.
`Fluorometry involves exposing a sample containing the
`fluorescent label or probe to stimulating (also called excita(cid:173)
`tion) radiation, such as a light source of appropriate wave(cid:173)
`length, thereby exciting the probe and causing fluorescence.
`The emitted radiation is detected using an appropriate detec(cid:173)
`tor, such as a photodiode, photomultiplier, charge-coupled 60
`device (CCD), or the like.
`Fluorometers for use with fluorescent-labeled samples are
`known in the art. One type of fluorometer is an optical reader,
`such as described by Andrews et al. in U.S. Pat. No. 6,043,
`880. A sample plate containing an array of samples is inserted 65
`in the optical reader, which exposes the samples to excitation
`light and detects the emitted radiation. The usefulness of
`
`2
`optical readers is limited by the need to remove the sample
`plate from the thermal cycler, making it difficult to monitor
`the progress of amplification.
`One improvement integrates the optical reader with a ther-
`5 ma! cycler, so that the sample plate may be analyzed without
`removing it from the thermal cycler or interrupting the PCR
`process. Examples of such combination devices are described
`in U.S. Pat. No. 5,928,907, U.S. Pat. No. 6,015,674, U.S. Pat.
`No. 6,043,880, U.S. Pat. No. 6,144,448, U.S. Pat. No. 6,337,
`10 435, and U.S. Pat. No. 6,369,863. Such combination devices
`are useful in various applications, as described, e.g., in U.S.
`Pat. No. 5,210,015, U.S. Pat. No. 5,994,056, U.S. Pat. No.
`6,140,054, and U.S. Pat. No. 6,174,670.
`Existing fluorometers suffer from various drawbacks. For
`15 instance, in some existing designs, different light sources and
`detectors are provided for different sample wells in the array.
`Variations among the light sources and/or detectors lead to
`variations in the detected fluorescent response from one well
`to the next. Alternatively, the light source and/or detector may
`20 be arranged in optical communication with more than one of
`the wells, with different optical paths to and/or from each
`well. Due to the different optical paths, the detected fluores(cid:173)
`cent response varies from one sample well to the next. To
`compensate for such variations, the response for each sample
`25 well must be individually calibrated. As the number of sample
`wells in an array increases, this becomes an increasingly
`time-consuming task, and errors in calibration may introduce
`significant errors in subsequent measurements.
`In addition, existing fluorometers generally are designed
`30 such that the light sources and detectors are fixed parts of the
`instrument. This limits an experimenter's ability to adapt a
`fluorometer to a different application. For instance, detecting
`a different fluorescent label generally requires using a differ(cid:173)
`ent light source and/or detector. Many existing fluorometers
`35 make it difficult for an experimenter to reconfigure light
`sources or detectors, thus limiting the variety of fluorescent
`labels that may be used.
`It is also difficult to perform concurrent measurements of a
`number of different fluorescent labels that may be present in
`40 a sample (or in different samples). As described above, to
`maximize the data obtained in an assay, experimenters often
`include multiple fluorescent labeling agents that have differ(cid:173)
`ent excitation and/or emission wavelengths. Each labeling
`agent is adapted to bind to a different target sequence, in
`45 principle allowing multiple target sequences to be detected in
`the same sample. Existing fluorometers, however, do not
`facilitate such multiple-label experiments. Many fluorom(cid:173)
`eters are designed for a single combination of excitation and
`emission wavelengths. Others provide multiple light sources
`50 and detectors to allow detection of multiple labels; however,
`these configurations often allow only one label to be probed at
`a time because the excitation wavelength of one label may
`overlap the emission wavelength of another label; excitation
`light entering the detector would lead to incorrect results.
`55 Probing multiple labels generally cannot be done in parallel,
`slowing the data collection process.
`Therefore, an improved fluorometer for a thermal cycler
`that overcomes these disadvantages would be desirable.
`
`BRIEF SUMMARY OF THE INVENTION
`
`Embodiments of the present invention provide fluores(cid:173)
`cence detection in a thermal cycling apparatus. According to
`one aspect of the invention, a fluorescence detection appara(cid:173)
`tus for analyzing samples located in a plurality of wells in a
`thermal cycler includes a support structure attachable to the
`thermal cycler and a detection module movably mountable on
`
`Agilent Exhibit 1203
`Page 10 of 17
`
`

`

`US 8,835,118 B2
`
`4
`FIG. 7 is a block diagram illustrating electrical connections
`for a lid assembly for a thermal cycling apparatus according
`to an embodiment of the present invention; and
`FIG. 8 is a flow diagram of a process for using a thermal
`5 cycler having a fluorescence detection system according to an
`embodiment of the present invention.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`3
`the support structure. The detection module includes an exci(cid:173)
`tation light generator and an emission light detector, both
`disposed within the detection module. When the support
`structure is attached to the thermal cycler and the detection
`module is mounted on the support structure, the detection
`module is movable so as to be positioned in optical commu(cid:173)
`nication with different ones of the plurality of wells.
`According to another aspect of the invention, the detection
`module may include two or more excitation light generators
`and two or more emission light detectors arranged to form 10
`two or more excitation/detection pairs. In one embodiment,
`the excitation/detection pairs are arranged such that each
`excitation/detection pair is simultaneously positionable in
`optical contact with a different one of the plurality of wells. In 15
`an alternative embodiment, excitation/detection pairs are
`arranged such that when a first one of the excitation/detection
`pairs is positioned in optical contact with any one of the
`plurality of wells, a different one of the excitation/detection
`pairs is not in optical contact with any one of the plurality of 20
`wells. In some embodiments, the detection module is detach(cid:173)
`ably mounted on the support structure, thereby enabling a
`user to replace the detection module with a different detection
`module.
`According to yet another aspect of the invention, a method
`for detecting the presence of a target molecule in a solution is
`provided. A plurality of samples is prepared, each sample
`containing a fluorescent probe adapted to bind to a target
`molecule. Each sample is placed in a respective one of a
`number of sample wells of a thermal cycler instrument, the
`thermal cycler instrument having a detection module mov(cid:173)
`ably mounted therein, the detection module including an
`excitation/detection charmel, the excitation/detection chan(cid:173)
`nel including an excitation light generator disposed within the
`detection module and an emission light detector disposed
`within the detection module. The thermal cycler instrument is
`used to stimulate a reaction, and the sample wells are scanned
`to detect a fluorescent response by moving the detection
`module and activating the excitation/detection charmel. Dur(cid:173)
`ing the scanning, the detection module is moved such that the 40
`excitation/detection channel is sequentially positioned in
`optical communication with each of the plurality of sample
`wells. Where the detection module includes multiple excita(cid:173)
`tion/detection pairs or charmels, charmels may be active in
`parallel or sequentially.
`The following detailed description together with the
`accompanying drawings will provide a better understanding
`of the nature and advantages of the present invention.
`
`An exemplary apparatus embodiment of the present inven(cid:173)
`tion will be described with reference to the accompanying
`drawings, in which like reference numerals indicate corre(cid:173)
`sponding parts. Methods of using the apparatus will also be
`described. It is to be understood that embodiments shown and
`described herein are illustrative and not limiting of the inven-
`tion.
`I. Exemplary Apparatus
`FIG. 1 is a perspective view of a thermal cycling apparatus
`100 according to an embodiment of the present invention.
`Apparatus 100 consists of a base unit 110 and a lid assembly
`112. Base unit 110, which may be of conventional design,
`provides power and control functions for a thermal cycling
`process via conventional electronic components (not shown),
`25 such as programmable processors, clocks, and the like. Base
`unit 110 also provides a user interface 116 that may include a
`keypad 118 and an LCD display screen 120, enabling a user to
`control and monitor operation of the thermal cycler. Base unit
`110 connects to an external power source (e.g., standard 120
`30 V ac power) via a power cable 121. Some examples of base
`unit 110 include the DNA Engine®, Dyad™, and Tetrad™
`thermal cyders sold by MJ Research, Inc., assignee of the
`present application.
`Lid assembly 112 includes a sample unit and a fluores-
`35 cence detection apparatus, disposed within a lid 122; these
`components will be described below. Lid 122 has a handle
`124 to aid in its placement on and removal from base unit 110,
`and ventilation holes 126. Lid 122 provides optical and ther-
`mal isolation for the components inside lid assembly 112.
`FIG. 2 is an exploded view of the inside of lid assembly
`112. Shown are a sample unit 202, a lid heater 204, and a
`fluorometer assembly 206. Sample unit 202 contains a num(cid:173)
`ber of sample wells 210 arranged in a regular array (e.g., an
`8x12 grid). In one embodiment, each sample well 210 holds
`45 a removable reaction vessel (not shown), such as a tube, that
`contains a nucleic acid sample to be tested, together with
`appropriate PCR reactants (buffers, primers and probes,
`nucleotides, and the like) including at least one fluorescent
`label or probe adapted to bind to or otherwise respond to the
`50 presence of a target nucleic acid sequence. The reaction ves(cid:173)
`sels are advantageously provided with transparent sample
`caps (not shown) that fit securely over the tops of the vessels
`to prevent cross-contamination of samples or spillage during
`handling. Reaction vessels may also be sealed in other ways,
`55 including the use of films such as Microseal®B (made by MJ
`Research, Inc.), wax products such as Chill-out™ (made by
`MJ Research, Inc.), or mineral oil. In an alternative configu(cid:173)
`ration, a removable sample tray (not shown) that holds one or
`more distinct samples at locations corresponding to sample
`60 wells 210 is used. The sample tray may also be sealed in any
`of the ways described above.
`Sample unit 202 also includes heating elements (e.g.,
`Peltier-effect thermoelectric devices), heat exchange ele(cid:173)
`ments, electrical connection elements for connecting the
`65 heating elements to base unit 110, and mechanical connection
`elements. These components (not shown) may be of conven(cid:173)
`tional design. Sample unit 202 also provides electrical con-
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a perspective view of a thermal cycling apparatus
`according to an embodiment of the present invention;
`FIG. 2 is an exploded view of a lid assembly for a thermal
`cycling apparatus according to an embodiment of the present
`invention;
`FIG. 3 is a bottom view of a fluorometer assembly for a
`thermal cycling apparatus according to an embodiment of the
`present invention;
`FIG. 4 is a top view of detection module according to an
`embodiment of the present invention;
`FIGS. SA-B are bottom views of detection modules
`according to alternative embodiments of the present inven(cid:173)
`tion;
`FIG. 6 is a schematic diagram of an excitation/detection
`pair for a detection module according to an embodiment of
`the present invention;
`
`Agilent Exhibit 1203
`Page 11 of 17
`
`

`

`US 8,835,118 B2
`
`5
`6
`nections for lid heater 204 and fluorometer assembly 206 via
`moving a translation stage 306 along the x direction (indi(cid:173)
`multiwire cables 212, which are detachably connected to
`cated by arrow). Limit switches 308 are advantageously pro(cid:173)
`connectors 214.
`vided to restrict the motion of translation stage 306 to an
`Lid heater 204 has holes 220 therethrough, matching the
`appropriate range, large enough to allow detection module
`size and spacing of the sample wells 210, and electronically 5
`234 to be placed in optical contact with any of the wells while
`controlled heating elements (not shown). Lid heater 204 is
`preventing translation stage 306 from contacting other system
`coupled to lid 122. The coupling mechanism (not shown) is
`components, such as stepper motor 302.
`advantageously movable ( e.g., lid heater 204 may be attached
`Translation stage 306 has a y-axis stepper motor 316 and a
`to lid 122 by a hinge) in order to provide access to fluorometer
`lead screw 318 mounted thereon. Stepper motor 316 operates
`assembly 206 when lid 122 is removed from sample unit 202. 10
`to tum lead screw 318, thereby moving shuttle 232 along the
`When lid 122 is in place on sample unit 202, supports 224
`y direction (indicated by arrow). Limit switches 320 are
`hold lid heater 204 in position. Lower portions 226 of sup(cid:173)
`advantageously provided to restrict the motion of shuttle 232
`ports 224 are advantageously designed to compress lid heater
`to an appropriate range, large enough to allow detection mod(cid:173)
`204 toward sample unit 202, thereby reducing the possibility
`ule 234 to be placed in optical contact with any of the wells,
`of sample evaporation during operation of apparatus 100. 15
`while preventing shuttle 232 from contacting other system
`components, such as stepper motor 316.
`This compression also allows reaction vessels of different
`sizes to be used. Lid heater 204 is used to control the tem(cid:173)
`Stepper motors 302, 316, lead screws 304, 318, and limit
`switches 308, 320 may be of generally conventional design. It
`perature of the sample caps ( or other sealants) of reaction
`vessels sample wells 210, in order to prevent condensation
`will be appreciated that other movable mountings may be
`from forming on the caps during thermal cycling operation. 20 substituted. For example, instead of directly coupling the
`Lid heater 204 advantageously includes one or more cali(cid:173)
`motors to the lead screws, indirect couplings such as chain
`bration elements 222 positioned between selected ones of
`drives or belt drives may be used. Chain drives, belt drives, or
`holes 220 or in other locations away from the holes, such as
`other drive mechanisms may also be used to position the
`near the periphery oflid heater 204. Calibration elements 222
`detection module without lead screws, e.g., by attaching a
`25 translation stage to the chain, belt, or other drive mechanism.
`provide a known fluorescence response and may be used to
`calibrate fluorescence detectors in fluorometer assembly 206.
`Other types of motors, such as servo motors or linear motors,
`Calibration elements 222 may be made, e.g., of a fluorescent
`may also be used. Different drive mechanisms may be used
`coating on a glass or plastic substrate, or they may consist of
`for different degrees of freedom.
`Shuttle 232 holds detection module 234 via connectors
`a plastic with a dye impregnated in it, fluorescent glass, or a
`330,331. Connectors 330,331 which may vary in design, are
`fluorescent plastic such as polyetherimide (PEI). Neutral- 30
`configured to support and align detection module 234 on the
`density or other types of filters may be placed over the fluo(cid:173)
`underside of shuttle 232. The connectors are advantageously
`rescent material in order to avoid saturating the fluorescence
`detectors. In general, any material may be used, provided that
`adapted to allow easy insertion and removal of detection
`module 234, to facilitate replacement of the detection mod-
`its fluorescence characteristics are sufficiently stable over
`ule. In one embodiment, connectors 330 provide mounting
`time with the application oflight (photo-bleaching) and heat. 35
`for a cylindrical member (not shown) that pivotably holds an
`To the extent practical, the effect of temperature on the fluo(cid:173)
`edge of detection module 234, while connectors 331 include
`rescence response is advantageously minimized. Where mul(cid:173)
`tiple calibration elements 222 are provided, different materi-
`ball plungers mounted on shuttle 232 that are insertable into
`corresponding receptacles on detection module 234. Electri(cid:173)
`als may be used for different ones of the calibration elements.
`In an alternative embodiment, lid heater 204 may be omitted, 40
`cal connections (not shown) between shuttle 232 and detec(cid:173)
`and calibration elements 222 may be disposed on the surface
`tion module 234 may also be provided, as will be described
`of sample unit 202.
`below.
`Sample unit 202 and lid heater 204 may be of conventional
`FIG. 4 is a top view of detection module 234. Detection
`module 234 includes fittings 420 that couple to corresponding
`design. Examples of suitable designs include sample unit and
`connectors 330 on the underside of shuttle 232, thereby secur(cid:173)
`lid heater components of the various Alpha™ modules sold 45
`ing detection module 234 in place so that it moves as a unit
`by MJ Research, Inc., assignee of the present application.
`Fluorometer assembly 206 includes a support frame or
`with shuttle 232. Detection module 234 also includes an
`platform 230 fixedly mounted inside lid 122. Movably
`electrical connector 424 that couples to a corresponding elec(cid:173)
`mounted on the underside of support frame 230 is a shuttle
`trical connector on the underside of shuttle 232, thereby
`232, which holds a detection module 234. Shuttle 232 is 50
`allowing control and readout signals to be provided to and
`obtained from detection module 234.
`movable in two dimensions so as to position detection module
`234 in optical communication with different ones of the
`FIG. SA is a bottom view of one embodiment of detection
`sample wells 210 in sample unit 202 through the correspond(cid:173)
`module 234, showing four openings 502, 504, 506, 508 for
`ing holes 220 in lid heater 204. Support frame 230 and sup(cid:173)
`four independently controlled fluorescent excitation/detec(cid:173)
`tion charmels (also referred to as "excitation/detection pairs")
`ports 224 are advantageously dimensioned such that when lid 55
`122 is positioned in base unit 110 and closed, detection mod(cid:173)
`arranged inside the body of detection module 234. Examples
`ule 234 is held in close proximity to lid heater 204; one of skill
`of excitation/detection channels will be described below. The
`spacing of openings 502, 504, 506, 508 corresponds to the
`in the art will appreciate that this arrangement reduces light
`spacing of sample wells 210. Thus, when opening 502 is
`loss between the sample wells and the detection module.
`FIG. 3 is a bottom view of fluorometer assembly 206, 60
`placed in optical communication with one of the sample wells
`showing a movable mounting of shuttle 232 and detection
`210, openings 504, 506, and 508 are each in optical commu-
`module 234. In this embodiment, translation stages driven by
`nication with a different one of the sample wells 210. Open(cid:173)
`stepper motors are used to move the shuttle 232, to which
`ings 502, 504, 506, 508 may simply be holes through the
`detection module 234 is detachably coupled, to a desired
`bottom surface of detection module 234, or they may be made
`position. Specifically, support platform 230 has an x-axis 65
`of any substance that has a high degree of transparency to the
`stepper motor 302 and a lead screw 304 attached thereto.
`excitation and detection light wavelengths of their respective
`Stepper motor 302 operates to turn lead screw 304, thereby
`channels.
`
`Agilent Exhibit 1203
`Page 12 of 17
`
`

`

`US 8,835,118 B2
`
`7
`FIG. SB is a bottom view of a detection module 234'
`according to an alternative embodiment of the invention. In
`this embodiment, four openings 512, 514, 516, 518 are pro(cid:173)
`vided, but they are arranged in a staggered fashion so that only
`one opening at a time may be in optical communication with
`any of the sample wells. This configuration is useful for
`reducing cross-talk between the excitation/detection pairs.
`FIG. 6 is a schematic diagram illustrating a configuration
`of optical elements for an excitation/detection channel (or
`excitation/detection pair) 600 according to an embodiment of
`the invention. Detection module 234 may include one or more
`instances of excitation/detection pair 600, each of which pro(cid:173)
`vides an independent fluorescence detection channel. Excita(cid:173)
`tion/detection pair 600 is arranged inside opaque walls 602,
`which provide optical isolation from other excitation/detec(cid:173)
`tion pairs that may be included in detection module 234, as
`well as from external light sources. An excitation light path
`604 includes a light-emitting diode (LED) or other light
`source 606, a filter 608, a lens 610, and a beam splitter 612. A
`detection light path 620 includes beam splitter 612, a filter
`624, a lens 626, and a photodiode or other photodetector 628.
`Beam splitter 612 is advantageously selected to be highly
`transparent to light of the excitation wavelength and highly
`reflective of light at the detection (fluorescent response)
`wavelength.
`The components of excitation light path 604 are arranged
`to direct excitation light of a desired wavelength into a reac(cid:173)
`tion vessel 616 held in a sample well 210 of sample block 202.
`The desired wavelength depends on the particular fluorescent
`labeling agents included in reaction vessel 616 and is con(cid:173)
`trolled by selection of an appropriate LED 606 and filter 608.
`Optical communication between the excitation/detection pair
`600 and reaction vessel 616 is provided by opening 502 in
`opaque walls 602 and a hole 220 through lid heater 204, as
`described above. To maximize light trans