(12‘; lNTERNATlONAL APPLECA’TTUN PUBLlSHED UNDER THE PATENT COOPERATEON TREATY (PCT)
`
`(19} Wnrld Intellectual Property Organization ‘
`International Bureau
`
`(43} l’nternetinna} Publication liaise
`11 January 2067 (11.01.2387)
`
`(51} International Patent Classification:
`Bil/IL 7/60 {2006.01}
`
`
`
`(ill) Internatienal Pulilieaiien Nnumber
`
`”WC Ziiiifiiiiifiil’i’i Al
`
`(81) Designated States (unless oiizeiwise indicaled, for every
`kind ofrmiimmi protecrien available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG‘, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE. DK, DM. DZ, EC, EE EG ES, Fl,
`GB. CD, (1E, GJJ, (TB/i. HR, HU, JD, IL, TN, 13, JP, KE,
`KG, KM, KN, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV,
`LY, MA, Ml), MG, MK, MN, I‘va, MK, MZ, NA, NG, Nl,
`NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG,
`SK, SL, SM, S‘i’, TJ, TM, TN, TR, Tl, TZ, UA, UG, US,
`UZ, VC, VN, YU. ZA, ZM. ZW.
`
`(84) Designated States (unless olherwise indicated. for every
`kind {if mginnai pmzeczipn available): ARTPO (BVV, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL. SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FT,
`ER, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO. SE, SI, SK, TR). CAPT (BF. BJ, CF, CG. CJ, CM, GA,
`GN, GQ, GW, ML, MK, NE, SN, TD, TG‘).
`Lieclaratiens under Rule 4.1”;
`-----
`as to applicants entitlement to appiyfor and be granted a
`patent (Rule 4.1 7(ii))f0r all designations
`as to the applicants entitlement to claim the priority (gfihe
`earlier application {Rule 4. I 7(iii),ljbr (iii designations
`Published:
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`-----
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`----- with inmrmztimmi search report
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`(21) International Application Number:
`PCTHJSZOOfi/Ol 0819
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`(22)
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`international Filing Date: 24 March 2006 (24.€)3.2{)06)
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`(25) Filing Language:
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`(26) Publication Language:
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`(30) Prini‘it‘y Data:
`l l/174,69’i
`
`English
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`English
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`5 July 2005 ((35.07.2005)
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`US
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`lI'S): 3M
`(71) Applicant (for all deszgmated States except
`ENNOVAT EVE PROPERTIES COBvll’ANY {USES};
`3111 Center, Post Office Box 33427, Saint Paul, Minnesota
`55133-3427 (US).
`
`inventors; BEDENGIL’SM, ‘Wiiliam D.,; 3111 Center, Post
`Office Box, 33427, Saint Paul, Minnesota 551333417,?
`(US). I.UDOWISE Peter EL:
`.imtTente. Pest OIfice
`Box 33427, Saint Paul Minnesota 551333477 (US).
`
`ROBOJ.,E,Barry W,”' 3m Center, Pns- Ofiice Box 33427,
`Saint Paul, Minnesota 55"1.. 5-3512? (US)
`
`(74) Agents: LAMBERT, Nancy ML, e1 al.; 3m Office OI En»-
`tellectnal Property Ceunsel, Post Office Box 334-27, Saint
`Paul, Minnesota SSJ 33—3427 (US).
`
`F{Irwin-letter codes and Other abbreviations, refer m the "Guiti
`ance News on Codes aiszbbrevimions" appearing at the begin—
`ning of each regular issue ofzhe PCT Gazette.
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`{‘3€57) Abstract: Techniques are described for the detection of multiple target species in real-time PCR (polymerase chain reaction).
`Fm example, a system cempnses a data acquisition device and a deter:Lion device coupled to the data acquisition device. The detec—
`tiion deviceincludes a mta‘ting disk having a plurality (1-If piecess chemeers l1;-wing a plurality of species that emit fluorescent light at
`diflerent wavelengths, The device Iuttnerinc lndes cl pluleliiy oi removable optical modules that "are optically cenIigureLl to excite
`the species and capture flame scent lightLemitted by the species 1t different W'avelengths Afilier optic: bundle conpled to the pli-rality
`of:emovable op-Lical nicdoles conveys; ti1ell uorescent light from the optical modules to a single detector. [he device feather includes
`' '1Lsting eleinent IUI testing um 01 n1me piocess chambLIs on th(3 disk in additloi1, the device may control the Ilow of fluidin the
`:
`disklpy locating and selectively opening valves separating chambets by heating the valves with a inset.
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`$3
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`we 2067/005077
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`FCT/iiszeee/smsm
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`s1“
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`HEATING ELEMENT FGR A RG’E‘ATING MULTIPLEX
`FLUORESCENCE DETECTION DEVECE
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`2J1
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`*i‘ECHNTECAL FEELD
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`The inventinn reistes te assaying systems and, more pai‘tieuiariy, techniques fer
`heating fluid for the detectien 0f muitipie target species using fluorescent dyes.
`
`BACKGRGUND
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`10
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`Optics} disc systems are often used to perform various biologiceig chemical or
`
`hie—eiiemieei assays. In a typical system, a. rotatable disc is used as a medium for
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`storing and processing fluid specimens? such as blood, plasma? serum, urine er other
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`fluid. In some cases the fluids within the disk may need to he meved from one
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`ieee‘tien to enether during the processing
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`15
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`One type of anaiysis is pei‘ymerase ehaih reaction (PCR): Whieh is often used
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`for nueieie acid sequence aneiysis. In partiouian PCR is often used for DNA
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`sequencing, ciening, genetic mapping, and other fonns efmieieie acid sequence
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`anaiysis.
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`in enerai. PCR relies on the ehiiitv 0f ENE neon in enzvmes to remain stable
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`a
`i 3’
`V
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`{\2 C}
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`at high temperatures. There are three major steps in PCR: denaturetion, annealing: and
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`extension, During the denaturetionj a iiquid sempie is heated at approximateiy 940C.
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`During this process, doubleetranded DNA “melts” open into single-stranded DNA.
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`During armeaiing, the singidsh‘zmded DNA is ooeied to approximately 54°C. At this
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`temperature, primers bind er ”annesi” to the ends of the DNA segments that are to be
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`25
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`repiiceted. During extension,- the sample is heated to 75°C. At this temperature,
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`enzymes add nucleotides add to the target sequence and eventueiiy a complementary
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`copy ef the DNA template is ibrmedu The new DNA strand beeemes a new target for
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`the next sequence 0f events, or “cycle.”
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`There are a number sf existing PCR instruments designed ta) determine ieveis of
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`30
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`specific DNA and RNA. sequences in the sampie during the PCR in real—time. Many of
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`the instrimients are based on the use of fluorescent dyes.
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`in paitieuiar, inehy
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`conventional read-time PCR instruments detect a fluorescent signal produced
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`proportienaiiy during ampiifioatien of a PCR product.
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`Conventional real«time PCR. instruments use different methods for detection of
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`different fluorescent dyes. For example, some conventional ”PCR instruments
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`incorporate white light sources with filter Wheels for spectrally resolving each dye. The
`white light sources are tungsten halogen bulbs, which have a lifetime maxirna of a few
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`5
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`thousand hours“ The filter wheels are typically complicated electromechanical parts that
`are susceptible to wear.
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`SUMMARY
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`10
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`15
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`in general, the invention relates to techniques for the detection of multiple target
`species in realntime PCR (polymerase chain reaction), referred to herein as multiplex
`PCR. in particular, a multiplex fluorescence detection device is described that
`
`incorporates a plurality of optical modules. Each of the optical modules may be
`
`optimized for detection of a respective fluorescent dye at a discrete wavelength band.
`in other words, the optical modules may be used to interrogate multiple, parallel
`reactions at different wavelengths The reaction may, for example, occur within a
`
`single process chamber (cg, well) of a rotating disk. Additionally, each optical
`
`module may he removable to quickly change the detection capabilities of the (levice.
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`The plurality of optical modules may be optically coupled to a single detector
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`by a mule-legged optical fiber bundle. ln this manner, multiplexing can be achieved by
`using a plurality of optical modules and a single detector, e.g., a photomultiplier tube.
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`20
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`The optical components in each optical module may be selected to iriaxirnize sensitivity
`and minimize the amount of spectral crosstalk, i.e., signals from one (lye on another
`
`optical module,
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`25
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`30
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`The device also includes a heating element for heating ' re disk or selectively
`heating one or more process chambers on the disk. The heating element includes an
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`energy source and a reflector for directing most of the emitted energy to a target on the
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`disk. Elliptical and spherical surfaces on the reflector may reflect light from a halogen
`bulb placed away from the axis of the reflector.
`
`in one embodiment, a device comprises a motor to rotate a dial: having a
`plurality of process chambers, wherein one or more process chambers contain a sample,
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`an energy source that emits electromagnetic Energy to heat one or more of the plurality
`
`of process chambers, and a reflector that includes a combination of spherical and
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`elliptical reflecting surfaces to reflect a portion of the electromagnetic energy to the
`disk
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`UH
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`In another ernhoailmenta a system. comprises a data acquisition device. The
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`system also comprises a detection device coupled to the data acquisition device,
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`wherein the detection device comprises a motor to rotate a disk having a plurality of
`process chambers, wherein one or more process chambers contain, a sample, an energy
`source that emits electromagnetic energy to heat one or non: of the plurality of process
`chamber; and a reflector that includes a combination of spherical and elliptical
`
`reflecting surfaces to reflect a portion of the electromagnetic energy to the disk.
`
`it)
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`in an additional embodiment, a method comprises rotating a disk having a
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`plurality of process chambers, wherein one or more process chamhers contain a sample,
`emittino‘ electromagnetic energy to heat the plurality of process chambers? and
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`reflecting a portion of the electromagnetic energy with a combination of spherical and
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`elliptical reflecting surfaces to the disk.
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`15
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`The invention may provide one or more advantages. For examplea the reflector
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`may direc . nearly 100 percent of the emitted energy to the dish. Directing all of the
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`energy to the disk may increase heating efficiency, decrease heating time and decrease
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`overall run time Moreover, the energy source does not need to physically contact the
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`disk or process chambers. which may decrease device complexity and operational
`costs.
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`20
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`While the device is he capable ofconducting realwtirne FOR: the device may be
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`capable of analyzing any type ot‘hiological reaction While it occurs. The device may be
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`able to modulate the temperature of each reaction independently or as a selected group,
`and the device may be able to support multiple stages of reactions hy including a valve
`between two or more chambers.
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`25
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`in some embodiments: the device may be portable and robust to allow operation
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`in remote areas or temporary laboratories. The device may include a data acquisition
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`computer for analyzing the reactions in realtirne, or the device may communicate the
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`data to another device through wired or wireless communication interfaces.
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`'l‘he details of one or more embodiments of the invention are set forth in the
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`accompanying drawings and the description below. Other features, obj ects, mid
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`advantages ofthe invention will be apparent from the description and drawingsg and.
`from the claims.
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`BRIEF DESCRIPTION GIT DRAWlNGS
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`U1
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`l, is a block diagram illustrating arr exemplary embodiment of a multiplex
`FIG»,
`fluorescence detection device,
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`HQ. 2 is a schematic diagram illustrating an exemplary detection module,
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`which may correspond to any of a plurality of detection modules of the fluorescence
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`detection device of Flt}, it
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`ill
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`FlG. 3 is a perspective diagram illustrating a front View of an exemplary set of
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`removable optical modules within the device housing,
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`FIG. 4 is an perspective diagram illustrating the exemplary set of removable
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`optical modules within the device housing.
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`FIG. 5 is perspective diagram illustrating a front sicle View of an exemplary set
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`l5
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`of removable optical modules having one module removed to expose a module
`eoimector.
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`FIG. 6 is perspective diagram illustrating the components Within an exemplary
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`main removable optical module.
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`FlGu 7 perspective diagram illustrating the components Within an exemplary
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`20
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`supplemental removable optical module.
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`FIG. 8 is an illustration of the side view of an exemplary set of removable
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`optical modules within the device housing with the laser valve control syatem located
`over a slot on the aisle.
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`FlGS. 9A and QB illustrate the chambers and vales of two exemplary disks that
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`may he used to hold samples within the detection device.
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`FIG. 10 is an exemplary illustration of a, heating element within an offmaxis
`reflector‘
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`FIG. ll is an exemplary ray diagram of the light emitted by a, heating element
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`as it reflects off of an open reflector to heat a disk.
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`F1G. l2 is ar- exemplary illustration of a heating element within an 011—5in3
`reflector.
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`FIG” 13 is an exemplary ray diagram of the light emitted. by a heating element
`as it reflects offof a closed reflector to heat: a disk.
`
`F1G. 14 is a block diagram illustrating an example embodiment of the multiplex
`fluorescence detection device in further detail.
`
`FIG. 15 is a block diagram of the a single detector coupled to tour optical fibers
`of the optical fiber bundle.
`
`FIG. 16 is a flow diagram illustrating exemplary operation of the multiplex
`fluoreseence detention device,
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`FIG. 17 is a flow diagram illustrating exemplary operation ofthe laser valve
`control. system for the detection device.
`
`F1G. 18 is a block diagram of a heating circuit that controls a heating element
`which treats a disk.
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`FIG. 19 is a flow diagram illustrating exemplary operation of the heating; circuit
`for heating a disk,
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`FIG-3 20 and 21 Show the absorption and emission spectra of commonly used
`fluorescent dyes that may be utilized for multiplex PCR.
`
`FIGS, 22A and 228 illustrate raw data sequired from two exemplary detection
`modules with a single detector during a PCR analysis.
`
`F1G. 23 is a graph that shows the data once adjusted for a time offset.
`F1G8, 24A and 24.3 Show a limit of detection (LSD) for the data received from
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`two exemplary detection modules.
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`DETAILED DESCRIPTIGN
`
`FIG. 1 is a block diagram illustrating an exemplary embodiment of e multiplex
`fluorescence detection device 10.
`in the illustrated example, device it) has four optical
`modules l6 that provide four “channels” for optical detection of four different dyes.
`in
`particular? device 10 has four optical modules 16 that excite different regions of
`rotating disk 13 at any given time, and colieet emitted fluorescent light er orgy at
`different wavelengths from the dyes. As a result, moduies 16 may be used to
`interrogate multiple, paraliei reactions occurring within sample 22.
`
`The multiple reactions may, for exznnple, occur simultaneously within a single
`chamber of a rotating disk 13. Each of optical moduies 15 interrogates sample 22 and
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`it)
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`PCT/USZiillti/{llfllfilg
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`collects fluorescent light energy at different wavelengths as the disk 13 rotates. For
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`example, excitation Sources within modules l6 may be sequentially activated for
`periods sufficient to collect data at the corresponding wavelengths. That is}, an optical
`module 16A may be activated for a period of time to collect data at a. first range of
`wavelengths selected for a first dye corresponding to a first reaction. The excitation
`
`source may then be deactivated, and an excitation source Within module 16.8 may be
`activated to interrogate sample 22 at a second range of wavelengths selected for a
`
`second dye oorreeponding to a second. reaction. This process continues until data has
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`been captured from all optical modules 16.
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`in one embodiment, each of the excitation
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`sources within optical modules id is activated for an initial period of approximately
`two seconds to reach steady state followed by an interrogation period which lasts; for
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`ltlnSG rotations of disk 13.
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`in other embodiments, the excitation sources may be
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`sequenced for shorter (cg, l or 2 milliseconds) or longer periods.
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`in some
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`embodiments, more than one optical module may be activated simultaneously for
`concurrent interrogation of sample 22 without stopping the rotation of dish 13,
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`Although a single sample 22 is: illustrated, disk 13 may contain a plurality of
`chambers holding samples. Optical modules 16 may interrogate some or all of the
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`tliilerent chambers at different wavelengths, in one embodiment, disk, 13 includes 96
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`chambers space around a circumference of disk ‘33,
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`illith a 96 chamber disk and four
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`20
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`optical modules: l6, device 10 may be capable of acquiring data from 384 different
`
`species.
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`in one embodiment, optical modules l6 include excitation. sources that are
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`inexpensive hgh power light emitting diodes (LED’S), which are commercially
`«2
`available in a variety of wavelengths and have long lifetimes (e.
`a, 100,000 hours or
`
`in another embodiment, conventional halogen bulbs or mercury lamps may be
`more).
`used as excitation sources.
`
`As illustrated in FlG. 1, each of optical modules 16 may be coupled to one leg
`of a fiber optic bundle 14. Fiber optic bundle 14 provides a flexible mechanism for
`
`collection of fluorescent Signals from optical modules 16 Without loss of sensitivity. In
`general, a fiber optic bundle comprises mu tiple optical fibers laid aide by side and
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`bonded together at the ends and encased in a flexible protective jacket. Alternatively,
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`fiber optic bundle l4 may comprise a smaller number of discrete, large diameter multiu
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`mode fibers, either glass or plastic, having a common end. For example, for a four~
`optical module device, fiber optic bundle 16 may comprise four discrete multimode
`
`fillSl‘S; each having a 1 mm core diameter. The common end of the bundle contains the
`four libero bound together. in this example, the aperture of detector l8 may be 8 mm
`which is more than sufficient for coupling; to the tour liberal
`
`In this example, fiber optic bundle l4 couples optical. modules iii to a single
`detector 18. The optical fibers carry the fluorescent light collected by optical modules
`16 and efiectively deliver the capuired light to detector lit.
`in one embodimenn
`detector l8 is a photomultiplier tube. in another embodiment: the detector may include
`multiple photomultiplier elements: one for each optical fihen within the single detector”
`in other embodiments; one or more solild~state detectors may be used.
`
`The use of a single detector 18 may be advantageous in that it allows use of a
`
`highly sensitive and possibly expensive detector (eg a photomultiplier), while
`maintaining a minimal coat in that only a Single detector need be used; A single
`detector is discussed herein; however? one or more detectors may be included for
`detecting a greater number of dyes. For example, four additional optical modules l6
`and a second detector maybe added to the system to allow for the detection. of eight
`different wavelengths emitted from one disk. An exemplary fiber optic bundle coupled
`to a single detector for use with rotating disk l3 is described in US, Patent Application
`Serial No. ll/174,755, entitled “MUL’l‘ll’lgEX FLUORESCENCE DETECTlON
`
`DEVl SE ill-"1V1N8 FIBER BUNDLE CGUPLING MUETIPLE OPTIC AL
`
`MODULES TO A COMMON DETECTOR,” filed on July 5, 2905"
`
`Optical modules 16 are removable from the device and easily interchangeable
`with other optical modules that are optimized for interrogation at different wavelengths.
`For example, optical modules l6 may be physically mounted Within locations of a
`
`module housing. Each of optical modules 16 may be easily inserted within a respective
`location ofthe housing along guides (e.g., recessed grooves} that mate with one or
`
`more marking (cg, guide pins) of the optical module. Each of optical modules l6 may
`be secured Within the carriage by a latch} magnet, screw or other factening device.
`Each optical module includes an Optical output port l:shown in FIGS. 6 and 7') for
`coupling to one leg of fiber optic bundle 14. The optical output port may have a
`
`threaded end coupled to a threaded coiniector of the leg. Alternatively, a form of
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`“quicloconnect” may he used {c.gw a. slidable connection having an o-«ring and a catch
`pin} that allows fiber optic bundle 14 to be slidably engaged and disengaged from the
`optical output port. Moreover, each ot‘optical modules 16 may have one or more
`electrical contacts pads or llex circuits for electronically coupling to control unit 23
`when fully inserted. Exemplary removable optical modules for use with rotating dislr
`l3 is described in US, Patent Application Serial No. ll/17-4y754, entitled
`
`“lleL’l‘lPl_,EX FLUORESCENCE DETECTlGN DEVECE HAVING REMOVABLE
`
`Ql’TlCAL MDDULESF filed on July 55 2005.
`
`The modular architecture of device l C- allows the device to be easily adapted for
`all of the fluorescent dyes used in a given analysis environment.” such as multiplex
`PCR. Other chemistries that may be used in device 10 include invader (Third Wave?
`Madison, Wisconsin), Transcripted—mediated Amplification {GenProbe, San Diego?
`California), fluorescence labeled enzyme linked immunosorhent assay (ElilSA) or
`fluorescence in site hybridization {'FlSli). The modular architecture of device 10 may
`provide another advantage in that the sensitivity of each optical module 16 can be
`
`optimized hy choice ofthe corresponding excitation source ifnot shown) and excitation
`
`and detection filters for a small specific target range of wavelengths in order to
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`selectively excite and detect a corresponding dye in the In ultiplex reaction.
`
`For purpose of example, device it) is illustrated in a ducolor multiplex
`arrangement, but more or less channels can he used with the appropriate fiber optic
`handle 14. This modular design allows a user to easily upgrade device it) in the field
`
`by simply adding another optical module 16 to device l O and inserting one leg offiher
`optic bundle ill into the new optical module. Optical modules l6 may have integrated
`electronics that identify the optical modules and download calibration data into an
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`45
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`internal control module or other internal electronics (ex e, control unit 23) of device 10.
`
`lo the example of FlG. 1, samples 22 are contained in chambers of disk 13,
`
`which is mounted on rotating platform 25 under the control of control unit 23. A slot
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`sensor trigger 27 provides an output signal utilized by control unit 23 for synchronizing
`data acquisition device ill with chamber position during disk rotation. Slot sensor
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`30
`
`trigger 27 may he a mechanical, electrical, magnetic, or optical sensor For example, as
`described in further detail below, slot sensor trigger 27 may include a light source that
`
`emits a beam of light to through a slot formed through disk 13 that is detected each
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`revolution of the disk. As another example, slot sensor trigger may sense reflected light
`for purposes of synchronizing t to rotation of disk l3 and data acquisition by ntoduleslo
`and detector lit.
`in other embodiments; disk l3 may include a tab, protrusion or
`reflective surface in addition to or in place of the slot. Slot sensor trigger 27 may use
`any physical structure or mechanism to locate the radial position of disk 13 as it rotates”
`
`thical modules l6 may be physically mounted above rotating plationn 25. As a
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`i
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`result, optical modules 16 are overlapped with dili‘erent chambers at any one time.
`Rotating platforms, base plates, thermal structures and other structures that may be used
`in connection with the present invention are described in US. Patent Application Serial
`No; l l/l74,757, entitled “SAMPLE PROCESSING DEVlCE COMPRESSION
`
`SYSTEMS AND METHODS,” filed on July 5, 2005.
`
`Detection device 10 also includes a heating element (not shown) for modulating
`the temperature of the sample 22 on disk l3, The heating element. may comprise a
`cylindrical halogen bulb contained within a reflective enclosure, The reflective
`
`cl‘iamher is she ted to focus radiation from the bulb onto a radial section of disk 13.
`
`Generally, the heated area of disk l3 would comprise an amiular ring as disk l3 spins,
`in this embodiment, the shape of the reflective enclosure may he a combination of
`
`in other embodiments,
`elliptical and spherical geometries that allow precise focusing.
`the reflective enclosure may be of a different shape or the bull) may broadly iniadiate a
`larger area.
`in other embodiments, the reflective enclosure may be shaped to focus the
`radiation front the bulb onto a single area of the disk 13,, such as a single process
`chamber containing a sample 22.
`
`In some embodiments, the heating element may heat air and force the hot air
`
`over one or more samples to modulate the temperature. Additionally, the samples may
`be heated directly by the dislr.
`in this case, the heating element may he located in
`platform 25 and thermally couple to disk 13. Electrical resistance within the heating
`element may heat a selected region of the disk as controlled by control unit 23. For
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`example, a region may contain one or more chambers, possibly the entire disk.
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`Alternatively, or in addition, device 10 may also includes a cooling component
`(not shown). A fan is included in device 10 to supply cold air, l,e,, room temperature
`air, to disk 13. Cooling may be needed to modulate the temperature ofthe sample
`appropriately and store samples after an experiment has completed.
`In other
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`embodiments the cooling component may include thermal coupling between platform
`25 and disk 13, as platform 25 may recluse its temperature when needed. For
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`example, some biological samples may he stored at 4 degrees Celsius to reduce enzyme
`activity or protein denaturing.
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`Detection device 10 me ' also he capable of controlling reaction species
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`contained Within a process chamber. For exmnple, it may be beneficial to load some
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`species in a process chamber to generate one reaction and later adding another species
`to the sample once the. first reaction has tennineted. A valve control system may be
`utilized to control a valve position separating an inner holding chamber from the
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`process chamber, thereby controlling the addition of species to the chamber during
`rotation otfdisk l3” The valve control system may be located within or mounted to one
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`of optical modules 16 or separate from the optical modules. Directly below the laser,
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`tinder disk 13, may he a laser sensor for positioning the laser relative to disk 13.
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`in one embodiment, the valve control system includes a near infrared (MIR)
`laser capable of being driven at two or more power levels in combination with a sensor.
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`Under a, lovvr power setting, the laser may he used for positioning disk. l3 and targeting
`select valves, eg.., by the sensor sensing the NIH. light emitted by the laser though a slot
`in disk 13‘ Once the targeted valve is rotated into position, control unit 23 directs the
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`laser to output a short burst of high power energy to heat the valve and open the
`targeted valve The burst of energy forms a void in the velvet e.g., hy piercing, melting
`or ahlating? causing the valve to open and allowing a, fluid to flow through a charmel
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`from an inner holding chamber to an outside process chamber.
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`in some etrihotlitnents,
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`disk 13 may contain a plurality of valves of various sizes and materials to generate a
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`plurality of reactions in sequence. More than one set of valve control system may he
`used, when utilizing a tlislt having multiple chamber valves. Ari exemplary loser
`homing valve control system for use with rotating disk 13 is described in US. Patent
`
`Application Serial No" lit/"174,957, entitled “ ’ALVE CQNTROL SYSTEM FORA
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`ROTATING WIL'l‘ll’LEX FLUORESCENCE DETECTION DEVlCE,” tiled on
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`July 5, 2005:
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`Data acquisition device 21 may collect data from device ll) for each (lye either
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`sequentially or in parallel.
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`in one embodiment, data acquisition system 2;? collects the
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`data from optical modules l6 in sequence, and corrects the spatial overlap by a trigger
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`delay for each one ofthe optical modules measured from the output signal received
`from slot sensor trigger 2 .
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`One application for device 10 is real~tirne PCR, but the techniques described
`herein may be extended to other platforms that utilize fluorescence detection at multiple
`wavelengths Device l0 may combine rapid thermal cycling; utilizing the heating
`element? and centiiiiigally driven niicroiluiciics for isolation, amplification, and
`detection of nucleic acids By making use ofninltiplex fluorescence detection, multiple
`target species may be detected and analyzed in parallel.
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`For realntinie PCRi fluorescence is used to measure the amount of amplification
`in one of thee general teclniiques The first techni' tie: is the use ofa dye, such as Sybr
`Green (Molecular Probes, Eugene, Oregon)? whose fluorescence increases upon
`binding to doublestranded DNA. The second teclniiqne uses ileorescently labeled
`probes whose fluorescence changes when bound to the amplified target sequence
`{hybridization probes? hairpin probes, etc). This technique is similar to using a double-
`stranded DNA binding dye:a but is more specific because the probe will bind only to a
`certain section ot‘tlie target sequence“ The third technique is the use of hydrolysis
`probes ('leqrnanm; Applied BioSysterns, Foster City Calii‘bmia)? in which the
`exonuclease activity ofthe polymerase enzyme cleaves a quencher molecule from the
`probe during the extension phase oi’PCRf, making it fluorescently active.
`in each of the. approaches, fluorescence is linearly proportional to the amplified
`target concentration. Data acquisition system 2i measures an output signal from.
`detector 18 {or alternatively optionally sampled and communicated by control unit 23)
`during the PCR reaction to observe the amplification in near real-time.
`ln multiplex.
`FOR, the multiple targets are labeled with different dyes that are measured
`independently. Generally speaking, each (lye will have different ebsorbance and
`emission spectra. For this reason, optical modules l6 may have excitation sources,
`lenses and related filters that are optically selected for interrogation of sample 22 at
`different wavelengths.
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`Some examples of suitable construction techniques or materials that may be
`adapted for use in connection with the present invention may be described in: eg,
`cominonlymassigned US. Fetent No. 6,734,4lll titled “ENHANCED SAMME
`
`PROCESSlNG DEVICES SYS'l‘lEM'S AND lle'l'ilODS” (Bedlnghem et al.) and US
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`Patent Application Publication No. US 2002/0064885 titled “SAMPLE PROCESSING
`DEVICES,” Other useable device constructions may be found in, e.g,, US. Provisional
`Patent Application Serial No 60/2l 4,508 filed on lime 28, 2000 and entitled
`
`“THERE/ML PROCESSlNC-l DEVlCES AND METHODS”; US. Provisional Patent
`
`Application Setial No, 60/214,642 filed on June 28, 2000 and entitled “SAMPLE
`
`PROCESSING DEVlCES, SYSTEMS AND METHQDS”; US. Provisional Patent
`
`Application Serial No, 60/237,072 filed on October 2, 2000 and entitled “SAlly/lPLE
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`PRGCESSING DEVlCES, SYSTEMS AND METHODS”; US. Provisional Patent
`
`Application Serial No. 60/260,063 filed on January 6, 2001 and titled “SAMPLE
`
`PRQCESSING DEVICE-S, SYSTEMS AND METHODS”; US, Provisional Patent
`
`Application Serial No, 60/284,637 filed on April l8, 200i and titled “ENHANCED
`
`SAMPLE l’ROCESSlNG DEVlCES, SYSTEMS AND METHODS”; autl US Patent
`
`Application Publication No. US 200210048533 titled “SAMPLE PROCESSING
`
`DEVlC-ES AND CARRIERS.” Other potential device constructions maybe found in,
`e.g., US Patent No. 6,627,159 titled “CEN'l‘RlFUGAL li‘lill,l...lNG OF SAMPLE
`PRGCESSING DER/ICES” (Bedinghem et ah).
`1
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`Eli}. 2 is a schematic diagram illustrating an exemplary optical module loA,
`which may correspond to any of optical modules iii of FIG. 1.
`lu this example, optical
`module l6A contains a l1lgll~p0W6I excitation source, LED 30, a colliniating lens 32, an
`excitation filter 34, a tlichroic filter 36, a focusing lens 38, a detection filter 40, and a
`lens 42 to focus the fluorescence into one leg cftihet optic bundle 14.
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`Consequently, the excitation light from LED 30 is colliinatecl by colliinating
`lens 32, filtered by excitation filter 34, transmitted through clichroic filter 36, and
`focused into the sample 22 by focusing lens 38. The resulting fluorescence emitted by
`the sample is collected by the same focusing lens 38, reflected off of Clichroic filter 36,
`and filtered by detection filte