(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`16 August 2007 (16.08.2007)
`
`
`
`(10) International Publication Number
`WO 2007/091230 Al
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`(51) International Patent Classification:
`BOIL 7/00 (2006.01)
`GOIN 35/08 (2006.01)
`BOIL 3/00 (2006.01)
`
`(21) International Application Number:
`PCT/AIE2007/000015
`
`(22) International Filing Date: 7 February 2007 (07.02.2007)
`
`(25) Filing Language:
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`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/765,670
`
`7 February 2006 (07.02.2006)
`
`US
`
`(71) Applicant (for all designated States except US): STOKES
`BIO LIMITED [IEE]; Shannon Arms, Henry Street,
`Limerick (TF).
`
`(72)
`(75)
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection 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,FI,
`GB, GD, GE, GH, GM, GT, HN, HR, HU,ID,IL,IN,IS,
`JP, KE, KG, KM,KN,KP, KR, KZ, LA, LC, LK, LR, LS,
`LT, LU, LV, LY, MA, MD, MG, MK, MN, MW, MX, MY,
`MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS,
`RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, T, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(34) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, 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, FI,
`FR, GB, GR,HU,IE,IS, IT, LT, LU, LV, MC, NL,PL, PT,
`RO, SE,SI, SK, TR), OAPI (BF, BJ, CE, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`(74) Agents: O’BRIEN, John, A.et al.; c/o John A OBrien
`& Associates, Third Floor, Duncairn House, 14 Carysfort
`Avenue, Blackrock, County Dublin (IE).
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes and Abbreviations" appearing at the begin-
`ning of each regularissue of the PCT Gazette.
`
`(54) Title: A MICROFLUIDIC ANALYSIS SYSTEM
`
`1,
`
`Sample
`
`(57) Abstract: A thermal cycling device (3) device a numberof fixed thermal zones (11, 12, 13) and a fixed conduit (10) passing
`throughthe thermal zones. A controller maintains each thermal zone includingits section of conduit (10) at a constant temperature. A
`scries of droplcts flows through the conduit (10) so that cach droplet is thermally cycled, and a detection system detects fluorescence
`from droplets at all of the thermal cycles. The conduit is in a single plane, and so a numberof thermal cycling devices may be
`arranged together to achieve parallelism. The flow conduit comprises a channel (17) and a capillary tube (10) inserted into the
`channel. The detection system may perform scans along a dircction to detect radiation from a plurality of cycles in a pass.
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`5
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`PCRThermocycling
`Perparation
`2|wate Na
`
`Inventors; and
`Inventors/Applicants (for US only}: DAVIES, Mark
`[GB/AIE]; 12 Ilarbour View Terrace, South Circular Road,
`Limerick (IE). DALTON, ‘ara [LE/LE|; Ashfort, Patrick-
`—_with international search report
`swell, County Limerick (IE).
`
`
`
`
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`WO2007/091230AdIINVRINNNENATATINNINTINTUMMAANTATAA
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`‘A Microfluidic Analysis System”
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`INTRODUCTION
`
`Field of the Invention
`
`The invention relates to analysis of samples to which thermal cycling is applied for
`
`nucleic acid amplification, such as in the quantitative polymerase chain reaction
`
`(qPCR).
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`Prior Art Discussion
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`Conventionally, nucleic acid amplification has involved providing an array of samples
`
`in an assay plate and thermally cycling the plate reaction vessel. This, however,
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`involves the laborious task of loading the samples and preparing a fresh assay well
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`plate.
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`for nucleic acid amplification, and
`is known to provide a thermal cycler
`It
`US85270183, W02005/075683, US6033880, and US6814934 describe thermal cycler
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`analysis systems.
`
`The prior systems suffer from being complex, both in physical and control terms. For
`example, in the system of US6033880 it is necessary to rotate heat exchangers into
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`desired positions, and in the system of US6814934 it is necessary to heat and cool a
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`reaction vessel.
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`The invention is directed towards providing an improved thermal cycler system in
`which a requirement to heat and cool a reaction vessel is avoided. Another object is to
`achieve improved detection efficiency.
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`SUMMARY OF THE INVENTION
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`there is provided a microfluidic analysis system
`According to the invention,
`comprising a thermal cycling device, the device having a plurality of fixed thermal
`zones and a fixed conduit passing through the thermal zones, a controller for
`maintaining each thermal zone including its section of conduit at a constant
`temperature, means for flowing a series of droplets through the conduit so that each
`droplet is thermally cycled, and a detection system for detecting electromagnetic
`radiation from droplets at a plurality of said thermal cycles.
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`In one embodiment, the conduit is in a single plane.
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`In one embodiment, the conduit comprises a channel.
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`In one embodiment, the thermal zones are mutually thermally insulated.
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`In one embodiment, the flow conduit comprises a channel and a capillary tube inserted
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`into the channel.
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`In one embodiment, the capillary has a circular cross-section.
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`In one embodiment, the channel and capillary are configured to receive a refractive
`index-matchingliquid in the channel andat least partly surrounding the capillary.
`
`In another embodiment, the channel has a depth greater than that of the capillary.
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`In one embodiment, the detection system comprises optics for focusing incidentlight
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`radiation.
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`In one embodiment,
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`the detection system comprises optics for filtering incident
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`radiation.
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`In one embodiment,
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`the detection system comprises optics for filtering emitted
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`radiation.
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`In another embodiment, the detection system performs scans along a direction to
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`detect radiation from a plurality of cycles in a pass.
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`In one embodiment, the detection system performs simultaneous detection of emitted
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`light from a plurality of cycles.
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`In one embodiment, there is an air gap between adjacent thermal zones.
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`In one embodiment, said air gap-is adjustable.
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`In one embodiment, the flow conduit passes through a hot thermal zone for a length
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`before a first cycle, providing a denaturation zone.
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`In another embodiment, the detection system comprises a plurality of optic fibres for
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`point illumination of each ofa plurality of cycles.
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`In one embodiment, the detection system comprises a plurality of optic fibres for point
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`detection of each of a plurality of cycles.
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`In one embodiment, the detection system comprises a rotating filter for cyclic filtering
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`of incident or emitted light.
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`In one embodiment, the conduit is in a serpentine pattern of multiple folds, each fold
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`extending through a plurality of thermal zones.
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`In a further embodiment, the system comprises a plurality of thermal cycling devices
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`arranged in parallel.
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`In one embodiment, the thermal cycling devices are interconnected to form a physical
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`unit.
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`In one embodiment, the detection system performs simultaneous detection of emitted
`light from a plurality of cycles from a plurality of thermal cycling devices.
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`DETAILED DESCRIPTION OF THE INVENTION
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`Brief Description of the Drawings
`
`The invention will be more clearly understood from the following description of some
`embodiments thereof, given by way of example only with reference to the
`accompanying drawings in which:-
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`Fig. 1 is a block diagram of an analysis system of the invention,
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`Fig. 2 is a plan view of a thermal cycler of the system having three thermal
`zones, Fig. 3 is a vertical cross section, and Fig. 4 is an end view ofthe thermal
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`cycler;
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`Fig. 5 is a perspective view of an alternative thermal cycler, having only two
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`thermal zones;
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`Fig. 6 is a diagram showing an arrangement with two exits, providing a choice
`of n cycles or n+p cycles;
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`Fig. 7 is a photograph showing droplets flowing in a number of cycles of the
`thermal cycler having three thermal zones;
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`Fig. 8 is a plot illustrating fluorescence characteristics for detection;
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`Fig. 9 is a block diagram of a detection system ofthe analysis system;
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`Fig. 10 is a pair of photographs, showing negative and positive fluorescence
`detection, from left to right;
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`Figs. 11 to 14 are diagrams showing alternative detection arrangements;
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`Figs. 15 and 16 are perspective views showing image capture via optic fibres;
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`Fig. 17 is a perspective view of a three-dimensional cycler for parallel
`amplification, and Fig. 18 is a cross-sectional plan view ofthis cycler;
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`Fig. 19 is a sample image ofpart of a detector array captured from the thermal
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`cycler of Fig. 17; and
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`Figs. 20 and 21 are viewsof arrays of windowsofthe cycler of Fig. 17.
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`Description of the Embodiments
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`technology.
`system of the invention is based on microfluidics
`An analysis
`Microfluidic devices themselves have dimensions ranging from several millimetres to
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`micrometers. Typically one of the components or dimensions of the device, such as a
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`channelin the device, is of the order of micrometers.
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`The polymerase chain reaction, or PCR, is a powerful technique used to amplify low
`concentrations of specific DNA sequencesto levels which may be detected. PCR can
`be used to achieve a billionfold increase in target sequence copy numberby thermally
`cycling a specific chemical mix. This makes the PCR method extremely sensitive as it
`can detect a single DNA molecule in a sample.
`
`Fig. 1 shows an analysis system 1 for PCR. It has a sample preparation stage 2, a
`thermal cycling stage 3 for PCR, a waste outlet 4, and a real time detection stage 5 to
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`achieve qPCR.
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`Fig. 2 shows the thermal cycler 3. It has a planar two dimensional serpentine channel
`10 which is machined into a block which is segmented into three thermal zones 11,
`12, and 13 separated by 1mm air gaps 15. The three thermal zones are controlled to
`achieve the three individual temperature zones required for the PCR reaction. Each
`thermal section is controlled by continuous temperature sensing and a PID feedback
`control system. Circular tubing is laid into a channel in a block of Al material to
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`ensure biocompatibility for the reaction. The circular tubing gives a smooth internal
`surface and has no sharp edgesto restrict the reaction. This results in stable, spherical
`sample droplets. The tubing is embedded in the machined channel which results in
`high heat transfer from the block to the sample.
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`10
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`Fig. 3 shows the machined channel 17 which contains the tubing 10 and a refractive
`index matching solution. The machined channel 17 enables the introduction of the
`refractive index matching solution 16 as it is considerably deeper than the diameter of
`the tubing 10. The solution 16 covers the remainder of the channel above the tubing
`10 and results in high accuracy detection through the tubing. An example of the
`refractive index matching with the tubing is the use of a glycerine dilution solution.
`The device is planar in design, which provides the ability for continuous detection
`throughout the thermal cycling process. This enables real time quantitative detection
`(termed “qPCR”). The assembly may be sealed using optical quality glass or thin film
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`adhesive.
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`Fig 4 showsthermalfoil heaters 18 for heating the blocks of the thermal zones 1] and
`13. The low temperature thermal zone 12 has a water channel 19 for maintaining a
`uniform low temperature. The thermal sections are controlled by temperature sensor
`monitoring and a PID feedback control system.
`
`The inlet to the analysis system 1 is connected to the PCR preparation system 2.
`During sample preparation the double-stranded DNA sample is combined with two
`oligonucleotide primers. The sample is segmented into droplets which are wrapped in
`immiscible oil. The oil avoids cross contamination between the sequential droplets
`and carry-over contamination within the device. This configuration avoids the need to
`purge the system between different samples. A queue of different droplets from the
`preparation system may be passed through the thermal cycler 3 directly. The block
`and tubing are stationary so only the wrapped samples and oil solution move in the
`thermal cycle system. Each thermal zone 11, 12, and 13, including the Al block and
`the embedded tubing 10,
`is an isothermal zone. Each zone is controlled to be
`isothermal with respect to time. The velocity of the sample through the device is
`defined by the control of the velocity of the carrier fluid. This is controlled by an
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`external pumping system. The velocity may then be varied to control the residency
`time of the sample in each temperature zone 11-13.
`
`The sample passes to the PCR thermal cycler 3 within the carrier fluid and through an
`initial denaturation zone 11(a) before commencementof thermal cycling. The sample
`passes into the high thermal section 11(a) where it is first separated into single
`stranded DNA in a process called denaturation at a temperature Ty.
`
`The sample flows through the device at a steady controlled velocity to the second
`temperature T., where the hybridisation process takes place, during which the primers
`anneal to the complementary sequences of the sample. Finally, as the sample flows
`through the third and medium temperature zone, Tm, the polymerase process occurs
`when the primers are extended along the single strand of DNA with a thermostable
`enzyme. The sample undergoes the same thermal cycling and chemical reaction asit
`passes through N amplification cycles of the complete thermal device. This results in a
`maximum two-fold amplification after each cycle and a total amplification of
`I(1+B)%
`whereI is the initial product, E is the efficiency of the reaction and N is the number of
`
`cycles.
`
`Example
`Fluorescent probes are contained in each sample droplet. The fluorescence levelis
`detected in each droplet at each cycle. This quantitative analysis provides information
`on the specific concentration in the sample.
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`The three thermal zones are controlled to have temperatures as follows:
`
`Zone 11 95°C (Ty),
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`Zone 12 55°C (Tx),
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`Zone 13 72°C (wu).
`
`The prepared sample droplets, wrapped in the carrier fluid, enter the inlet to the
`thermal cycler at the controlled velocity. The sample then passes to the PCR thermal
`cycler 3 within the carrier fluid and through the initial denaturation zone 11(a) before
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`thermal cycling. The initial preheat is an extended zone to ensure the sample has
`denatured successfully before thermal cycling. The requirement for a preheat zone and
`the length of denaturation time required is dependent on the chemistry being used in
`the reaction. The samples passinto the high temperature zone, of approximately 95°C,
`where the sample is first separated into single stranded DNA in a process called
`denaturation. The sample then flowsto the low temperature zone 12, of approximately
`55°C, where the hybridisation process takes place, during which the primers anneal to
`the complementary sequences of the sample. Finally, as the sample flows through the
`third medium temperature zone 13, of approximately 72°C, the polymerase process
`occurs when the primers are extended along the single strand of DNA with a
`thermostable enzyme. The sample undergoes the same thermal cycling and chemical
`reaction as it passes through each thermal cycle of the serpentine pattern. The total
`number of cycles in the device is easily altered by an extension of block length and
`tubing. The system 1 has a total cycle number of 30 in this embodiment. The device
`may be extended to a longer thermal cycler or a combination of two thermal cyclers to
`achieve a greater cycle number.
`
`Referring to Fig. 5, in a cycler 20 there are two temperature zones 21 and 23,
`separated by an insulated air gap 24 to provide the correct temperatures zones
`necessary for the PCR reaction. The zone 21 is heated by a thermal foil heater 22, and
`the zone 23 is heated by natural convection from the top block 21. Again, the two
`zones including the embedded tubingare stationary throughout the reaction and hence
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`isothermal with respect to time.
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`The section temperatures are:
`
`Zone 21, 95°C (Ty),
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`Zone 23, 60°C (Ty),
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`The position of the lower block may be adjusted by increasing the insulation gap 24.
`This adjusts the temperature of the zone 23. The tubing protrudes below the edge of
`the bottom aluminium block whenit is laid in the channel, providing an inspection
`window. This is advantageous for the quantitative detection as it provides optical
`
`access to the tubing in two planes.
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`The prepared sample droplets, wrapped in the carrier fluid, enter the inlet to the
`thermal cycler at the controlled velocity. Different droplets are queued in the sample
`preparation device and flow into the thermal cycler in a queue of droplets. A
`suggested optimum configuration for droplet stability, and to avoid contamination,is a
`droplet diameter of approximately 400um, and a spacing of the same distance. The
`wrapped nature ofthe droplets enables continuous flow of alternative droplets without
`any contamination. This also removes therequirement to purge the system after each
`reaction. The sample then passes to the PCR thermal cycler within the carrier fluid
`and through an initial preheat zone before entering the thermal cycling. The preheat
`zone is necessary for some chemistry for activation and also to ensure the sample is
`fully denatured before the thermal cycling reaction begins. The preheat dwell length
`results in approximately 10 minutes preheat of the droplets at the higher temperature.
`The sample continues into the high temperature zone, of approximately 95°C, where
`the sampleis first separated into singlestranded DNA in a process called denaturation.
`The sample then flows through the device to the low temperature zone, of
`approximately 60°C, where the hybridisation process takes place, during which the
`primers anneal to the complementary sequences of the sample. Finally the polymerase
`process occurs when the primers are extended along the single strand of DNA with a
`thermostable enzyme. The sample undergoes the same thermal cycling and chemical
`reaction as it passes through cach thermal cycle of the complete device. The total
`number of cycles in the device is easily altered by an extension of block length and
`tubing. The system has a total cycle number of 50 in this embodiment. The device
`may be extended to a longer thermal cycler or a combination of two thermal cyclers to
`achieve a greater cycle number. Real time detection is applied to the device to provide
`quantitative polymerase chain reaction (qPCR). This involves the use of fluorescent
`probes such as SYBR Green or Taqman probes.
`
`For a larger cycle number, or an optional extension to the cycle number, the device
`"may be divided into two sections; one with n cycles and one with p cycles as shown in
`Fig. 6. The combination of the two devices enables a PCRtotal cycle numberof n, p
`or (n+p) depending on the tubing configuration and the heater control. Each block
`may be separately controlled to allow for individual use or combined use. Therefore,
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`the cycle numberof the device may be varied for greater versatility.
`°
`Case 1: Block 2 is thermally controlled and block 1
`is uncontrolled (no
`temperature input). The sample may then enter block 1, flow through the device
`and exit the thermal cycler at exit 2 following p cycles.
`Case 2: The two blocks are thermally controlled. Then the sample enters block
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`°
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`e
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`1, flows through the device and exits at exit 2 after (n+p) cycles.
`Case 3: The tubing is changed to use exit 1. The sample enters block 1, flows
`through block 1 and then exits at exit 1 following n cycles.
`
`Fig. 7 shows a photograph of segmented droplets flowing though the thermal cycler
`shown in Fig.2. The system allows for the quadruplicate amplification of a sample.
`The design avoids cross contamination between successive samples and the planar
`device allowsfull field detection during the thermal cycling.
`
`A suggested optimum configuration for droplet stability, and to avoid contamination,
`is a droplet diameter of approximately 400um and a spacing of the same distance.
`This configuration is suggested for the tubing used in this embodiment which has an
`internal diameter of 400 xm. The wrapped nature of the droplets enables continuous
`flow of alternative droplets without any contamination. This also removes the
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`requirement to purge the system.
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`Detection System
`Quantitative PCR, or Q-PCR,is a variant of the basic PCR technique. The present Q-
`PCR methods use fluorescent probes to monitor the amplification process as it
`progresses. The SYBR Green 1 dye is commonly used for the fluorescent detection of
`double-stranded DNA generated during PCR. The dye exhibits a peak excitation
`maximum at 497 nm and a peak emission maximum at 520 nm. Taqman probes may
`also be used which are a more target specific probe. The Taqman probes have
`different excitation and emission wavelengths but one example is the FAM labelled
`probe which has a peak excitation of 488nm and an emission of 520nm.
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`Through the analysis of the cycle-to-cycle change in fluorescence signal important
`information regarding the DNA sample maybe obtained. This is done by illuminating
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`the sample and detecting the resulting fluorescence. Different product concentration
`will demonstrate fluorescence amplification at difference cycle numbers. Through the
`analysis of the behaviour of the sample the characterisationis possible.
`
`Fig 8 demonstrates an example of a fluorescence amplification curve. This was
`demonstrated using a Taqman probe. There islittle change in the fluorescent signal
`after the first number of thermal cycles. This defines the baseline for the amplification
`plot. Fluorescence intensity levels above this baseline represent amplification of PCR
`product. A fluorescent threshold can be fixed above this baseline that defines the
`threshold cycle, or Ct, for each reaction. The threshold cycle is defined as the
`fractional cycle number at which the fluorescence passes above a fixed threshold. Ct is
`observed in the early exponential stages of amplification. The higherthe starting DNA
`template concentration, the sooner a significant increase in fluorescence is observed.
`Therefore the starting DNA concentration may be determined | by the real
`time
`fluorescent detection of the amplifying sample.
`
`Referring to Fig. 9 the detection system 5 comprises:
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`30,
`31,
`32,
`34,
`35,
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`36,
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`37,
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`light source;
`optics for focusing the incident light;
`filters for filtering the incident light;
`focusing optics for focusing fluorescence emitted by the sample;
`filter optics forfiltering the emitted fluorescence;
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`sensor electronics; and
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`processing electronics.
`
`The choice of light source is dependent on the remainder of the detection system but
`there are many options including filtered white light, specific wavelength laser or laser
`diode. Fibre optics may also be incorporated for light transport. The filtering is
`dependent on the light source and detection system but commercially available filter
`components may be used.
`
`If a detection indicator is used this will be provided in the sample preparation system.
`The use of SYBR green fluorescence is demonstrated in Fig. 10. This demonstrates
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`the use ofthe fluorescence for the amplification detection in the tubing used in the
`thermal cycler. The increase of fluorescence with increased sample amplification may
`be seen from the images.
`
`The detection sensor used is dependent on the field of view required and the
`illumination wavelength chosen. Detector options include CCD, CMOS, photodiode
`
`and photomultipliers
`
`As the choice and combination of elements chosen are dependent on the overall
`detection system design and implementation a number of systems are outlined below.
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`b.
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`Cc.
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`to
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`In summary, the system amplifies a DNA sample in a polymerase chain reaction
`comprising the following steps:
`a.
`Introducing spherical droplets of sample contained in an immiscible carrier
`fluid to the thermal cycler
`Passing the sample through circular tubing to provide a smooth internal surface
`and no sharp edges allowing for most stable, spherical droplets.
`Controlling the three thermal zones for successful reaction
`Controlling the carrier fluid velocity by an external pumping system to achieve
`the target residency times in the thermal zones
`thermally controlled zones
`Passing the sample through the (three)
`successfully achieve DNA sample amplification.
`Repeating step e the necessary numberoftimes to achieve the desired sample
`amplification
`The quantitative detection of the amplification process.
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`e.
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`f.
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`g.
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`The device is planar in design, enabling continuous quantitative PCR and multiple
`levels for any desired level of parallelism.
`
`The channel design enables manipulation for refractive index matching within the
`device for high qualitydetection. Also, the channel design results in high heat transfer
`efficiency by embedding the tubing within the channel. As the droplets are wrapped in
`an immiscible oil, sequential sample contamination or cross-over contamination
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`within the device is avoided.
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`Each thermal zone is controlled by continuous temperature sensing and a PID
`feedback control system. In the embodiments there are 30 cycles and the particular
`temperatures defined achieved successful denaturation, annealing and hybridisation
`
`reactions.
`
`Fig. 11 shows a full field detection system 40 which allows real time detection
`without any moving parts. The system 40 comprises an illuminator 41 and lenses 42
`illuminating the cycler 20, and a filter 43 for impingement of emission onto a detector
`44. This enables global measurement of the full thermal cycler 20 or the specific
`measurement at localised points along the thermal cycler. This is demonstrated in a
`view of the detection system in Fig.12, in which individual measurements are taken
`for a linear series of points P. The detection measurement point in each cycle is
`dependent on the fluorescent probes used for qPCR. Some probes fluoresce at any
`point in the reaction whilst others only fluoresce at the annealing/extension stage.
`
`Figs. 13 and 14 are scanning detection systems for two alternative configurations.
`These systems also allow real time detection by moving the relative positions of the
`detection system and the thermal cycler. In the system of Fig. 13 a positioning stage
`45 moves the cycler 20, whereas in the system of Fig. 14 a positioning system 46
`
`movesthe illuminator 41 and the detector 44.
`
`Whilst the above describes a single thermal cycler, the same movement may be
`applied to multiple thermal cyclers by simple adding detection and illumination
`points. The angle of illumination and detection, or orientation of the optical fibers,
`may alsobe altered to facilitate multiple thermal cycler real time detection.
`
`Figs. 15 and 16 show another quantitative detection configuration, 50. Optical fibers
`are placed at each loop of the tubing in the block. A set of fibers 51 are placed
`vertically below the thermal cycler 20 and the fiber ends are perpendicular to the
`tubing. This bundle is attached to a light source 52 which excites the fluorescent
`particles contained in the droplets as they pass the fiber ends. Another bundle of
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`fibers, 53, are placed horizontally at the front of the thermal cycler with the fiber ends
`perpendicular to the tubing. This fiber bundle 53 collects the emitted light from the
`fluorescent particles in the droplet as they pass the fiber ends. The other end of the
`fiber bundle is detected by a camera 54 for detection of the droplet fluorescence. An
`example of a detected fiber array is shown in Fig. 19. The continuous acquisition of
`the fiber bundle image provides the quantitative detection of droplet fluorescence at
`each individual fiber position. A filter wheel 55 may be used for alternative detection
`of different
`fluorescent probes. For example,
`there are probes with excitation
`wavelengths which are appropriate to use the same excitation source. However,
`different detection bandwidths will enable the detection of different probes
`individually. A filter wheel, a spectrometer or an alternative method of wavelength
`separation will successfully achieve this goal.
`
`Referring to Figs. 17 and 18, the throughput may also be increased by operating a
`bank 60 of thermal cyclers 61 -64 in parallel. A planar system can achieve series
`sampling of w samples andthe parallel configuration can contain y parallel levels. The
`continuous multi layered thermal cycler 60 resulis in the product (w x y) sample
`capability. Such a PCR test of the whole genome of any living form, including the
`human, could be addressed, which would have applications beyond diagnosis, in many
`fields of pure and applied science. Fig. 18 shows a part of a cross-section through the
`cycler, in the direction of the arrows XIX-XIX of Fig. 17. This shows the blocks 66
`and 67 and the tubing 68. The tubing where it is exposed provides an array of
`inspection windows69.
`
`All detection techniques may be applied to a multiple thermal cycler system for
`quantitative detection. The protruding tubing array for a multiple thermal cycler
`system, as shown in Fig. 17, can be seen in Fig. 20. Fig. 20 shows inspection windows
`69 for a full 4 x 50 cycle system and Fig. 21 showsa detailed view of a small array of
`inspection windows 69 more closely. The measurement points may be illuminated by
`full field illumination or point illumination by high speed scanning or fiber optics. The
`detection may be carried out the same way, by full field, scanning or simultaneous
`point detection.
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`The invention improves upon current well based technology for the quantitative
`amplification of nucleic acids. In that technology the reagents and sample are loaded
`into a multi-well plate that is then thermally cycled, with each cycle approximately
`doubling the
`target
`number. The
`resulting
`fluorescent
`intensity increases
`proportionally so that, with calibration, the amplification can be monitored with time.
`Standard techniques are then available to calculate the number of targets initially
`present, whichis the required output for qPCR.
`
`In this invention the data set is again three dimensional, monitoring over the x, y plane
`and with time. The advantage over the well plate is that when plate amplification is
`complete the plate must be cleaned or disposed with, and a new plate primed and
`loaded onto the thermal cycling plate.
`In the invention the data is provided
`continuously for as long as droplets are fed into the thermal cycler. Because there is
`no carryover the system can be used continuously.
`
`The geometric arrangement of the capillary tubing in the thermal cycler allows for
`serial processing, a procession of droplets, parallel processing and an array of closely
`packed capillary tubes. The rate of production of datais dependant upon the following
`factors:
`
`1.
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`2
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`3.
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`4
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`The droplet length (c. 0.5mm)
`
`The droplet spacing (c. 1.5mm)
`
`The droplet velocity (c. 1mm/s)
`
`The numberofparallel lines.
`
`Typical values are given in brackets. The possible degree of parallelism is very great.
`Using 0.8mm outside diameter tubing, 100 parallel lines could only take up 80mm of
`transverse width.
`
`following the time when the first droplets have completed
`Using data above,
`amplification, the system will produce an amplification curve every 0.02 seconds, or
`180,000 curves per hour. This is far greater than anything available. Typical high-end
`systems at present with 384 well plates would need to process 469 plates to achieve
`the samedataset.
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`The following are some applications of the invention:
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`Rare target detection
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`Multiple assay analysis
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`Multiple sample/assay analysis
`