U.S. patent application number 14/110635 was filed with the patent office on 2014-05-01 for biological detection system and method of use.
This patent application is currently assigned to STOKES BIO LIMITED. The applicant listed for this patent is Mauro Aguanno, Brian Barrett, Brian Chawke, Kieran Curran, Damian Curtin, Tara Dalton, Mark Davies, Xiaona Hou, David Kinahan, Damien King, Mark Korenke, Conor McCarthy, David McGuire, Michael Sayers, Noel Sirr, Ryan J. Talbot. Invention is credited to Mauro Aguanno, Brian Barrett, Brian Chawke, Kieran Curran, Damian Curtin, Tara Dalton, Mark Davies, Xiaona Hou, David Kinahan, Damien King, Mark Korenke, Conor McCarthy, David McGuire, Michael Sayers, Noel Sirr, Ryan J. Talbot.
Application Number | 20140120604 14/110635 |
Document ID | / |
Family ID | 46052862 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120604 |
Kind Code |
A1 |
Aguanno; Mauro ; et
al. |
May 1, 2014 |
Biological Detection System and Method of Use
Abstract
Provided herein is a biological detection system and method of
use wherein the biological detection system comprises at least one
mixer or liquid bridge for combining at least two liquid droplets
and an error correction system for detecting whether or not proper
mixing or combining of the two component droplets have
occurred.
Inventors: |
Aguanno; Mauro; (Singapore,
SG) ; Barrett; Brian; (Cashel, IE) ; Chawke;
Brian; (Askeaton, IE) ; Curran; Kieran;
(Limerick, IE) ; Curtin; Damian; (Tralee, IE)
; Dalton; Tara; (Limerick, IE) ; Davies; Mark;
(Limerick, IE) ; Hou; Xiaona; (Annacotty, IE)
; Kinahan; David; (Dublin, IE) ; King; Damien;
(Terenure, IE) ; Korenke; Mark; (Richmond, VA)
; McCarthy; Conor; (Ballineen, IE) ; McGuire;
David; (Raheen, IE) ; Sayers; Michael;
(Tralee, IE) ; Sirr; Noel; (Mungret, IE) ;
Talbot; Ryan J.; (Stamford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aguanno; Mauro
Barrett; Brian
Chawke; Brian
Curran; Kieran
Curtin; Damian
Dalton; Tara
Davies; Mark
Hou; Xiaona
Kinahan; David
King; Damien
Korenke; Mark
McCarthy; Conor
McGuire; David
Sayers; Michael
Sirr; Noel
Talbot; Ryan J. |
Singapore
Cashel
Askeaton
Limerick
Tralee
Limerick
Limerick
Annacotty
Dublin
Terenure
Richmond
Ballineen
Raheen
Tralee
Mungret
Stamford |
VA
CT |
SG
IE
IE
IE
IE
IE
IE
IE
IE
IE
US
IE
IE
IE
IE
US |
|
|
Assignee: |
STOKES BIO LIMITED
Limerick
IE
|
Family ID: |
46052862 |
Appl. No.: |
14/110635 |
Filed: |
April 6, 2012 |
PCT Filed: |
April 6, 2012 |
PCT NO: |
PCT/US2012/032554 |
371 Date: |
December 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61473551 |
Apr 8, 2011 |
|
|
|
Current U.S.
Class: |
435/287.2 |
Current CPC
Class: |
G01N 2021/6441 20130101;
B01L 3/502715 20130101; G01N 2021/6439 20130101; C12Q 1/686
20130101; G01N 35/00623 20130101; B01L 2300/0867 20130101; B01L
2200/143 20130101; B01F 13/0071 20130101; G01N 21/6428 20130101;
B01L 3/502784 20130101; G01N 2201/062 20130101; B01L 7/52 20130101;
B01L 7/525 20130101; B01L 2300/0654 20130101; B01F 13/0005
20130101; B01F 15/00214 20130101; G01N 2035/00465 20130101; B01L
2300/06 20130101 |
Class at
Publication: |
435/287.2 |
International
Class: |
G01N 21/64 20060101
G01N021/64; B01F 13/00 20060101 B01F013/00 |
Claims
1. A system for detecting a biological target comprising: a first
liquid input for providing a first liquid; a second liquid input
for providing a second liquid; at least one mixer in fluid
communication with the first liquid input and the second liquid
input, wherein the mixer is configured to segment the first liquid
into at least one first liquid droplet and the second liquid into
at least one second liquid droplet and to create a mixed droplet
from the first liquid droplet and the second liquid droplet; and at
least one detector, wherein the detector is configured to detect
the presence or absence of the at least one first liquid droplet
and the at least one second liquid droplet in the mixed
droplet.
2. The system of claim 1 comprising a third liquid input in fluid
communication with the mixer wherein the mixer is configured
segments the third liquid into at least one third liquid
droplet.
3. The system of claim 2 wherein the mixer mixes the first liquid
droplet, the second liquid droplet, and the third liquid droplet to
form the mixed droplet.
4. The system of claim 3 wherein the first liquid comprises a first
fluorescent dye, the second liquid comprising a second fluorescent
dye, and the third liquid comprising a third fluorescent dye, each
of the first, second, and third fluorescent dyes emitting
fluorescence upon excitation wherein the fluorescence emitted from
each is spectrally resolvable from the fluorescence emitted from
the others.
5. The system of claim 1 wherein the system comprises a
thermocycler.
6. (canceled)
7. The system of claim 1 wherein the system comprises a fluid
charging apparatus.
8. The system of claim 7 wherein the fluid charging apparatus is a
static bar.
9. The system of claim 8 wherein the static bar comprises an
electrode.
10.-23. (canceled)
24. A system for detecting proper mixing of at least three liquids,
comprising: a conduit system comprising at least a main conduit for
carrying a mixed sample droplet; a mixed sample droplet in the main
conduit and comprising a first liquid, a second liquid, and a third
liquid, the first liquid comprising a first fluorescent dye, the
second liquid comprising a second fluorescent dye, and the third
liquid comprising a third fluorescent dye, each of the first,
second, and third fluorescent dyes emitting fluorescence upon
excitation wherein the fluorescence emitted from each is spectrally
resolvable from the fluorescence emitted from the others; and an
optical detection system comprising an excitation source for
irradiating the mixed sample droplet in the main conduit and a
detector for detecting emissions from the mixed sample droplet to
determine whether each of the first, second, and third fluorescent
dyes is present in the mixed sample droplet.
25. The system of claim 24, wherein the mixed sample droplet is
encompassed by a carrier fluid that is substantially immiscible
with the mixed sample droplet.
26. The system of claim 24, wherein the conduit system further
comprises a first auxiliary conduit containing therein the first
liquid, a second auxiliary conduit containing therein the second
liquid, and a third auxiliary conduit containing therein the third
liquid; wherein the first, second, and third auxiliary conduits
intersect with the main conduit at a junction configured to form
the mixed sample droplet.
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. The system of claim 24, wherein the detector comprises a first
detector for detecting emission from the first fluorescent dye, a
second detector for detecting emission from the second fluorescent
dye, and a third detector for detecting emission from the third
fluorescent dye.
32. The system of claim 31, wherein the first detector, the second
detector, and the third detector each comprise a camera.
33. (canceled)
34. (canceled)
35. The system of claim 24, further comprising a signal processing
system for monitoring information generated by the detector and
determining whether proper mixing of the first liquid, the second
liquid, and the third liquid has occurred in the mixed sample
droplet.
36. The system of claim 24, wherein the first and second dyes
comprise a passive reference dye and the third dye comprises a
reporter dye.
37. The system of claim 24, further comprising a train of droplets
including the mixed sample droplet, in the main conduit, the train
of droplets comprising carriages each comprising a plurality of
spaced apart droplets, wherein a first spacing is provided between
adjacent droplets within each carriage, and the carriages are
spaced apart from adjacent carriages by a second spacing that
differs from the first spacing.
38. The system of claim 24 further comprising: a conduit support
board that holds the main conduit; and an excitation source support
board that holds the excitation source; wherein the conduit support
board and the excitation source support board are disposed parallel
to each other such that the main conduit and the excitation source
are aligned with each other and at least a portion of the main
conduit is exposed to radiation emitted from the excitation
source.
39. The system of claim 38, wherein the conduit support board holds
a plurality of main conduits and the excitation source support
board holds a plurality of excitation sources.
40. The system of claim 24, further comprising a plurality of
conduit support boards, a plurality of excitation boards, and a
housing in which the plurality of conduit support boards and the
plurality of excitation support boards are retained.
41. The system of claim 24, further comprising a fiber optic cable
connected to the detector and configured to receive fluorescent
emissions from the main conduit.
42.-44. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to detection of biological
components.
BACKGROUND OF THE INVENTION
[0002] Polymerase chain reaction (PCR) systems or thermocyclers
typically include a sample block, a heated cover, and heating and
cooling elements. These components are then controlled or monitored
by an onboard control system. Real-time PCR systems or
thermocyclers generally also include an optical detection system
for detecting electromagnetic radiation emitted by one or more
probes attached to a nucleic acid sample. Real-time PCR systems can
additionally include an external computer or control system for
controlling and monitoring system components and analyzing data
produced by the optical detection system.
[0003] Current standard PCR systems and real-time PCR systems are
well-based systems. These systems receive samples in a sample
support device that includes a plurality of wells. The samples are
prepared or mixed with reagents before being loaded into the PCR
system. The PCR system then cycles the temperatures of the samples
in the wells. Additionally, real-time PCR systems monitor the
samples in the wells for electromagnetic or fluorescent
emissions.
[0004] As the uses and need for genetic and genomic information
have increased, so has the need for PCR amplification and analysis.
In particular, it has become increasingly important to improve the
throughput of PCR systems. Although each generation of PCR systems
can cycle the temperatures of samples slightly faster, the
technology has not kept up with the performance improvements of
other genetic and genomic analysis instruments. For example,
deoxyribonucleic acid (DNA) sequencing instruments are advancing to
the point where sample preparation and PCR amplification are the
most limiting steps in terms of time and cost for sequencing
experiments.
[0005] In addition, the reliance of current PCR systems on
well-based technology limits the overall throughput of these
systems. Current systems can cycle the temperatures of samples in
approximately 40 minutes. Using the largest well-based sample
support device with 384 wells, therefore, produces a maximum
overall sample throughput of about 500 samples per hour. Further,
current PCR systems receive samples already prepared or mixed in
the sample support device. Therefore these systems are dependent on
the time consuming and sometimes manual step of well-based sample
preparation.
SUMMARY OF THE INVENTION
[0006] A system for detecting a biological target comprising a
first liquid input for providing a first liquid, a second liquid
input for providing a second liquid, at least one mixer in fluid
communication with the first liquid input and the second liquid
input, wherein the mixer is configured to segment the first liquid
into at least one first liquid droplet and the second liquid into
at least one second liquid droplet and to create a mixed droplet
from the first liquid droplet and the second liquid droplet, and at
least one detector, wherein the detector is configured to detect
the presence or absence of the at least one first liquid droplet
and the at least one second liquid droplet in the mixed droplet. In
some embodiments the system may comprise a third liquid input in
fluid communication with the mixer wherein the mixer is configured
segments the third liquid into at least one third liquid droplet.
The system may then mix the first liquid droplet, the second liquid
droplet, and the third liquid droplet to form the mixed droplet.
Additionally, in some embodiments, the first liquid comprises a
first fluorescent dye, the second liquid comprising a second
fluorescent dye, and the third liquid comprising a third
fluorescent dye, each of the first, second, and third fluorescent
dyes emitting fluorescence upon excitation wherein the fluorescence
emitted from each is spectrally resolvable from the fluorescence
emitted from the others. In some embodiments, the system may
include at least one of a thermocycler, an auto sampler, a fluid
charging apparatus, such as for example a static bar which may or
may not include and electrode.
[0007] Further provided herein is a method for detecting proper
mixing of at least three liquids, comprising mixing together a
first liquid, a second liquid, and a third liquid, each being
miscible with the others, to form a mixed sample droplet, the first
liquid comprising a first fluorescent dye, the second liquid
comprising a second fluorescent dye, and the third liquid
comprising a third fluorescent dye, each of the first, second, and
third fluorescent dyes emitting fluorescence upon excitation
wherein the fluorescence emitted from each is spectrally resolvable
from the fluorescence emitted from the others, moving the mixed
sample droplet in a conduit, irradiating the mixed sample droplet
in the conduit with an excitation source; and detecting emissions
from the mixed sample droplet to determine whether each of the
first, second, and third fluorescent dyes is present in the mixed
sample droplet.
[0008] Provided herein is a method for detecting a droplet in
system comprising moving the mixed sample droplet in a conduit;
irradiating the mixed sample droplet in the conduit with an
excitation source; and detecting emissions from the mixed sample
droplet to determine whether each of the first, second, and third
fluorescent dyes is present in the mixed sample droplet. In some
embodiments of the method, the first liquid comprises a first
droplet, the first droplet is encompassed by a carrier fluid that
is substantially immiscible with the first liquid, the second
liquid comprises a second droplet, the second droplet is
encompassed by the carrier fluid, the third liquid comprises a
third droplet, and the third droplet is encompassed by the carrier
fluid. The mixed sample droplet may be encompassed by a carrier
fluid that is substantially immiscible with the mixed sample
droplet. The mixed sample droplet may be formed at an intersection
of the conduit with three other conduits, each of the other
conduits containing therein the first liquid, the second liquid,
and the third liquid, respectively. The excitation source may
include one or more LEDs. The excitation source comprises one or
more blue LEDs, each blue LED emitting an excitation beam having a
single wavelength that excites each of the first, second, and third
fluorescent dyes. The detecting comprises detecting emission from
the first fluorescent dye using a first detector, detecting
emission from the second fluorescent dye using a second detector,
and detecting emission from the third fluorescent dye using a third
detector. In some embodiments, the method may further comprise
tracking the mixed sample droplet as it moves in the conduit and
accepting or rejecting data generated by downstream processing of
the mixed sample droplet based on the emissions detected.
Additionally, the method may further comprising forming a train of
droplets including the mixed sample droplet and detecting emissions
from each droplet of the train of droplets. In some embodiments,
the method may further comprising forming a train of droplets
including the mixed sample droplet, the train of droplets
comprising carriages each comprising a plurality of spaced apart
droplets, wherein a first spacing is provided between adjacent
droplets within each carriage, and the carriages are spaced apart
from adjacent carriages by a second spacing that differs from the
first spacing. Additionally the methods provided herein may include
determining, based on the detected emissions, that proper mixing of
the first liquid, second liquid, and third liquid has occurred in
the mixed sample droplet; and gathering data from downstream
processing of the mixed sample droplet. Alternatively, the method
may comprise determining, based on the detected emissions, that
improper mixing of the first liquid, second, liquid, and third
liquid has occurred in the mixed sample droplet; and recording
occurrence of an error; forming a new mixed sample droplet from the
first liquid, the second liquid, and the third liquid; and ignoring
data generated by downstream processing of the mixed sample
droplet. In some embodiments, the first and second dyes comprise a
passive reference dye and the third dye comprises a reporter
dye.
[0009] Further provided herein is a system for detecting proper
mixing of at least three liquids, comprising: a conduit system
comprising at least a main conduit for carrying a mixed sample
droplet; a mixed sample droplet in the main conduit and comprising
a first liquid, a second liquid, and a third liquid, the first
liquid comprising a first fluorescent dye, the second liquid
comprising a second fluorescent dye, and the third liquid
comprising a third fluorescent dye, each of the first, second, and
third fluorescent dyes emitting fluorescence upon excitation
wherein the fluorescence emitted from each is spectrally resolvable
from the fluorescence emitted from the others; and an optical
detection system comprising an excitation source for irradiating
the mixed sample droplet in the main conduit and a detector for
detecting emissions from the mixed sample droplet to determine
whether each of the first, second, and third fluorescent dyes is
present in the mixed sample droplet. The mixed sample droplet may
be encompassed by a carrier fluid that is substantially immiscible
with the mixed sample droplet. The system of claim 24, wherein the
conduit system further comprises a first auxiliary conduit
containing therein the first liquid, a second auxiliary conduit
containing therein the second liquid, and a third auxiliary conduit
containing therein the third liquid; wherein the first, second, and
third auxiliary conduits intersect with the main conduit at a
junction configured to form the mixed sample droplet. The
excitation source may include one or more LEDs or one or more
lasers or one or more light sources which may have different
wavelengths or the same wavelength. Additionally the detector may
include a first detector for detecting emission from the first
fluorescent dye, a second detector for detecting emission from the
second fluorescent dye, and a third detector for detecting emission
from the third fluorescent dye. The first detector, the second
detector, and the third detector may each comprise a camera, such
as a digital camera or a spectral camera, with or without filters.
The system may further comprise a signal processing system for
monitoring information generated by the detector and determining
whether proper mixing of the first liquid, the second liquid, and
the third liquid has occurred in the mixed sample droplet and the
first and second dyes comprise a passive reference dye and the
third dye comprises a reporter dye. The system may further
comprising a train of droplets including the mixed sample droplet,
in the main conduit, the train of droplets comprising carriages
each comprising a plurality of spaced apart droplets, wherein a
first spacing is provided between adjacent droplets within each
carriage, and the carriages are spaced apart from adjacent
carriages by a second spacing that differs from the first spacing.
Additionally, the system may include a conduit support board that
holds the main conduit; and an excitation source support board that
holds the excitation source; wherein the conduit support board and
the excitation source support board are disposed parallel to each
other such that the main conduit and the excitation source are
aligned with each other and at least a portion of the main conduit
is exposed to radiation emitted from the excitation source, where
the conduit support board may or may not holds a plurality of main
conduits and the excitation source support board holds a plurality
of excitation sources. Furthermore, the system may include a
plurality of conduit support boards, a plurality of excitation
boards, and a housing in which the plurality of conduit support
boards and the plurality of excitation support boards are retained.
In some embodiments, the system includes a fiber optic cable
connected to the detector and configured to receive fluorescent
emissions from the main conduit.
[0010] Also provided herein is a control unit comprising a
processor programmed to carry out a method, the method comprising:
mixing together a first liquid, a second liquid, and a third
liquid, each being miscible with the others, to form a mixed sample
droplet, the first liquid comprising a first fluorescent dye, the
second liquid comprising a second fluorescent dye, and the third
liquid comprising a third fluorescent dye, each of the first,
second, and third fluorescent dyes emitting fluorescence upon
excitation wherein the fluorescence emitted from each is spectrally
resolvable from the fluorescence emitted from the others; moving
the mixed sample droplet in a conduit; irradiating the mixed sample
droplet in the conduit with an excitation source; and detecting
emissions from the mixed sample droplet to determine whether each
of the first, second, and third fluorescent dyes is present in the
mixed sample droplet. The processor may be a computer.
[0011] Further provided herein is a computer readable medium
comprising a program stored thereon, the program comprising a set
of instructions for carrying out a method, the method comprising:
mixing together a first liquid, a second liquid, and a third
liquid, each being miscible with the others, to form a mixed sample
droplet, the first liquid comprising a first fluorescent dye, the
second liquid comprising a second fluorescent dye, and the third
liquid comprising a third fluorescent dye, each of the first,
second, and third fluorescent dyes emitting fluorescence upon
excitation wherein the fluorescence emitted from each is spectrally
resolvable from the fluorescence emitted from the others; moving
the mixed sample droplet in a conduit; irradiating the mixed sample
droplet in the conduit with an excitation source; and detecting
emissions from the mixed sample droplet to determine whether each
of the first, second, and third fluorescent dyes is present in the
mixed sample droplet.
INCORPORATION BY REFERENCE
[0012] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0014] FIG. 1 is a schematic diagram of a flap valve opening method
in accordance with various embodiments;
[0015] FIG. 2 is a schematic diagram of a liquid/plate handling
system in accordance with various embodiments;
[0016] FIG. 3 is a bottom view parametric model drawing of an
ionizing electrode assembly;
[0017] FIG. 4a is a bottom view of a ground electrode; FIG. 4b is a
side view of the ground electrode shown in FIG. 4a; FIG. 4c is a
perspective top view parametric model drawing of the ground
electrode shown in FIGS. 4a and 4b;
[0018] FIG. 5 is a perspective bottom view parametric model drawing
of the electrode assembly;
[0019] FIG. 6 is a schematic diagram showing a system for high
throughput PCR amplification and analysis;
[0020] FIGS. 7A & 7B illustrate various embodiments of a liquid
bridge;
[0021] FIG. 8 is one embodiment of a support structure for a for
conduits entering a liquid bridge;
[0022] FIGS. 9A and 9B show examples of stacked liquid bridges;
[0023] FIGS. 10A and 10B show examples of liquid bridge
substrates;
[0024] FIG. 11 shows one example of a shared oil cavity;
[0025] FIG. 12 shows one embodiment of a post bridge diagnostic
system;
[0026] FIG. 13 is one embodiment of a schematic diagram of a side
view of a system for detecting spectral and spatial information in
a continuous flow PCR system;
[0027] FIG. 14 is a block diagram that illustrates a computer
system;
[0028] FIG. 15 is a schematic diagram of a system that includes one
or more distinct software modules that perform a method for high
throughput PCR amplification and analysis;
[0029] FIG. 16 is a schematic diagram showing how files are
transferred between a graphical user interface (GUI) and an
instrument, in accordance with various embodiments;
[0030] FIG. 17 is a flowchart showing a method for uploading a file
using a file transfer protocol (FTP) server, in accordance with
various embodiments; and
[0031] FIG. 18 is a schematic of one embodiment of the system.
DETAILED DESCRIPTION OF THE INVENTION
Instrument
[0032] Provided herein is a PCR instrument, in some embodiments a
continuous flow 96-line PCR instrument, capable of sampling
different fluids or gases from different locations. In some
embodiments, the instrument samples the same fluid or different
fluids from at least two separate locations. In some embodiments,
three different samples may be combined. In some embodiments, the
sample may be combined with different fluids such as, for example,
a master-mix, a sample and a primer and/or probe simultaneously
located separately, and mixing these fluids in a micro-channel
geometry, for example in a liquid bridge or other suitable mixer,
to form a mixed droplet. The mixed droplet may then flow downstream
to a thermocycler where the contents of the droplet may undergo
further processing. In some embodiments, the droplet may contain,
for example at least one template of a nucleic acid, which may then
be subject to conditions for amplification. The droplets and their
contents may then pass a data-acquisition system where a parameter
of interest may be measured from the droplet such as, for example,
concentration or intensity of a dye or multiple dyes located in the
droplet, viscosity of the droplet, volume of the droplet, turbidity
of the droplet, optical density of the droplet, size of the
droplet, or any other suitable parameter.
[0033] The instrument provided herein may be comprised from several
different individual components, each of which is discussed further
herein.
[0034] Auto-Sampler
[0035] In various embodiments, the system provided for herein may
include a fluid sampling device. In some embodiments, the fluid
sampling device may be automatic. In some embodiments, the
auto-sampling device may be configured to withdraw segmented plugs
or droplets of fluid from a fluid vessel. The fluid may be charged
fluid or may be a fluid with no charge. The withdrawal and
acquisition of fluid may be performed in either a continuous
operation or batch operation mode. For example, fluid sampling
devices may comprise, for example, the devices described in United
States Patent Application Publication Nos. 2010/0304443 and
2010/0294048, which are incorporated by reference in its entirety
herein. The devices described in United States Patent Application
Publication Nos. 2010/0304443 and 2010/0294048 may be configured to
withdraw segmented fluid samples from a vessel, wherein the
segmented fluid samples are surrounded by an immiscible carrier
fluid.
[0036] In some embodiments, the fluid sampling devices may further
include at least one robotics system to control the fluid sampling
devices. The robotics systems may control movement of the sampling
devices to control sample acquisition from the fluid vessel. In
some embodiments, the driving force for the withdrawal of charged
fluid by a fluid sampling device may be provided by one or more
pumps. An exemplary pump is shown in International Patent
Application Publication No. WO 2007/091229, which is incorporated
by reference in its entirety herein. In some embodiments, the fluid
sampling devices may be configured to withdraw fluid using the
hydrostatic siphoning effect described in United States Patent
Application Publication No. 2010/0120635, which is incorporated by
reference in its entirety herein.
[0037] In some embodiments, provided herein is a method for
generating small volumes of droplets. In some embodiments, droplets
of a small volume may be generated using pick-up heads as
previously described in United States Patent Application
Publication Nos. 2010/0304443 and 2010/0294048, which are
incorporated by reference in their entirety. Provided herein is a
system that operates under continuous flow such that a first fluid
is in continuous contact with the passageway through which the
fluid travels such that the first fluid segments a second fluid
into discrete volumes and surrounds the second fluids, thereby
preventing contact of the second fluid with the passageway. The
continuous flow of the system enables collecting a sample located
in different wells without drawing air into the system. For
example, in some embodiments, a sample is drawn into the system by
moving a sample pick-up heads from well-to-well using a continuous
flow of a fluid, such as, for example, an oil, such that air is not
drawn into the system. In some embodiments the fluid or fluids may
be drawn into the system with or without the use of sheathing
fluid. In some embodiments, the pick-up heads may include a
protective barrier that is configured to prevent air from entering
into the system, such as for example, a flap valve. In some
embodiments, the protective barrier may be opened and closed using
robotic control, pressure, movement, or any other suitable
mechanism for opening and closing the protective barrier. In some
embodiments, the pick-up heads may draw-up and/or segment a sample
fluid using a both sheath fluid and a flap valve. In an embodiment
where the sheath tube may be used, the system may comprise a larger
bore tube which may be fitted around at least one sampling tube.
The sheath tube may provide for a sheath fluid that wraps the at
least one sampling tube in oil. The continuous flow of oil into the
sheathing tube may match or slightly exceed the flow of the sample
being drawn into the system. In such an embodiment, the tips of the
sample tubes of the continuous flow lines may be wrapped in oil
providing for a continuous flow system. The sheath fluid may be
controlled by at least two independent sheathing pumps, and in some
embodiments, may be controlled by at least three independently
controlled sheathing pumps. Such a system allows the sample pick-up
heads to be moved freely from well to well without drawing any air
into the system.
[0038] FIG. 1 is a schematic diagram of a flap valve opening method
1300, in accordance with various embodiments. In order to
facilitate the use of flap valves/sheathing (which needs to be
opened before sampling can take place) the tips are mounted on a
double Z-axis. The secondary axis 1320 is mounted on the primary
axis 1310. The sheathing/flap valves are mounted on primary axis
1310 while the tips are mounted on secondary axis 1320.
[0039] In step 1 of method 1300, in air the robotic head moves over
the required wells.
[0040] In step 2, primary axis 1310 lowers the tips (sheathing and
secondary axis 1320) into the oil overlay which covers the sample
in each well.
[0041] In step 3, secondary axis 1320 then extends the tips
(pushing the valves open) so the tip is over the sample.
Simultaneously primary axis 1310 rises by an equal distance. The
combined effect is that secondary axis 1320 is stationary in space
while primary axis 1310 moves upwards. Combined with the geometry
of the flap-valves, this movement allows an extra 30 .mu.l volume
of sample be used in each (96-wellplate) well.
[0042] In step 4, secondary axis 1320 lowers further into the well
and completes opening of the flap valve. The secondary axis 1320
pauses until triggered to sample.
[0043] In step 5, at the precise time required, secondary axis 1320
dips into the fluid and draws up approximately 75 nl of fluid
(sample/primer-probe, master mix approx. 150 nl). The amount of
fluid drawn depends on the flow-rate used and the time the tip is
within the fluid.
[0044] In step 6, the tip then retracts from the sample and pauses
ready to sample again if required. If the next sample is needed
from a neighboring well (or a plate-change) the tip retracts into
the sheathing and the primary axis 1310 then moves the sampling
head out into the air. The sheathing motion is a reverse of the
unsheathing motions.
[0045] FIG. 2 is a schematic diagram of a liquid/plate handling
system 1400, in accordance with various embodiments. In system
1400, the liquid/plate handling provides movement along 15 axes.
For reference, system 1400 is divided into three sampling systems
and one plate handling system. The directions of motion of each
stage are shown by arrows. Note that the sampling arm of the
multi-lumen unit is shown. However, for clarity, the sampling arms
of the master-mix unit and single-tip unit are rendered invisible.
Additionally the master mix unit is mounted on the roof of the
enclosure. The individual axes are: [0046] Single-tip Sampling
[0047] X-axis [0048] Y-axis [0049] Primary Z-axis (Z1) [0050]
Secondary Z-axis (Z2) [0051] Multi-lumen Sampling [0052] X-axis
[0053] Y-axis [0054] Primary Z-axis (Z1) [0055] Secondary Z-axis
(Z2) [0056] Rotational Axis [0057] Master-mix Sampling [0058]
X-axis [0059] Primary Z-axis (Z1) [0060] Secondary Z-axis (Z2)
[0061] Plate handling [0062] Y-axis [0063] X1-axis
(Tray1--Single-tip) [0064] X2-axis (Tray2--Multi-lumen)
[0065] The single-tip system consists of 96 tips each of which can
enter a single well on a 96-well or 384-well plate. Therefore
system 1400 can sample from a 96-well plate in a single movement or
a 384-well plate in four movements. The multi-lumen system consists
of four bundles of 24-tips. All 24 lines in each bundle can enter a
single well. Each line in the bundle is arrayed against one of the
single-tip lines--meeting in a bridge. In some embodiments, the
liquid bridge line flows directly into the thermocycler. In some
embodiments, the output from a liquid bridge may be captured into a
containment unit and then further processed in the container unit
or withdrawn from the containment unit for further processing. The
multi-lumen head is mounted on a rotational unit. Therefore through
four rotations and dips, four wells on Tray 2 (Multi-lumen side)
may be arrayed against an entire 96-well plate. Similarly 16
robotic movements (four multi-lumen rotations times four single-tip
movements) can permit four wells on Tray 2 be arrayed against an
entire 384-well plate.
[0066] When exposing the tip of a sampling device to different
samples carry-over between tips may occur. In some embodiments over
Sample carryover/contamination may occur when the sampling tubes
are exposed to concentrated samples. Sample contamination may be
reduced by minimizing the area of the sampling tube exposed to the
sample. The area of the sampling tube exposed to the sample may be
minimized in various ways. In some embodiments, a reduced portion
of a sampling tube may extend from a sheath tube. Such a tube in
tube embodiment may be fabricated by inserting at least a portion
of the sampling tube in a sheath tube. In some embodiments, the
sampling tube may be etched on the exterior surface. The sampling
tube may then be placed inside a sheath tube. In some embodiments,
the interior surface of the sheath tube may be etched as well.
Placing the sampling tube in the sheath tube may create at least a
partial seal which prevents the exterior surface of the sampling
tube from being contaminated. In some embodiments, friction between
the inner tube and outer tube causes a seal between the two tubes.
In some embodiments, an adhesive may be used to form a seal between
the sampling tube and sheath tube. Examples of adhesives that may
be used include, for example, glue, epoxy, putty, or any other
suitable adhesive. Once a partial seal has been formed between the
sampling tube and sheath tube, the combined tube may be cut to
expose the distal end of the sampling tube. In some embodiments,
the combined tube may be laser cut to ensure a smooth finish on the
distal end of the tip. Creating a nested sampling tube structure
may lead to a reduction in the cross-sectional area of the wall of
up to 92%.
[0067] Static Charging of Droplets
[0068] In various embodiments, a fluid charging system configured
to charge a fluid contained in a fluid vessel comprises an ionizing
electrode and a ground electrode. The ionizing electrode and the
ground electrode may be positioned adjacent to the fluid vessel.
The ionizing electrode and the ground electrode may be opposed so
that the fluid vessel is positioned between the ionizing electrode
and the ground electrode. The ionizing electrode and the ground
electrode are configured to produce an ion field that contacts
fluid contained in the fluid vessel, thereby charging the fluid.
Various embodiments of static charging systems are described in
copending U.S. application Ser. No. ______ (Attorney Docket No.
LT00400 PRO), entitled "System and Method for Charging Fluids",
which is incorporated by reference in its entirety.
[0069] In various embodiments, a method for charging a fluid
contained in a vessel comprises producing an ion field between an
ionizing electrode and a ground electrode. A fluid-containing
vessel may be positioned adjacent to and between the ionizing
electrode and the ground electrode. The ion field produced by the
ionizing electrode and the ground electrode contacts the fluid
contained in the fluid vessel, thereby charging the fluid. The
devices, systems, and methods disclosed herein may be used to
produce a net charge in various fluids.
[0070] Fluid charged in the devices, systems, and methods disclosed
herein may be mixed with other fluids after being charged. The net
charge carried by the fluids charged in the devices, systems, and
methods disclosed herein may increase the extent of the mixing of
fluids in downstream devices, systems, and methods. For instance,
fluids carrying a net charge may exhibit improved mixing with other
miscible fluids when mixed with fluid plugs or droplets in an
immiscible carrier fluid in microfluidic system. In this manner,
the net charge may decrease undesirable static electric effects
observed in microfluidic systems that can adversely affect fluid
mixing.
[0071] In various embodiments, a fluid charging system may comprise
one or more fluid sampling devices configured to withdraw charged
fluid from the fluid vessel, such as described and illustrated
below. In various embodiments, the fluid sampling devices may
comprise one or more tubes, such as, for example, capillary tubes,
configured to withdraw charged fluid from the fluid vessel. In
various embodiments, the fluid sampling devices may comprise one or
more sheaths, wherein each sheath surrounds one or more tubes, such
as, for example, capillary tubes, configured to withdraw charged
fluid from the fluid vessel. In various embodiments, the one or
more fluid sampling devices may be in continuous or discontinuous
fluid communication with the fluid vessel.
[0072] Fluid charging systems including fluid sampling devices may
further include at least one robotics system to control the fluid
sampling devices. The robotics systems may control movement of the
sampling devices to control sample acquisition from the fluid
vessel. In various embodiments, the driving force for the
withdrawal of charged fluid by a fluid sampling device may be
provided by one or more pumps. An exemplary pump is shown in
International Patent Application Publication No. WO 2007/091229,
which is incorporated by reference herein. In various embodiments,
the fluid sampling devices may be configured to withdraw fluid
using the hydrostatic siphoning effect described in United States
Patent Application Publication No. 2010/0120635, which is
incorporated by reference herein.
[0073] In various embodiments, the ionizing electrode may comprise
an emitter plate and one or more emitter pins connected to the
emitter plate. The emitter plate may be made of a conductive
metallic material, such as, for example, a stainless steel alloy.
The emitter pins may be made of a metallic or ceramic material
comprising tungsten. For instance, the emitter pins may comprise
tungsten carbide, such as, for example, emitter pins made of
tungsten carbide or a cemented tungsten carbide (cement) composite
material. Alternatively, the emitter pins may be made of a metal
alloy comprising tungsten, for example.
[0074] In operation, electrical current delivered to the ionizing
electrode concentrates at the tips of the emitter pins and ionizes
atoms and/or molecules comprising the surrounding air or other
gaseous atmosphere, producing an ion cloud. The ion cloud emits
from the emitter pins and moves toward the ground electrode along a
static electric field established between the ionizing electrode
and the ground electrode in accordance with the physical principles
of static electricity. This produces an ion field between the
ionizing electrode and the ground electrode. The polarity of the
ion field is the same as the polarity of the electrical current
provided to the ionizing electrode. Although the ion fields
illustrated in the figures presented herein are shown with a
positive polarity (+) symbol, it is understood that, in various
embodiments, the ion field may be of negative polarity. Materials
contacting the ion field become charged at the same polarity as the
ion field.
[0075] A fluid charging system is provided for charging fluids to
be mixed with other fluids in a microfluidic system. The system
includes a Fraser Model 7330 static generator connected to an
ionizing electrode assembly via a high voltage cable. The ionizing
electrode assembly includes a cross-shaped electrode comprising a
cross-shaped stainless steel emitter plate and five (5) tungsten
emitter pins. The ionizing electrode is connected to a 100 megaohm
resistor unit via a high voltage lead. The ionizing electrode
assembly has the dimensions and configuration shown in FIG. 3
(dimensions in millimeters). A ground electrode comprises a
circular aluminum static ground plate that sits in a non-conductive
acrylic holder. The ground electrode has the dimensions and
configuration shown in FIGS. 4a, 4b, and 4c (dimensions in
millimeters). The ground electrode is connected to the ground lug
on the static generator.
[0076] In various embodiments, a fluid vessel may be positioned
between and adjacent to the electrodes so that the ion field
contacts fluid contained within the fluid vessel, thereby charging
the fluid. The emitter pins may be connected to the side of the
emitter plate that faces an open top end of a fluid vessel, which
facilitates contact between the field produced by the ionizing
electrode and fluid contained in the fluid vessel to charge the
fluid. Although the ionizing electrodes illustrated in certain
figures presented herein are shown positioned adjacent an open top
end of a fluid vessel, it is understood that, in various
embodiments, the ionizing electrodes may be positioned adjacent to
any region or end of an open or closed fluid vessel, provided the
ionizing electrodes and ground electrodes are mutually positioned
in a spaced apart relationship.
[0077] FIG. 5 shows the ionizing electrode and the ground electrode
positioned adjacent to a glass fluid vessel. The ionizing electrode
and the ground electrode are opposed so that the fluid vessel is
positioned between the ionizing electrode and the ground electrode.
The glass fluid vessel has an outer diameter that substantially
matches the diameter of the ground electrode, the ground electrode
and holder being dimensioned to seat and support the fluid vessel.
Four (4) fluid sampling devices are positioned through open
quadrant regions of the emitter plate of the ionizing electrode.
The four (4) fluid sampling devices include sheaths surrounding
tubes configured to withdraw fluid from the fluid vessel.
[0078] In various embodiments, a fluid sampling device may be in
fluid communication with a fluid dispensing device configured to
dispense charged and/or mixed fluids to vessels, such as, for
example, eppendorf tubes, vials, beakers, flasks, centrifuge tubes,
capillary tubes, cryogenic vials, bags, channels, cups, containers,
microtiter plates, microcards, and the like. The transport of
charged fluids from the fluid vessel to other vessels may be
accomplished, for example, using pumps, hydrostatic pressure,
capillary forces, and the like.
[0079] In various embodiments, the fluid charging systems disclosed
herein may be used to provide charged fluid to microfluidic
processing networks and systems. A charged fluid may be mixed with
other fluids in a microfluidic processing network or system.
Microfluidic processing networks and systems in which fluids may be
mixed are described, for example, in United States Patent
Application Publication Nos. 2005/0092681, 2005/0272144,
2008/0277494, 2010/0015606, 2010/0029512, 2010/0109320, and
2010/0297748, which are all incorporated by reference herein. The
fluid charging systems disclosed herein may be in fluid
communication with microfluidic processing networks and systems
such as those described in these documents.
[0080] In various embodiments, the fluid charging systems disclosed
herein may be used to provide charged fluid to microfluidic
processing networks and systems comprising liquid bridges. United
States Patent Application Publication Nos. 2008/0277494,
2010/0015606, 2010/0029512, 2010/0109320, and 2010/0297748, which
are all incorporated by reference herein, describe microfluidic
processing networks and systems comprising liquid bridges. A liquid
bridge is a device in which liquid droplets are formed. The
droplets formed in a liquid bridge are enveloped in an immiscible
carrier fluid. Generally, a liquid bridge is formed by an inlet in
communication with a chamber that is filled with immiscible carrier
fluid. The carrier fluid is immiscible with fluid droplets flowing
through the inlet into the chamber. The fluid droplets expand until
they are large enough to span a spatial gap between the inlet and
an outlet in communication with the chamber. Droplet formation is
accomplished, for example, by adjusting flow rate or by joining one
or more additional fluid droplets to a first fluid droplet,
resulting in formation of an unstable liquid bridge between the
inlet and the outlet that subsequently ruptures from the inlet.
After rupturing from the inlet, the fluid droplet enters the
outlet, surrounded by the carrier fluid from the chamber.
[0081] The fluid charging systems disclosed herein may be
configured to provide charged fluid to a liquid bridge. For
example, a fluid sampling device of a fluid charging system may be
in fluid communication with a liquid bridge. In various
embodiments, a liquid bridge may be configured to segment a charged
fluid into droplets. In various embodiments, a liquid bridge may be
configured to mix droplets of charged fluid with droplet of other
fluid (that may be uncharged or charged, for example, as described
herein) that is miscible with the charged fluid. As used herein,
the term "droplet" refers to a relatively small microfluidic
quantity or plug of liquid as it is suspended and/or flows in an
immiscible carrier liquid in a conduit or chamber, such as, for
example, in a microfluidic processing network or system.
[0082] Further provided herein is a method of mixing droplets using
electrostatic charging of droplets. In some embodiments a charged
droplet, for example a statically charged droplet, may be directed
toward a second droplet. The droplet may be charged using the
method and system provided herein. The second droplet may be
charged or uncharged. As the charged droplet approaches the second
droplet, the charged droplet may induce charge separation in the
awaiting second droplet. The charge separation may then cause the
charged droplet and the second droplet to become more attracted to
each other and may facilitate the combining of the two droplets.
The charge separation in the second droplet, together with the
charged droplet may cause the two droplets to mix in a more
efficient manner than when both droplets are uncharged. In some
embodiments, a charged droplet may be combined with at least two
droplets, in which at least one droplet may be charged. In some
embodiments, a first droplet may be charge and the second and third
droplets may be uncharged. In some embodiments, a first droplet and
a second droplet may be charged and the third droplet may be
uncharged.
[0083] In some embodiments, the droplets may be charged or
uncharged to prevent droplets from combining. In some embodiments,
charging droplets may be useful in sorting droplets by preventing
droplets from combining or by dictating which path the droplet may
flow through.
[0084] Other embodiments, of fluid charging systems are described
in copending U.S. Ser. No. ______ (Atty Docket: LT00400 PRO), which
is incorporated herein by reference in its entirety.
[0085] Fluid Pumping System
[0086] FIG. 6 is a schematic diagram showing a system 200 for high
throughput PCR amplification and analysis, in accordance with
various embodiments. System 200 includes PCR system 210 and
processor 220. PCR system 210, in turn, includes liquid handling
system 230, fluid pumping system 240, post-bridge detection system
250, thermocycler 260, and endpoint detection system 270. The
system 200 operates under the principal of continuous flow. A
constant flow of oil is maintained through the thermocycler (TC
line 242) and this flow of oil carries mixed droplets. It is
required that the flow upstream of the liquid-bridges (from
sample-tips to bridges) be faster than the flow through the
thermocycler in order to meet throughput demands. A draft line 241
is fitted to the bridge and bleeds off excess oil. The TC line 242
and the draft line 241 both operate at fixed flow rates. It is
required that these lines be controlled as the addition of droplets
to the lines increases the pressure drop along each line. The
combined flow in the TC line 242 and draft line 241 equals that of
the master-mix, sample and primer-probe lines.
[0087] In addition the pumping system incorporates a number of
subsystems for priming the system with oil and bleeding it of air.
FIG. 6 shows a general schematic (for a single line system) showing
the TC Line 242, the Draft Line 241 and where the hardware
components are located.
[0088] If a PCR system operates under continuous flow, moving the
system through air to move from well-to-well would cause air to be
drawn into the system. This is avoided through the use of
sheathing/flap valves. These larger bore tubes are fitted around
the sampling tubes and wrap them in oil. The continuous flow of oil
into the sheathing (driven by 3 independent sheathing pumps)
matches (or slightly exceeds) the flow being drawn into the system
tips insuring that the continuous flow lines are always wrapped in
oil. Hence the tips can move freely from well to well without
drawing any air into the system.
[0089] Liquid Bridge Technology
[0090] In various embodiments, a liquid bridge configured to
segment charged fluid withdrawn from a fluid vessel into droplets
comprises a first inlet port in fluid communication with a fluid
sampling device, a second inlet port in fluid communication with a
source of immiscible fluid, an outlet port, and a chamber. The
inlet ports and the outlet port open into the chamber and may be
structured and positioned so that fluid instability in fluid
droplets formed between the first inlet port and the outlet port
segments the fluid withdrawn from the fluid vessel into fluid
droplets separated by the immiscible fluid. The fluid droplets may
be withdrawn from the chamber through the outlet port.
[0091] In various embodiments, a liquid bridge configured to mix
charged fluid withdrawn from a fluid vessel in a fluid charging
system with one or more additional fluids that may be miscible with
the charged fluid comprises a first inlet port in fluid
communication with the fluid sampling device, one or more
additional inlet ports in fluid communication with sources of the
one or more additional fluids, an outlet port, and a chamber. The
inlet ports and the outlet port open into the chamber and may be
structured and positioned so that first fluid droplets formed at
the first inlet port contact and mix with one or more additional
fluid droplets formed at the one or more additional inlet ports,
thereby forming unstable funicular bridges of mixed fluid. The
unstable funicular bridges rupture, thereby forming mixed fluid
droplets separated by immiscible carrier fluid that are withdrawn
from the chamber through the outlet port. The net charge carried by
the fluid withdrawn from the fluid vessel improves the mixing of
the charged fluid with the one or more additional fluids. One
embodiment of a liquid bridge is shown in FIGS. 7A & 7B (from
Stokes LB Patent application). A more detailed description of a
liquid bridge may be found in United States Patent Applications
Nos. 2008/0277494 and 2010/0029512 and PCT Application Nos.
PCT/IE07/000013 and PCT/US10/24180, each of which is incorporated
by reference in their entirety.
[0092] In various embodiments, a liquid bridge configured to mix
charged fluid withdrawn from a fluid vessel in a fluid charging
system with one or more additional fluids that are miscible with
the charged fluid comprises a chamber, one or more inlet ports, a
first outlet port, and a second outlet port. The inlet ports and
the outlet ports may open into the chamber. An inlet port may be in
fluid communication with a fluid sampling device and sources of one
or more additional fluids. The inlet ports may serially provide
fluid droplets of the charged fluid withdrawn from the fluid vessel
and the one or more additional fluids, wherein the droplets may be
separated by an immiscible carrier fluid. The first outlet port may
be configured to withdraw a portion of the immiscible carrier fluid
entering the chamber. The inlet ports and the outlet ports may be
structured and positioned so that trailing droplet transporting
through the inlet port contact and mix with leading droplets formed
at the inlet port in the chamber, thereby forming mixed fluid
droplets that may be withdrawn from the chamber through the second
outlet port separated by immiscible carrier fluid. The net charge
carried by the fluid withdrawn from the fluid vessel improves the
mixing of the charged fluid with the one or more additional
fluids.
[0093] In some embodiments the liquid bridge comprises at least two
channels, tubes, capillaries, or any other suitable conduit for
providing fluid communication between a reservoir containing a
fluid and a liquid bridge. In some embodiments, the mechanism may
be PTFE tubes. The PTFE tubes may be layered so that multiple tubes
are stacked one on top of the other. In some embodiments, the
conduits may be stacked using a support. In some embodiments, the
support or washboard may smooth. Alternatively, the washboard may
be on a support/washboard that has undulations. The undulating
pattern may create undulations in the conduits themselves, wherein
these undulations may then provide natural stops for the droplets
flowing in the tubes or conduits, for example sample and assay
droplets. In such a manner, the droplets may be stopped or held in
position prior to entry into the liquid bridge. The holding of
holding/stopping of the droplets may allow for all necessary
droplets to come into the liquid bridge prior to being mixed
together. In some embodiments, 10 individual PTFE parts may be
used. In some embodiments, one PTFE coated aluminum part may be
used. An example of a washboard/support may be found seen in FIG.
8. In some embodiments, the liquid bridge my further include a PTFE
tube having an internal diameter that widens to slow the droplet
speed as it approaches the liquid bridge.
[0094] In some embodiments, the liquid bridge may be a single
bridge. In some embodiments, the bridges may be stacked by placing
individual bridges on one another. FIGS. 9A & 9B show examples
of an isolated stacked liquid bridge and a stacked liquid bridge as
connected to the system, respectively. In some embodiments, any
number of bridges may be combined to form a stacked liquid bridge
having a single bridge cavity. In some embodiments, a single cavity
may be constructed from any suitable number of bridges. In some
embodiments, a single cavity may include at least 2, at least 4, at
least 8, at least 12, at least 16, at least 20, at least 24, at
least 30, at least 50, at least 75, at least 90, at least 96, at
least 120 bridges.
[0095] In some embodiments a combined liquid bridge cavity may be
formed by machining a substrate containing at least one "bridge".
Such a substrate may be a precision machined substrate, such as a
polycarbonate piece onto which features are assembled. The
substrate may be machined such that features are on both sides of
the substrate to aid in stacking of substrates. FIG. 10A shows a
single substrate with four bridges. FIG. 10B shows multiple
substrates stacked upon each other.
[0096] The capillaries, tubes, channels or other suitable mechanism
for providing for fluid communication between a reservoir and the
bridge may be generated by thermoforming the PTFE capillaries. The
substrate may be designed to have curved channel paths into which
tubing is bonded. Thermoforming involves placing tubes into
geometric paths similar to the paths found in the substrate,
applying a stop band at a precise location, heating to 240 degrees
Celsius for 30 minutes and then cooling the tubes. When removed,
the tube retains the shape of the path. The preformed tubing can
then be assembled into the substrate quickly. The stop bands formed
on the tubes prevents the tubing tips from protruding too far into
the liquid bridge.
[0097] The stacked liquid bridge may have a geometry that includes
a shared oil cavity as seen in FIG. 11. As seen in FIG. 11, each
"bridge" of the stacked bridge comprises a washboard 1110, inlet
tubes 1130 and outlet tube 1140. The bridges include a shared oil
cavity 1100 in which oil is free to flow between the bridges.
Additionally, blockers 1120 may be present to prevent movement of
droplets between bridges. However, oil is free to flow in the
shared oil cavity. Therefore, the blockers may be spaced such that
they may restrict movement or loss of droplets during mixing.
Additionally, in some embodiments, the blockers may serve to close
off the mixing zone. The spacing between the blockers and the
liquid bridge is small enough to prevent droplet loss from the
liquid bridge into the shared oil cavity but to allow oil to flow
into the liquid bridge.
[0098] The droplet stream leaving the liquid bridge or bridges (in
the case of a stacked liquid bridge) may be divided into packets.
The droplet stream may be divided based upon the time-stamp at
which the robotics takes a sample. For convenience these packets
are called carriages. The use of carriages--where the spacing
between carriages is at least twice that between droplets--permits
easier identification of individual droplets and indeed easy
identification of errors in the droplet stream. For example droplet
2 of carriage 2 (with 5 droplets per carriage) may be identified
more easily than droplet 12 of a continuous stream. Similarly
errors can be easily identified. If only 4 droplets are present in
a carriage of 5 then it is clear an error has occurred (droplet
merging); if 6 are present then a droplet has not mixed or has
mixed and then split into two.
[0099] Further description of the structure and operation of
segmenting liquid bridges and mixing liquid bridges is presented in
United States Patent Application Publication Nos. 2008/0277494 and
2010/0029512, which are incorporated by reference herein.
[0100] Post-Bridge Error Correction
[0101] In some embodiments of the system provided herein, the
system further comprises a post-bridge diagnostic system. In some
embodiments, the system may be used to detect normal droplets,
missing droplets, unmixed droplets, merged droplets. In some
embodiments the system may be used to detect the presence of air in
a droplet carriage. FIG. 12 is an example of a post bridge droplet
diagnostic system.
[0102] In some embodiments, the post-bridge detection system is a
post-bridge error correction detection system. In some embodiments,
the post-bridge correction system may include at least one light
emitting diode (LED). In some embodiments, the system may include
an array of blue light emitting diodes (LEDs) illuminating the
output line from the bridges (between the liquid bridges and the
thermocycler). Any suitable excitation source may be used including
but not limited to, LEDs, laser, or any other suitable excitation
source. In some embodiments, at least one, at least two, at least
three detectors may be used to monitor light emitted from a
droplet. In some embodiments, the detectors may be PMTs, or
cameras, or detector arrays. In some embodiments, three cameras
(for example Basler cameras) may used to monitor the fluorescent
wavelengths excited by the blue LEDs. In some embodiments, at least
one, at least two, at least three wavelengths may be monitored. In
some embodiments, the components, dyes, fluorescence emission
detected may be from the same is each droplet exiting the detection
system. In some embodiments, two of the emissions detected from the
droplet may be of the same wavelength and a third may be of a
different wavelength. For example purposes only, the system may be
able to detect FAM/VIC in the primer-probes droplet, ROX in the
Master-Mix droplet and a third dye (i.e. ALEXA) added to the sample
droplet as a reference. If the detection system picks up all three
wavelengths from a droplet, then this is considered a mixed and
valid droplet. However in some cases the bridges will not mix a
droplet correctly. This is found by determining that one or more of
the components are missing from the main droplet. In the event an
error occurs with a single droplet (or carriage) then this droplet
(or the entire carriage) will be re-sampled. Additionally, the
detection system may be used to measure a level of fluorescence in
a droplet to aid in sorting of droplets based on the droplet
contents.
[0103] In some embodiments of the system, the post-bridge detection
system may include at least one LED or an array of LEDs
illuminating the output line from the at least one liquid bridge or
at least one stacked liquid bridge but before the thermocycler. In
some embodiments, the detection system uses any suitable excitation
source, including but not limited to lasers, including both
fiber-coupled and freespace, electromagnetic radiation, white
light, filtered light Opposite the LEDs are fibers running to an
array. One cameras (Basler) monitors the fiber-array and detects
droplets passing the LEDs through variations in light intensity.
The system may then count the number of droplets in a carriage and
compare this to the number expected. If the numbers do not match an
error will be indicated and the carriage will be re-sampled.
[0104] In some embodiments, the droplet stream leaving the bridges
may be divided into packets or carriages (based upon the time-stamp
at which the robotics takes a sample. A carriage is defined when
the spacing between carriages or droplet trains is at least twice
the spacing between droplets. By dividing the droplets into
carriages, identification of individual droplets and identification
of errors in the droplet stream may be facilitated. For example
purposes, Droplet 2 of Carriage 2 (with 5 droplets per carriage)
may be identified more easily than Droplet 7 of a continuous
stream. Similarly errors can be easily identified. If only 4
droplets are present in a carriage of 5 then it is clear an error
has occurred (droplet merging); if 6 are present then a droplet has
not mixed or has mixed and then split into two.
[0105] In some embodiments, the number of droplets in a
carriage/droplet stream is known and/or expected. In some
embodiments, the number of droplets in a carriage is unknown. In
some embodiment, any change in the number of droplets in a carriage
indicates an error, including more droplets than expected, less
droplets than expected or a droplet from one carriage being present
in a second carriage. The presence, absence or dual droplet errors
may then be resolved by indicating that an error has occurred. This
indication may be that the whole droplet stream/carriage is
erroneous, the carriage and the adjacent carriage are erroneous,
one droplet in the carriage is erroneous, or multiple droplets in
the carriage are erroneous.
[0106] In some embodiments, the system may droplet detection may be
done using fluorescent detection. In some embodiments instead of
detecting the fluorescence emissions the absorbance of the droplet
can be detected. In such an embodiment, a single camera, LEDs and
related fibers may be used to first count all droplets in a
carriage. In some embodiments, the detection system may then be
used to also acquire the peak width which corresponds to the length
of a droplet. In some embodiments, the system may acquire the
diameter of the droplet, the volume of the droplet, size of the
droplet or any other suitable parameter. In such an embodiment, if
a carriage has more or less droplets than a predetermined amount of
droplets/carriage then the carriage fails and is rejected and
re-sampled. In some embodiments, the length of the droplet will
vary depending on how many of component droplets are present and
then combined by a mixer or liquid bridge into a mixed droplet. In
some embodiments, the carriage may be analyzed and the standard
deviation calculated. The standard deviation may then be divided by
the mean of the droplets in a carriage. In some embodiments, the
carriage may passed or fail if the result of the calculation is
either below or above a set threshold. The carriage is re-sampled
if it is failed. In some embodiments, error detection may occur
using a single droplet as opposed to a droplet carriage.
[0107] In some embodiments, the passage of a droplet between the
light source and the detection system may be used as a detection
method. The passage of a droplet between the light source and the
detection system may cause a unique signature away from baseline
measure. Although this signature may vary, in some embodiments, the
observed signal may be a sharp spike (sometimes followed by a
signal slightly above baseline) and then a sharp trough. The
leading edge of the droplet is focusing light intensity onto the
detector, resulting in the spike. The slightly higher signal may be
a result of the difference in refractive index between the oil and
aqueous signal. The trailing edge of the droplet may focus light
intensity away from the detector resulting in a sharp trough. As
the droplet clears the detector the baseline returns to normal. In
some embodiments, the leading edge of the droplet may lead to light
being directed away from the detector and the trailing edge
focusing light on the detector. In some embodiments, the signal may
be a peak, a trough, both a peak and trough, or an increase in
signal from baseline wherein the signal plateaus for a period of
time followed by a return in signal to baseline or a decrease from
baseline, a period of plateau, following by an increase back to
baseline. In some embodiments, air droplets or any other type of
droplet may be identified using the methods and system described
herein based on the unique signature of the air droplets.
[0108] In some embodiments, the light source is white light or
filtered light and a non-filtered detection source is used. The
wavelength of the light used may or may not affect the shape of the
droplet signatures detected. In some embodiments, the system may be
used to determine if all the components have combined into a mixed
droplet based on the spacing between the spikes which may correlate
to the width of the droplets.
[0109] In some embodiments, fluorescence or absorbance detection
may be used in conjunction with and applied to end-point
measurements. This may act as an additional quality control measure
to pick up any errors that are not caught by the post-bridge error
detection. In such a manner, the combination of factors may be used
to highlight any suspect data.
[0110] In some embodiments, the endpoint detection system may
include a free-space spectrograph system. In some embodiments, the
acquisition hardware is a Hamamatsu Orca camera. The 96
thermocycler lines are illuminated by a 488 nm laser-line. This
laser-line is imaged by the spectrograph/camera and resolved into
its constituent wavelengths. Appropriate wavelengths may then be
measured according to the contents of the droplets. Droplets may be
identified based upon the time-stamp generated by the post-bridge
detection module and raw fluorescent data is then generated for
droplet. In some embodiments, spectral compensation may then be
applied to compensate for dye bleed through. In some embodiments,
other methods of compensation may be used to compensate for dye
bleed through, including background/baseline subtraction, or any
other suitable method of compensation.
[0111] In order to maintain the high throughput of a continuous
flow PCR system, the PCR system needs to be able to detect
fluorescence in two or more micro-channels at the same time.
Measuring fluorescence across two or more micro-channels imposes a
number of limitations on an endpoint detection system.
[0112] For example, as the number of number of micro-channels is
increased, the field of view of the detector also needs to
increase. These micro-channels can be closely bundled or aligned
together in an array of transparent micro-channels or tubes.
However, a wall of some thickness has to be maintained between
tubes to prevent crosstalk between adjacent micro-channels. As a
result, the field of view of the detector is a function of the tube
diameter and tube array wall thickness. In order to maintain a high
fluorescence collection efficiency from the tubes on the edges of
the tube array, an increased beam length can be used. Increasing
the beam length from the tube array to the detector may increase
the overall physical size of the endpoint detection system.
[0113] In some embodiments, the system may be able to detect
spectral information from two or more micro-channels in a single
time step. However, in order to assign that spectral information to
the correct sample, the particular tube emitting that spectral
information may be located in the tube array. As result, the
detection system may provide spatial information in addition to
spectral information.
[0114] FIG. 13 is a schematic diagram of a side view of a system
3300 for detecting spectral and spatial information in a continuous
flow PCR system, in accordance with various embodiments. System
3300 includes laser 3310, line generator 3320, tube array 3330,
imaging lens 3340, spectrograph 3350, and imager 3360. Laser 3310
emits incident beam of electromagnetic radiation 3311.
[0115] Line generator 3320 receives incident beam 3311 from laser
3310. Line generator 3320 transforms incident beam 3311 into
incident line of electromagnetic radiation 3321. In other words,
line generator 3320 converts the power distribution of incident
beam 3311 from a non-uniform distribution to a uniform
distribution. Line generator 3320 is a Powell lens, for example. In
various embodiments, line generator 3320 is a diffractive line
generator.
[0116] Tube array 3330 receives incident line 3321 from line
generator 3320. Tube array 3330 includes one or more transparent
tubes in fluid communication with one or more micro-channels of a
PCR system. In various embodiments, one or more optical elements
3322 are placed between line generator 3320 and tube array 3320 to
steer incident line 3321 from line generator 3320 to tube array
3330. As shown in FIG. 13, one or more optical elements 3322 allow
system 3300 to be package in an overall smaller volume, for
example. In various embodiments, mirror 3325 is also placed between
line generator 3320 and tube array 3330 to steer incident line 3321
from line generator 3320 to tube array 3330. Mirror 3325 allows
tube array 3330 to be positioned horizontally in system 3300, for
example.
[0117] Imaging lens 3340 receives reflected electromagnetic
radiation 3331 from tube array 3330 and focuses reflected
electromagnetic radiation 3331. In various embodiments, one or more
optical elements (not shown) are placed between tube array 3330 and
imaging lens 3340 to steer reflected electromagnetic radiation 3331
from tube array 3330 to imaging lens 3340. In various embodiments,
mirror 3325 is placed between tube array 3330 and imaging lens 3340
to steer reflected electromagnetic radiation 3331 from tube array
3330 to imaging lens 3340. Imaging lens 3340 is a wide-iris lens
with a variable aperture, for example. In various embodiments,
imaging lens 3340 includes one or more optical filters (not shown).
The one or more optical filters remove reflection of incident line
3321 from reflected electromagnetic radiation 3331, for
example.
[0118] Spectrograph 3350 receives the focused reflected
electromagnetic radiation (not shown) from the imaging lens 3340.
Spectrograph 3350 detects a spectral intensity from the focused
reflected electromagnetic radiation. In some embodiments,
spectrograph 3350 can detect spectral wavelengths between 400 and
800 nanometers, for example. In some embodiments, the spectrograph
may be such that it can detect any suitable wavelength.
[0119] Imager 3360 receives the focused reflected electromagnetic
radiation from imaging lens 3340. Imager 3360 detects a location of
the spectral intensity. Imager 3360 is a CCD camera, for
example.
[0120] In various embodiments, system 3300 also includes a
processor (not shown). The processor receives the spectral
intensity from spectrograph 3350 and receives the location from
imager 3360. The processor determines an intensity value for a
sample moving through tube array 3330 from the spectral intensity
and the location.
Thermocycling
[0121] In some embodiments, the system may be in fluid
communication with a thermocycler. In some embodiments, the system
operates under the principal of continuous flow. In some
embodiments, a constant flow of oil may be maintained through the
thermocycler (TC Line) and this flow of oil may carry mixed
droplets. In some embodiments, the flow upstream of the
liquid-bridges (from sample-tips to bridges) is faster than the
flow through the thermocycler in order to meet throughput demands.
A Draft Line may be fitted to the bridge which bleeds off excess
oil. The TC Line and the Draft Line both operate at fixed flow
rates. It is required that they be controlled as the addition of
droplets to the lines increases the pressure drop along each line.
The combined flow in the TC Line and Draft Line equals that of the
master-mix, sample and primer-probe lines. In addition the pumping
system will incorporate a number of subsystems for priming the
system with oil and bleeding it of air. FIG. 6 shows a general
schematic (for a single line system) of showing the TC Line, the
Draft Line and where the hardware components are located. Also
shown in FIG. 7 is a schematic of our proposed software
architecture.
[0122] The thermocycler consists of 4 24-line thermocyclers. Each
block is preceded by a pre-heat block. Each block will be
maintained at its set-point using PID control.
[0123] Electrowetting/Vaporization
[0124] In some embodiments of the system, thermocycler may be used
thermal blocks may be used with or without other materials to
manipulate the thermal gradient of samples as they enter/exit
thermal zones. In some embodiments, the use of an in-line water
bath to provide a thermal step and more effective electrical
discharge of tubing prior to entering the preheat stage of the
thermal cycler. In some embodiments, a water bath may be used to
replace the existing preheat block.
[0125] In some embodiments, any static charge on the tubing
entering the preheat section of the thermocycler may be controlled
through the use of static generators and/or electrical circuits. In
some embodiments, electrically conductive PTFE tubing may be used
to provide more effective electrical discharge of the tubing. In
some embodiments, non-anodized components in thermocycler
assemblies may be used to provide more effective electrical
discharge of the tubing. In some embodiments, additives may be
added to the oil to increase electrical conductivity, or the use of
an alternative oil with better electrical conductivity. In
addition, in some embodiments, surfactants may be used to
manipulate the droplet-oil interfacial tension which would provide
a more resistant interface to vaporization. In some embodiments of
the system, the system may allow for pumping/processing of samples
through the thermocycler under positive pressure. In some
embodiments, environmental control of humidity/local external
pressure on the system may be controlled to produce less favourable
conditions for vaporization.
Detection
[0126] In some embodiments of the system, endpoint detection may
occur. In some embodiments, the system may include real-time
detection. In some embodiments, the system may include a free-space
Spectrograph system. For example, the acquisition hardware may be a
Hamamatsu Orca camera. The 96 thermocycler lines may illuminated by
a 488 nm laser-line. This laser-line may be imaged by the
spectrograph/camera and resolved into its constituent wavelengths.
In some embodiments, appropriate wavelengths may be measured
according to the contents of the droplets. Droplets may be
identified based upon the time-stamp generated by the post-bridge
detection module and raw fluorescent data may then be generated for
droplet. Spectral compensation may then be applied to compensate
for dye bleed through.
[0127] In another embodiment a single detector could be used, i.e a
single camera and filter wheel or a spectral camera based system
similar to our end-point system with the addition of optical
fibers. In some embodiments, the detector may be a spectrograph,
filterwheel/camera combo, acousto-optical tunable filter and
camera, photo-diode, photo-diode array, PMT, as well as
CCD/CMOS/digital cameras.
[0128] Droplet Dispensing or Collection
[0129] Using a microfluidic valve and a liquid bridge, the flow of
droplets can be controlled. For example purposes only, a droplet of
interest could be identified where the droplet is located in a
train of droplets. In such an embodiment, the droplet of interest
could be identified based on an optical detection system, wherein
the optical detection system may identify the droplet of interest
based on any suitable parameter, including but not limited to dye
color and/or concentration, turbidity, optical density, viscosity,
charge, polarity, light diffraction of diffusion, or any other
suitable parameter. Once the droplet of interest has been
identified by the detection system, a signal may be sent to a
microfluidic valve located upstream from the detection system to
direct the flow of the droplet through the system. In some
embodiment, the valve may be in fluid communication with at least
two microfluidic channels, wherein a single droplet is permitted to
travel through the channel at any given time. In some embodiments
the valve may be in fluid communication with at least three
microfluidic channels. In some embodiments, one of the microfluidic
channels may be in fluid communication with a collection system for
collecting the droplets. The droplet switching system may works as
follows. In some embodiments, a droplet generated by the liquid
bridge is sent through a primary microfluidic channel where a
parameter of interest may be detected. The droplet may be an
aqueous droplet surrounded by an immiscible fluid, such as oil.
Alternatively, the droplet may be an emulsion droplet of an oil
droplet surrounded by an immiscible fluid, wherein the immiscible
fluid is an aqueous fluid. The characteristics of the droplet
determine if the system dictates that the droplet is sent down
secondary channel A to be collected or through secondary channel B
for either further processing or for collection as waste. Once the
droplet passes through the valve into its proper secondary channel,
a new droplet passes through the detection system. Again, the
system may detect the presence or absence of a parameter of
interest and direct the second droplet to its proper secondary
channel. In some embodiments, there is a 30 second delay between
the droplets for the droplet detection system.
[0130] Optics
[0131] The optics of the system is such that the system can
simultaneously measure from 96 channels. A suitable embodiment of
the optical system may be found in U.S. patent application Ser. No.
______ (Atty Docket: LT00398 PRO) entitled Optical System and
Method of Use, which is incorporated by reference in its
entirety.
[0132] FIG. 13 is a schematic diagram of a side view of a system
3300 for detecting spectral and spatial information in a continuous
flow PCR system, in accordance with various embodiments. System
3300 includes laser 3310, line generator 3320, tube array 3330,
imaging lens 3340, spectrograph 3350, and imager 3360. Laser 3310
emits incident beam of electromagnetic radiation 3311.
[0133] Line generator 3320 receives incident beam 3311 from laser
3310. Line generator 3320 transforms incident beam 3311 into
incident line of electromagnetic radiation 3321. On other words,
line generator 3320 converts the power distribution of incident
beam 3311 from a non-uniform distribution to a uniform
distribution. Line generator 3320 is a Powell lens, for example. In
various embodiments, line generator 3320 is a diffractive line
generator.
[0134] Tube array 3330 receives incident line 3321 from line
generator 3320. Tube array 3330 includes one or more transparent
tubes in fluid communication with one or more micro-channels of a
PCR system. In various embodiments, one or more optical elements
3322 are placed between line generator 3320 and tube array 3320 to
steer incident line 3321 from line generator 3320 to tube array
3330. As shown in FIG. 13, one or more optical elements 3322 allow
system 3300 to be package in an overall smaller volume, for
example. In various embodiments, mirror 3325 is also placed between
line generator 3320 and tube array 3330 to steer incident line 3321
from line generator 3320 to tube array 3330. Mirror 3325 allows
tube array 3330 to be positioned horizontally in system 3300, for
example.
[0135] Imaging lens 3340 receives reflected electromagnetic
radiation 3331 from tube array 3330 and focuses reflected
electromagnetic radiation 3331. In various embodiments, one or more
optical elements (not shown) are placed between tube array 3330 and
imaging lens 3340 to steer reflected electromagnetic radiation 3331
from tube array 3330 to imaging lens 3340. In various embodiments,
mirror 3325 is placed between tube array 3330 and imaging lens 3340
to steer reflected electromagnetic radiation 3331 from tube array
3330 to imaging lens 3340. Imaging lens 3340 is a wide-iris lens
with a variable aperture, for example. In various embodiments,
imaging lens 3340 includes one or more optical filters (not shown).
The one or more optical filters remove reflection of incident line
3321 from reflected electromagnetic radiation 3331, for
example.
[0136] Spectrograph 3350 receives the focused reflected
electromagnetic radiation (not shown) from the imaging lens 3340.
Spectrograph 3350 detects a spectral intensity from the focused
reflected electromagnetic radiation. Spectrograph 3350 can detect
spectral wavelengths between 400 and 800 nanometers, for
example.
[0137] Imager 3360 receives the focused reflected electromagnetic
radiation from imaging lens 3340. Imager 3360 detects a location of
the spectral intensity. Imager 3360 is a CCD camera, for
example.
[0138] In various embodiments, system 3300 also includes a
processor (not shown). The processor receives the spectral
intensity from spectrograph 3350 and receives the location from
imager 3360. The processor determines an intensity value for a
sample moving through tube array 3330 from the spectral intensity
and the location.
Software
[0139] FIG. 14 is a block diagram that illustrates a computer
system 100, upon which embodiments of the present teachings may be
implemented. Computer system 100 includes a bus 102 or other
communication mechanism for communicating information, and a
processor 104 coupled with bus 102 for processing information.
Computer system 100 also includes a memory 106, which can be a
random access memory (RAM) or other dynamic storage device, coupled
to bus 102 for determining base calls, and instructions to be
executed by processor 104. Memory 106 also may be used for storing
temporary variables or other intermediate information during
execution of instructions to be executed by processor 104. Computer
system 100 further includes a read only memory (ROM) 108 or other
static storage device coupled to bus 102 for storing static
information and instructions for processor 104. A storage device
110, such as a magnetic disk or optical disk, is provided and
coupled to bus 102 for storing information and instructions.
[0140] Referring to FIG. 6 is a schematic diagram showing a system
200 for high throughput PCR amplification and analysis, in
accordance with various embodiments. System 200 includes PCR system
210 and processor 220. PCR system 210, in turn, includes liquid
handling system 230, fluid pumping system 240, post-bridge
detection system 250, thermocycler 260, and endpoint detection
system 270.
[0141] Processor 220 is in communication with PCR system 210.
Processor 220 can include, but is not limited to, a computer, a
microprocessor, a microcontroller, an application specific
integrated circuit (ASIC), or any device capable of executing
instructions and sending and receiving data or control
communications.
[0142] Processor 220 instructs liquid handling system 230 to obtain
a plurality of samples and a plurality of reagents for a PCR
experiment. In various embodiments, processor 220 instructs liquid
handling system 230 to pipette samples from a first sample support
device (not shown) located on tray 231 of liquid handling system
230, pipette assay reagents from a second sample support device
(not shown) located on tray 232 of liquid handling system 230, and
pipette a master mix reagent from vessel 233.
[0143] Processor 220 instructs fluid pumping system 240 to maintain
a continuous flow of a transport fluid through a plurality of
micro-channels. The transport fluid or oil is a passive buffer for
carrying samples around system 200. FIG. 6 shows a single
micro-channel of the plurality of micro-channels. This single
micro-channel or tube includes draft line 241 and thermocycler line
242. Draft line 241 is used to bleed off excess transport fluid and
maintain the continuous flow of a transport fluid through the
micro-channel at a constant flow rate. Thermocycler line 242 is
used to carry mixed samples through system 200.
[0144] Processor 220 instructs fluid pumping system 240 to maintain
a continuous flow of a transport fluid in order to receive the
plurality of samples and the plurality of reagents from liquid
handling system 230 as droplets in the plurality of micro-channels.
The continuous flow of a transport fluid by fluid pumping system
240 draws a sample droplet from tip 235 of liquid handling system
230 up through line 245 of fluid pumping system 240. Similarly, the
continuous flow of a transport fluid by fluid pumping system 240
draws an assay reagent droplet from tip 236 of liquid handling
system 230 up through line 246 of fluid pumping system 240 and
draws a master mix reagent droplet from tip 237 of liquid handling
system 230 up through line 247 of fluid pumping system 240, for
example.
[0145] Junction 249 is an exemplary liquid bridge for mixing
samples and reagents for a single micro-channel. Lines 245, 246,
and 247 meet at junction 249. Through precise timing control,
processor 220 instructs liquid handling system 230 to select
sample, assay reagent, and master mix droplets using tips 235, 236,
and 247 at specific times so that fluid pumping system 240 draws
these droplets to junction 249 at the same time. Because sample,
assay reagent, and master mix droplets reach junction 249
simultaneously, they are mixed as they are moving with the
continuous flow of transport fluid. The mixture produces a mixed
sample droplet. This mixed sample droplet leaves junction 249 and
enters thermocycler line 242. The mixed sample droplet continues
moving with the continuous flow of transport fluid at a constant
flow rate in thermocycler line 242.
[0146] In order to determine if each mixed sample droplet is mixed
correctly, processor 220 receives one or more post-bridge detection
values for each mixed sample droplet of the plurality of mixed
sample droplets from post-bridge detection system 250. Post-bridge
detection system 250, for example, detects mixed sample droplets in
thermocycler line 242 at precise time steps selected by processor
220. In various embodiments, post-bridge detection system 250 is an
optical system that includes one or more sources of illumination
and one or more cameras. In various embodiments, one camera is used
and the one or more post-bridge detection values include the
intensity of electromagnetic radiation absorbed or reflected by
each mixed sample droplet.
[0147] In various embodiments, three cameras are used by
post-bridge detection system 250. The one or more post-bridge
detection values received by processor 220 then include a first
intensity of electromagnetic radiation emitted by a first dye of a
sample of each mixed sample droplet, a second intensity of
electromagnetic radiation emitted by a second dye of an assay
reagent of each mixed sample droplet, and a third intensity of
electromagnetic radiation emitted by a third dye of a master mix
reagent of the mixed sample droplet. In various embodiments, the
one or more post-bridge detection values also include a time stamp
of the mixed sample droplet so the processor can identify the
sample and reagents used to create the mixed sample droplet.
[0148] In various embodiments, processor 220 instructs liquid
handling system 230 to re-sample a sample and an assay reagent of a
mixed sample droplet, if processor 220 determines from the one or
more post-bridge detection values that the mixed sample droplet is
mixed incorrectly. In other words, if processor 220 determines that
the one or more post-bridge detection values that the mixed sample
droplet are not indicative of a proper mixture, processor instructs
liquid handling system 230 to re-sample the sample and reagents
used to create the mixed sample droplet.
[0149] Finally, processor 220 receives from endpoint detection
system 270 one or more endpoint detection values for each mixed
sample droplet of the plurality of mixed sample droplets. Processor
220 uses the one or more endpoint detection values to analyze the
PCR experiment. In various embodiments, endpoint detection system
270 is also an optical detection system. Endpoint detection system
270 is a hyperspectral imaging system that determines both spatial
and spectral information, for example. Therefore, in various
embodiments, the one or more endpoint detection values include the
location of a micro-channel and a spectral intensity value detected
from that micro-channel. The location of the micro-channel allows
processor 220 to identify the mixed sample droplet and the spectral
intensity value detected provides a measure of the result of the
PCR experiment.
[0150] FIG. 15 is a schematic diagram of a system 400 that includes
one or more distinct software modules that perform a method for
high throughput PCR amplification and analysis, in accordance with
various embodiments. System 400 includes liquid handling module
410, fluid pumping module 420, post-bridge detection module 430,
thermocycler module 440, and endpoint detection module 450.
[0151] In order to enable system operation the following software
controlled elements are present: fluid pumping system, liquid
handling/plate handling system, post-bridge detection,
thermocycler, endpoint detection, and ancillary equipment. The
fluid pumping system includes five flow sensors, five pumps and
more than 40 level sensors and valves. The liquid handling/plate
handling system includes a plate stacker, a barcode reader, and a
15 axis sampling unit. The post-bridge detection includes three
Basler cameras. The thermocycler includes four 24-line temperature
controlled thermocyclers (TCs) each with separate denaturation
blocks. The endpoint detection includes one Hamamatsu Orca camera
and one laser.
[0152] In order to enable the system operation the following
software controlled elements are present: [0153] Fluid Pumping
System [0154] a. 5.times. Flow Sensors [0155] b. 5.times. Pumps
[0156] c. 40+Level Sensors and Valves [0157] Liquid Handling/Plate
Handling System [0158] a. OEM Plate Stacker [0159] b. Barcode
Reader [0160] c. 15 axis sampling unit [0161] Post-bridge Detection
[0162] a. 1.times. Basler Cameras [0163] Thermocycler [0164] a. 4
24-line temperature controlled TCs each with separate denaturation
block [0165] Endpoint Detection [0166] a. 1.times. Hamamatsu Orca
Camera [0167] b. 1.times. Laser [0168] Ancillary Equipment [0169]
a. LT00399 entitled High-throughput qPCR Control and Analysis
System, filed Dec. ______, 2010, and which is incorporated by
reference in its entirety.
[0170] In some embodiments, the system may be controlled using two
different ASCII.csv files. The command file will be titled in the
format BARCODETRAY1_BARCODETRAY2_cmds.csv while the volume file
will be titled BARCODETRAY1_vols.csv. The command file contains a
list of well combinations which will be sampled by the instrument.
The volume file contains information pertaining to the contents
(volume and components) of each well on the plate. On receiving a
RUN command the instrument will read the barcodes of each plate
present. It will search for matching command and volume files and
if present will process this project. Results will be outputted in
the form BARCODETRAY1_BARCODETRAY2_rslts.csv.
[0171] In FIG. 15 waypoints P1 through to P6 are shown. Both trays
T1 and T2 can access all 6 waypoints. In our current iteration P1
and P6 not used, P2 is used for barcode reading, P3 for
upstack/downstack into Hotel 1 on the plate-changer, P4 the same
for Hotel 2 and P5 will be used by Monsanto robots to load and
unload plates.
[0172] Graphical User Interface (GUI)
[0173] In some embodiments the system may provide for interaction
between the GUI and the instrument. In some embodiments, the
interaction includes commands to control the plate stacker and also
the transfer of files. In some embodiments, to transfer files an
FTP setup is used. There is an FTP server that stores files and
waits for clients to connect to it. The GUI acts as a client to
connect to the FTP server and transfer files. The instrument can
also connect to the same FTP server and transfer files.
To control the plate stacker a custom TCP interface is used. The
instrument acts as a server and waits for the GUI to connect to it.
After a connection is established predefined TCP commands may be
sent and received to control the instrument.
[0174] FTP
[0175] Command files and volume files can be created and modified
using the GUI. These files can then be transferred to the
instrument. The files are transferred using an FTP server. This
process is illustrated in FIG. 16. FIG. 16 is a schematic diagram
showing how files are transferred between a graphical user
interface (GUI) and an instrument, in accordance with various
embodiments. Command files and volume files can be created and
modified using the GUI. These files can then be transferred to the
instrument. The files are transferred using an FTP server.
[0176] FIG. 17 is a flowchart showing a method for uploading a file
using a file transfer protocol (FTP) server, in accordance with
various embodiments. To upload a file, the GUI sends a TCP command
to the instrument asking it for the address of the FTP server. Once
the instrument has responded with this information, the GUI
connects to the instrument and uploads a file. If the file already
exists on the FTP server the user is asked if they want to keep it
or overwrite it.
[0177] To download a file, the GUI sends a TCP command to the
instrument asking it for the address of the FTP server. Once the
instrument has responded with this information, the GUI connects to
the instrument and presents a list of files available for
downloading. The user selects a file, and the GUI then downloads it
to a predefined location on the local computer.
[0178] To upload a file the GUI sends a TCP command to the
instrument asking it for the address of the FTP server. Once the
instrument has responded with this information the GUI connects to
the instrument and uploads a file. If the file already exists on
the FTP server the user is asked if they want to keep it or
overwrite it.
[0179] To download a file the GUI sends a TCP command to the
instrument asking it for the address of the FTP server. Once the
instrument has responded with this information the GUI connects to
the instrument and presents a list of files available for
downloading. The user selects a file and the GUI then downloads it
to a predefined location on the local computer.
[0180] Plate Changing
[0181] The plate stacker allows the user of the instrument to load
multiple plates at once and run them without having to explicitly
load and run each plate combination individually. The stacker is
divided into two compartments. Each compartment is loaded with
plates. At run time the user tells the GUI which combinations to
run. The GUI doesn't know which plates are in the stacker. Through
a series of TCP commands instructing the instrument to transfer
plates between the stacker and the instrument proper, and barcode
the plates, the GUI can instruct the instrument to run all the
selected combinations.
[0182] The optics of the system is such that the system can
simultaneously measure from 96 channels. A suitable embodiment of
the optical system may be found in U.S. patent application Ser. No.
______ (Atty Docket: LT00399 PRO) entitled "High-throughput qPCR
Control and Analysis System", which is incorporated by reference in
its entirety.
[0183] Further provided herein is a method for detecting proper
mixing of at least three liquids, comprising mixing together a
first liquid, a second liquid, and a third liquid, each being
miscible with the others, to form a mixed sample droplet, the first
liquid comprising a first fluorescent dye, the second liquid
comprising a second fluorescent dye, and the third liquid
comprising a third fluorescent dye, each of the first, second, and
third fluorescent dyes emitting fluorescence upon excitation
wherein the fluorescence emitted from each is spectrally resolvable
from the fluorescence emitted from the others, moving the mixed
sample droplet in a conduit, irradiating the mixed sample droplet
in the conduit with an excitation source; and detecting emissions
from the mixed sample droplet to determine whether each of the
first, second, and third fluorescent dyes is present in the mixed
sample droplet.
[0184] Provided herein is a method for detecting a droplet in
system comprising moving the mixed sample droplet in a conduit;
irradiating the mixed sample droplet in the conduit with an
excitation source; and detecting emissions from the mixed sample
droplet to determine whether each of the first, second, and third
fluorescent dyes is present in the mixed sample droplet. In some
embodiments of the method, the first liquid comprises a first
droplet, the first droplet is encompassed by a carrier fluid that
is substantially immiscible with the first liquid, the second
liquid comprises a second droplet, the second droplet is
encompassed by the carrier fluid, the third liquid comprises a
third droplet, and the third droplet is encompassed by the carrier
fluid. The mixed sample droplet may be encompassed by a carrier
fluid that is substantially immiscible with the mixed sample
droplet. The mixed sample droplet may be formed at an intersection
of the conduit with three other conduits, each of the other
conduits containing therein the first liquid, the second liquid,
and the third liquid, respectively. The excitation source may
include one or more LEDs. The excitation source comprises one or
more blue LEDs, each blue LED emitting an excitation beam having a
single wavelength that excites each of the first, second, and third
fluorescent dyes. The detecting comprises detecting emission from
the first fluorescent dye using a first detector, detecting
emission from the second fluorescent dye using a second detector,
and detecting emission from the third fluorescent dye using a third
detector. In some embodiments, the method may further comprise
tracking the mixed sample droplet as it moves in the conduit and
accepting or rejecting data generated by downstream processing of
the mixed sample droplet based on the emissions detected.
Additionally, the method may further comprising forming a train of
droplets including the mixed sample droplet and detecting emissions
from each droplet of the train of droplets. In some embodiments,
the method may further comprising forming a train of droplets
including the mixed sample droplet, the train of droplets
comprising carriages each comprising a plurality of spaced apart
droplets, wherein a first spacing is provided between adjacent
droplets within each carriage, and the carriages are spaced apart
from adjacent carriages by a second spacing that differs from the
first spacing. Additionally the methods provided herein may include
determining, based on the detected emissions, that proper mixing of
the first liquid, second liquid, and third liquid has occurred in
the mixed sample droplet; and gathering data from downstream
processing of the mixed sample droplet. Alternatively, the method
may comprise determining, based on the detected emissions, that
improper mixing of the first liquid, second, liquid, and third
liquid has occurred in the mixed sample droplet; and recording
occurrence of an error; forming a new mixed sample droplet from the
first liquid, the second liquid, and the third liquid; and ignoring
data generated by downstream processing of the mixed sample
droplet. In some embodiments, the first and second dyes comprise a
passive reference dye and the third dye comprises a reporter
dye.
EXAMPLES
Example 1
[0185] In some embodiments of the system, an alternative approach
to post-bridge diagnostic detection may occur. In such an
embodiment of an alternative approach for the post bridge error
correction a single camera is used and the droplet time peak width
(corresponding to droplet length) is detected. Using the droplet
peak width approach and incorporating a +/-7% tolerance, erroneous
droplet carriages can be identified. Carriages of 9 droplets (3
reactions in triplicate) were used.
[0186] A droplet count check is used to pass or fail a carriage.
Then standard deviation of the 9 droplet carriage is then
calculated. If the standard deviation is above a set threshold
based on a set tolerance, then the carriage is rejected. The
results of which are presented in Tables 1, 2, and 3.
TABLE-US-00001 TABLE 1 Droplet Types Weighting (droplet
length/time) Standard Droplet 10 MasterMix & Sample (MM&GA)
8.5 MasterMix 7 Sample/Gene Assay (GA) 1.5 Note: Weighting is based
on the percentage size of the droplet from initial viewing of video
evidence.
TABLE-US-00002 TABLE 2 Main Premixing 1 Droplet 1 Sample & 3
This case will Failure Droplet 2 GA MM 7 cause a droplet Events
error count No Droplet 1 Sample 1.5 This case will Mixing Droplet 2
GA 1.5 cause a droplet Droplet 3 MM 7 count error Droplet This case
will Splitting cause a droplet (rare) count error Premixing 2
Droplet 1 Sample (or 8.5 This case will Droplet 2 GA) & MM 11.5
not cause error Standard Droplet & Sample
TABLE-US-00003 TABLE 3 Premixing 1 Allow a Allow a (Droplet Allow a
Perfect tolerance of tolerance of Count & tolerance of Droplet
# Carriage +/-5% +/-7% Premixing 2 Width) +/-10% 1 10 10.5 10.7 10
3 10 2 10 10.5 10.7 10 7 9 3 10 10.5 10.7 10 10 11 4 10 9.5 9.3 8.5
10 9 5 10 9.5 9.3 11.5 10 10 6 10 10.5 10.7 10 10 11 7 10 9.5 9.3
10 10 9 8 10 9.5 9.3 10 10 10 9 10 10.5 10.7 10 10 11 10 Standard 0
0.527046277 0.737864787 0.75 2.309401077 0.866025404 Deviation Pass
Pass Pass Fail Fail Fail
Example 2
Schematic of One Embodiment of the System
[0187] FIG. 18 shows one embodiment of a schematic of the system
described herein.
[0188] Graphical User Interface Module:
[0189] This part of the program is seen by the user to access the
machine and all components of the program.
[0190] The layout of the GUI module will be dictated by the
functionality of the main program: [0191] From SB: [0192] Is
machine setup required before each run? Or intermittently? [0193]
Is image calibration required before each run? Or intermittently?
[0194] From Monsanto: [0195] Does the machine need to have random
access--well by well access--or will the machine access entire
plates for every run? [0196] Should results be plotted on screen or
generated in the background? [0197] What format are Monsanto
databases in? What information from the databases should be passed
through the program to the Report Generation step?
[0198] Image Calibration Module/Intensity Data Module
[0199] These modules will be based on existing Matlab code. They
are used to take fluorescent intensity data from the cameras and
format it into a series of numerical values suitable for data
analysis. [0200] Technology: [0201] Imaging Source Cameras or
alternatives: [0202] Driver Compatibility [0203] Method of
Detection [0204] Fibres or alternatives (e.g. Current Digital
Setup) [0205] Three Cameras [0206] Endpoint only or Entry/Exit
Measurements [0207] Other Issues?
[0208] System Controls Module (Command List/Stage Movements)
[0209] This section of the program will control the flow rates with
the platform, and will also control the positioning stages in order
to generate a series of mixed droplets in the correct order.
Pumps/Sensors:
[0210] Will the system run using 8 (or 12) HNP Pump or Sensor
Combinations: [0211] Thermocycler Line [0212] Primary Draft Line
[0213] TAQ Draft Line [0214] Alternative Architecture [0215]
Alternative Components [0216] Large Pump with Flow Control
Valves
Stages
[0216] [0217] Equipment: [0218] Standa Stages [0219] Alternative
Stages [0220] More Expensive [0221] More Robust [0222] Faster
[0223] Require Drivers [0224] Dip Heights [0225] High-speed dipping
[0226] Incremental dipping [0227] Sensor measuring interface [0228]
Secondary Dipping/Wrapped Tip [0229] Interdependence of flow-rates
and dip-times. Lock in flow-rates.
Method
[0229] [0230] Analysis in duplicate/triplicate [0231] Location of
NTCs [0232] How will droplets be identified [0233] Carriage Spacing
[0234] Spiked Droplets [0235] Effect stage movements
[0236] Data Analysis Module
[0237] This module will be based on existing Matlab code. The
module will take in intensity and time data which has been
formatted correctly. It will analyse this data looking for discrete
droplets. These droplets will then be associated with a
PRIMER/SAMPLE pair which is also loaded into the program. The
intensities of FAM/VIC will then be calculated and reports
generated in the correct format. Errors in carriages (too many/too
few droplets) will be reported [0238] Measurement Locations [0239]
After Mixer [0240] Cycle 7 [0241] Cycle 42 [0242] Data-stream form
[0243] Trains/Carriages using spacing [0244] Trains/Carriages using
spiked drops [0245] Method of Data Analyse [0246] Endpoint
Intensities [0247] Normalise using ROX [0248] Normalise using ROX
and Cycle 7 [0249] Format for report generation [0250] VIC vs. FAM
plots [0251] Table of Boolean Data [0252] Exception Handling
[0253] Report Generation Module
[0254] This module outputs formatted data both to files and to the
GUI. [0255] Format of the output files/data [0256] What is
recorded--what is discarded [0257] Compatible with the Monsanto
Database [0258] Exception Reports
[0259] Exception Handling
[0260] This module is used as a link between the data-analysis
module and the stage control module. It will also monitor the
performance of the physical components of the system and take
appropriate action. [0261] Droplet Stream Error [0262] Not enough
droplets per carriage [0263] Action e.g. Repeat Carriage and Log
[0264] Action e.g. Increment dipping tip down [0265] Too many
droplets per carriage [0266] Action e.g. Repeat Carriage and Log
[0267] Action e.g. Reduce Primer Dip time [0268] No droplets
detected [0269] Action e.g. Abort Run [0270] Other possible errors
[0271] Component Error [0272] Stage Motion not detected [0273] Flow
rates outside tolerances [0274] Flow sensor noise-free
[0275] Overall System Architecture:
[0276] The system will be required to run off one PC. Architecture
must permit components to reach this PC and be connected to it. In
a lab environment it would be advantages to have as few exposed
cables as possible linking the PC to the platform. [0277] List of
Components: [0278] Powerful PC for Data Analysis and Report
Generation [0279] Sufficient Ports/Connectors to handle all
components: [0280] Example: [0281] 3.times. Firewire Cameras [0282]
8.times.RS-232 for 8 sensors [0283] 1.times.RS-232 for 8 pumps
[0284] 2.times.USB for 6 stages
[0285] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *