U.S. patent application number 12/093132 was filed with the patent office on 2008-11-13 for microfluidic analysis system.
This patent application is currently assigned to STOKES BIO LIMITED. Invention is credited to Tara Dalton, Mark Davies.
Application Number | 20080280331 12/093132 |
Document ID | / |
Family ID | 37946144 |
Filed Date | 2008-11-13 |
United States Patent
Application |
20080280331 |
Kind Code |
A1 |
Davies; Mark ; et
al. |
November 13, 2008 |
Microfluidic Analysis System
Abstract
A thermal cycling device (3) device a number of fixed thermal
zones (11, 12, 13) and a fixed conduit (10) passing through the
thermal zones. A controller maintains each thermal zone including
its section of conduit (10) at a constant temperature. A series of
droplets flows through the conduit (10) so that each 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 number of 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 direction to detect
radiation from a plurality of cycles in a pass.
Inventors: |
Davies; Mark; (Limerick,
IE) ; Dalton; Tara; (Limerick, IE) |
Correspondence
Address: |
COOLEY GODWARD KRONISH LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Assignee: |
STOKES BIO LIMITED
Shannon Arms, Limerick
IE
|
Family ID: |
37946144 |
Appl. No.: |
12/093132 |
Filed: |
February 7, 2007 |
PCT Filed: |
February 7, 2007 |
PCT NO: |
PCT/IE2007/000015 |
371 Date: |
May 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60765670 |
Feb 7, 2006 |
|
|
|
Current U.S.
Class: |
435/91.2 ;
435/287.2 |
Current CPC
Class: |
B01L 2300/185 20130101;
B01L 2300/0867 20130101; G01N 2201/0833 20130101; B01L 3/502715
20130101; B01L 2400/0487 20130101; C12Q 1/686 20130101; B01L
2300/0838 20130101; B01L 3/502784 20130101; B01L 2300/0654
20130101; B01L 7/525 20130101; G01N 2201/0826 20130101; B01L 3/5027
20130101; B01L 2200/0673 20130101; B01L 2300/18 20130101; B01L
2300/1822 20130101; G01N 21/6428 20130101; B01L 2300/0627
20130101 |
Class at
Publication: |
435/91.2 ;
435/287.2 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C12M 1/02 20060101 C12M001/02 |
Claims
1-23. (canceled)
24. A microfluidic analysis system 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 the conduit
at a constant temperature, a pumping system 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.
25. The microfluidic analysis system of claim 24, wherein the
conduit comprises a channel with a circular cross-section.
26. The microfluidic analysis system of claim 25, wherein the
conduit comprises a channel and a capillary tube inserted into the
channel.
27. The microfluidic analysis system of claim 26, wherein the
capillary has a circular cross-section.
28. The microfluidic analysis system of claim 26, wherein the
channel and capillary are configured to receive a refractive
index-matching liquid in the channel and at least partly
surrounding the capillary.
29. The microfluidic analysis system as claimed in claim 28,
wherein the channel has a depth greater than that of the
capillary.
30. The microfluidic analysis system of claim 26, wherein the
conduit is in a single plane.
31. The microfluidic analysis system of claim 26, wherein the
thermal zones are mutually thermally insulated.
32. The microfluidic analysis system of claim 26, wherein the
detection system comprises optics for focusing incident light
radiation.
33. The microfluidic analysis system of claim 26, wherein the
detection system comprises optics for filtering incident
radiation.
34. The microfluidic analysis system of claim 26, wherein the
detection system comprises optics for filtering emitted
radiation.
35. The microfluidic analysis system of claim 26, wherein the
detection system performs scans along a direction to detect
radiation from a plurality of cycles in a pass.
36. The microfluidic analysis system of claim 26, wherein the
detection system performs simultaneous detection of emitted light
from a plurality of cycles.
37. The microfluidic analysis system of claim 26, wherein there is
an air gap between adjacent thermal zones.
38. The microfluidic analysis system of claim 26, wherein said air
gap is adjustable.
39. The microfluidic analysis system of claim 26, wherein the
conduit passes through a hot thermal zone for a length before a
first cycle, providing a denaturation zone.
40. The microfluidic analysis system of claim 26, wherein the
detection system comprises a plurality of optic fibers for point
illumination of each of the plurality of cycles.
41. The microfluidic analysis system of claim 40, wherein the optic
fibers are placed at each loop of the capillary tube.
42. The microfluidic analysis system of claim 26, wherein the
detection system comprises a plurality of optic fibers for point
detection of each of the plurality of cycles.
43. The microfluidic analysis system of claim 42, wherein the optic
fibers are placed at each loop of the capillary tube.
44. The microfluidic analysis system of claim 26, wherein the
detection system comprises a rotating filter for cyclic filtering
of incident or emitted light.
45. The microfluidic analysis system of claim 26, wherein the
conduit is in a serpentine pattern of multiple folds, each fold
extending through a plurality of thermal zones.
46. The microfluidic analysis system of claim 26, wherein the
microfluidic analysis system comprises a plurality of thermal
cycling devices arranged in parallel.
47. The microfluidic analysis system of claim 26, wherein the
detection system performs simultaneous detection of emitted light
from a plurality of cycles from a plurality of thermal cycling
devices.
48. The microfluidic analysis system of claim 26, wherein the
microfluidic analysis system comprises two or more of the thermal
cyclic devices, allowing parallel processing of droplet trains.
49. The microfluidic analysis system of claim 26, further
comprising a pumping system maintaining the flow of droplets
through the conduit.
50. A method of performing a nucleic acid amplification reaction,
the method comprising: a) providing a biological sample; b)
segmenting the sample into droplets which are wrapped in an
immiscible oil; c) directing the flow of the droplets in oil though
a conduit passing through a plurality of thermal zones under
conditions sufficient for the amplification reaction to occur; and
d) detecting an output of the amplification reaction in one or more
droplets.
51. The method of claim 50, wherein the conduit comprises a
capillary tube inserted into the channel.
52. The method of claim 50, wherein said detecting is performed
throughout multiple cycles of the amplification reaction.
53. The method of claim 50, wherein the plurality of zones
comprises at least three different thermal zones.
54. The method of claim 50, wherein the detecting is performed by
detecting fluorescence signal emitted from the droplets.
55. The method of claim 50, wherein the detecting is performed
using a plurality of optic fibers for light transport.
56. The method of claim 50, wherein the droplet length is about 0.5
mm.
57. The method of claim 50, wherein the droplet diameter is about
400 .mu.m.
58. The method of claim 49, wherein the droplet spacing is about
1.5 mm.
59. The method of claim 49, wherein the droplet velocity is about 1
mm/s.
60. A device adapted to perform the method of claim 46.
61. A method of performing a nucleic acid amplification reaction,
the method comprising: a) creating a flow of spherical droplets of
sample contained in an immiscible carrier fluid; b) passing the
flow through a circular tubing in a thermal cycler; c) controlling
three thermal zones in said thermal cycler; d) controlling the
carrier fluid velocity by an external pumping system; e) passing
the sample through the thermal zones allowing the nucleic acid
amplification reaction to occur in the droplets; f) optionally,
repeating step e); and g) detecting of the amplification
reaction.
62. The method of claim 61, wherein the carrier fluid is an
oil.
63. The method of claim 61, wherein the amplification reaction is a
polymerase chain reaction.
Description
FIELD OF THE INVENTION
[0001] 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).
PRIOR ART DISCUSSION
[0002] Conventionally, nucleic acid amplification has involved
providing an array of samples in an assay plate and thermally
cycling the plate reaction vessel. This, however, involves the
laborious task of loading the samples and preparing a fresh assay
well plate. It is known to provide a thermal cycler for nucleic
acid amplification, and U.S. Pat. No. 5,270,183, WO2005/075683,
U.S. Pat. No. 6,033,880, and U.S. Pat. No. 6,814,934 describe
thermal cycler analysis systems.
[0003] The prior systems suffer from being complex, both in
physical and control terms. For example, in the system of U.S. Pat.
No. 6,033,880 it is necessary to rotate heat exchangers into
desired positions, and in the system of U.S. Pat. No. 6,814,934 it
is necessary to heat and cool a reaction vessel.
[0004] 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.
SUMMARY OF THE INVENTION
[0005] According to the invention, there is provided a microfluidic
analysis system 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.
[0006] In one embodiment, the conduit is in a single plane.
[0007] In one embodiment, the conduit comprises a channel.
[0008] In one embodiment, the thermal zones are mutually thermally
insulated.
[0009] In one embodiment, the flow conduit comprises a channel and
a capillary tube inserted into the channel.
[0010] In one embodiment, the capillary has a circular
cross-section.
[0011] In one embodiment, the channel and capillary are configured
to receive a refractive index-matching liquid in the channel and at
least partly surrounding the capillary.
[0012] In another embodiment, the channel has a depth greater than
that of the capillary.
[0013] In one embodiment, the detection system comprises optics for
focusing incident light radiation.
[0014] In one embodiment, the detection system comprises optics for
filtering incident radiation.
[0015] In one embodiment, the detection system comprises optics for
filtering emitted radiation.
[0016] In another embodiment, the detection system performs scans
along a direction to detect radiation from a plurality of cycles in
a pass.
[0017] In one embodiment, the detection system performs
simultaneous detection of emitted light from a plurality of
cycles.
[0018] In one embodiment, there is an air gap between adjacent
thermal zones.
[0019] In one embodiment, said air gap is adjustable.
[0020] In one embodiment, the flow conduit passes through a hot
thermal zone for a length before a first cycle, providing a
denaturation zone.
[0021] In another embodiment, the detection system comprises a
plurality of optic fibres for point illumination of each of a
plurality of cycles.
[0022] In one embodiment, the detection system comprises a
plurality of optic fibres for point detection of each of a
plurality of cycles.
[0023] In one embodiment, the detection system comprises a rotating
filter for cyclic filtering of incident or emitted light.
[0024] In one embodiment, the conduit is in a serpentine pattern of
multiple folds, each fold extending through a plurality of thermal
zones.
[0025] In a further embodiment, the system comprises a plurality of
thermal cycling devices arranged in parallel.
[0026] In one embodiment, the thermal cycling devices are
interconnected to form a physical unit.
[0027] In one embodiment, the detection system performs
simultaneous detection of emitted light from a plurality of cycles
from a plurality of thermal cycling devices.
DETAILED DESCRIPTION OF THE INVENTION
Brief Description of the Drawings
[0028] 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:
[0029] FIG. 1 is a block diagram of an analysis system of the
invention;
[0030] FIG. 2 is a plan view of a thermal cycler of the system
having three thermal zones,
[0031] FIG. 3 is a vertical cross section, and
[0032] FIG. 4 is an end view of the thermal cycler;
[0033] FIG. 5 is a perspective view of an alternative thermal
cycler, having only two thermal zones;
[0034] FIG. 6 is a diagram showing an arrangement with two exits,
providing a choice of n cycles or n+p cycles;
[0035] FIG. 7 is a photograph showing droplets flowing in a number
of cycles of the thermal cycler having three thermal zones;
[0036] FIG. 8 is a plot illustrating fluorescence characteristics
for detection;
[0037] FIG. 9 is a block diagram of a detection system of the
analysis system;
[0038] FIG. 10 is a pair of photographs, showing negative and
positive fluorescence detection, from left to right;
[0039] FIGS. 11 to 14 are diagrams showing alternative detection
arrangements;
[0040] FIGS. 15 and 16 are perspective views showing image capture
via optic fibres;
[0041] FIG. 17 is a perspective view of a three-dimensional cycler
for parallel amplification, and FIG. 18 is a cross-sectional plan
view of this cycler;
[0042] FIG. 19 is a sample image of part of a detector array
captured from the thermal cycler of FIG. 17; and
[0043] FIGS. 20 and 21 are views of arrays of windows of the cycler
of FIG. 17.
DESCRIPTION OF THE EMBODIMENTS
[0044] An analysis system of the invention is based on
microfluidics technology. Microfluidic devices themselves have
dimensions ranging from several millimetres to micrometers.
Typically one of the components or dimensions of the device, such
as a channel in the device, is of the order of micrometers.
[0045] The polymerase chain reaction, or PCR, is a powerful
technique used to amplify low concentrations of specific DNA
sequences to levels which may be detected. PCR can be used to
achieve a billionfold increase in target sequence copy number by
thermally cycling a specific chemical mix. This makes the PCR
method extremely sensitive as it can detect a single DNA molecule
in a sample.
[0046] 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 achieve qPCR.
[0047] 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 1 mm 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 ensure biocompatibility for the reaction. The circular
tubing gives a smooth internal surface and has no sharp edges to
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.
[0048] 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
adhesive.
[0049] FIG. 4 shows thermal foil heaters 18 for heating the blocks
of the thermal zones 11 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.
[0050] 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 external pumping system. The velocity may then be
varied to control the residency time of the sample in each
temperature zone 11-13.
[0051] The sample passes to the PCR thermal cycler 3 within the
carrier fluid and through an initial denaturation zone 11(a) before
commencement of 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
TH.
[0052] The sample flows through the device at a steady controlled
velocity to the second temperature T.sub.L, 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, T.sub.M, 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 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(11+E).sup.N
where I is the initial product, E is the efficiency of the reaction
and N is the number of cycles.
EXAMPLE
[0053] Fluorescent probes are contained in each sample droplet. The
fluorescence level is detected in each droplet at each cycle. This
quantitative analysis provides information on the specific
concentration in the sample.
[0054] The three thermal zones are controlled to have temperatures
as follows: [0055] Zone 11 95.degree. C. (T.sub.H), [0056] Zone 12
55.degree. C. (T.sub.L), [0057] Zone 13 72.degree. C. (.sub.M).
[0058] 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 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 pass into the high temperature zone,
of approximately 95.degree. C., where the sample is first separated
into single stranded DNA in a process called denaturation. The
sample then flows to the low temperature zone 12, of approximately
55.degree. 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.degree. 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.
[0059] 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 tubing are stationary
throughout the reaction and hence isothermal with respect to
time.
[0060] The section temperatures are: [0061] Zone 21, 95.degree. C.
(T.sub.H), [0062] Zone 23, 60.degree. C. (T.sub.L),
[0063] 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 when it 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.
[0064] 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 400 .mu.m,
and a spacing of the same distance. The wrapped nature of the
droplets enables continuous flow of alternative droplets without
any contamination. This also removes the requirement 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.degree. C., where the sample is first separated into single
stranded DNA in a process called denaturation. The sample then
flows through the device to the low temperature zone, of
approximately 60.degree. 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 each 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.
[0065] 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 PCR total cycle number of 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, the cycle number of the device may be
varied for greater versatility. [0066] 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. [0067] Case 2: The two
blocks are thermally controlled. Then the sample enters block 1,
flows through the device and exits at exit 2 after (n+p) cycles.
[0068] 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.
[0069] 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 allows full field detection during the thermal cycling.
[0070] A suggested optimum configuration for droplet stability, and
to avoid contamination, is a droplet diameter of approximately 400
.mu.m 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 .mu.m. The wrapped nature of the droplets
enables continuous flow of alternative droplets without any
contamination. This also removes the requirement to purge the
system.
Detection System
[0071] 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 488 nm and an emission of 520 nm.
[0072] Through the analysis of the cycle-to-cycle change in
fluorescence signal important information regarding the DNA sample
may be obtained. This is done by illuminating 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 characterisation is possible.
[0073] FIG. 8 demonstrates an example of a fluorescence
amplification curve. This was demonstrated using a Taqman probe.
There is little 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 higher the 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.
[0074] Referring to FIG. 9 the detection system 5 comprises:
[0075] 30, light source;
[0076] 31, optics for focusing the incident light;
[0077] 32, filters for filtering the incident light;
[0078] 34, focusing optics for focusing fluorescence emitted by the
sample;
[0079] 35, filter optics for filtering the emitted
fluorescence;
[0080] 36, sensor electronics; and
[0081] 37, processing electronics.
[0082] 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.
[0083] 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 the use of the
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.
[0084] 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
[0085] 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.
[0086] In summary, the system amplifies a DNA sample in a
polymerase chain reaction comprising the following steps: [0087] a.
Introducing spherical droplets of sample contained in an immiscible
carrier fluid to the thermal cycler [0088] b. Passing the sample
through circular tubing to provide a smooth internal surface and no
sharp edges allowing for most stable, spherical droplets. [0089] c.
Controlling the three thermal zones for successful reaction [0090]
d. Controlling the carrier fluid velocity by an external pumping
system to achieve the target residency times in the thermal zones
[0091] e. Passing the sample through the (three) thermally
controlled zones to successfully achieve DNA sample amplification.
[0092] f. Repeating step e the necessary number of times to achieve
the desired sample amplification [0093] g. The quantitative
detection of the amplification process.
[0094] The device is planar in design, enabling continuous
quantitative PCR and multiple levels for any desired level of
parallelism.
[0095] The channel design enables manipulation for refractive index
matching within the device for high quality detection. 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 within the device is avoided.
[0096] 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.
[0097] 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.
[0098] 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 moves the illuminator 41 and the detector
44.
[0099] 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 also be
altered to facilitate multiple thermal cycler real time
detection.
[0100] 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 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.
[0101] 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
and the parallel configuration can contain y parallel levels. The
continuous multi layered thermal cycler 60 results 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 windows 69.
[0102] 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.times.50 cycle system and FIG. 21 shows a 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.
[0103] 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, which is the required output for qPCR.
[0104] 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.
[0105] 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 data is dependant upon
the following factors: [0106] 1. The droplet length (c. 0.5 mm)
[0107] 2. The droplet spacing (c. 1.5 mm) [0108] 3. The droplet
velocity (c. 1 mm/s) [0109] 4. The number of parallel lines.
[0110] Typical values are given in brackets. The possible degree of
parallelism is very great. Using 0.8 mm outside diameter tubing,
100 parallel lines could only take up 80 mm of transverse
width.
[0111] Using data above, following the time when the first droplets
have completed 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 same data set.
[0112] The following are some applications of the invention: [0113]
Rare target detection [0114] Multiple assay analysis [0115]
Multiple sample/assay analysis [0116] End point qualitative
detection
[0117] The invention is not limited to the embodiments described
but may be varied in construction and detail. For example, the
overall pattern of the flow conduit may not be serpentine.
Alternatively, the thermal zones may be thermally controlled by
flow of hot water rather than directly by heaters in the hotter
zones. Also, a thermoelectric cooler may be used for one or more
cooler zones. Further, the flow conduit may not be in a repeated
pattern. Instead, it may be straight or curved, passing through a
plurality of sets of thermal zones to provide cycles. Also, the
detection may not involve fluorescence detection. It may
alternatively involve detection of other parts of the
electromagnetic spectrum such as change of light polarisation,
depending on the desired detection technology.
* * * * *