U.S. patent application number 14/173974 was filed with the patent office on 2014-06-05 for manipulating droplet size.
This patent application is currently assigned to Raindance Technologies, Inc.. The applicant listed for this patent is Raindance Technologies, Inc.. Invention is credited to Darren Roy Link, Benjamin J. Miller, Qun Zhong.
Application Number | 20140154695 14/173974 |
Document ID | / |
Family ID | 48135176 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140154695 |
Kind Code |
A1 |
Miller; Benjamin J. ; et
al. |
June 5, 2014 |
MANIPULATING DROPLET SIZE
Abstract
The invention generally relates to methods and systems for
manipulating droplet size. In certain aspects, the invention
provides methods for manipulating droplet size that include forming
droplets of aqueous fluid surrounded by an immiscible carrier
fluid, and manipulating droplet size during the forming step by
adjusting pressure exerted on the aqueous fluid or the carrier
fluid.
Inventors: |
Miller; Benjamin J.;
(Littleton, MA) ; Zhong; Qun; (Lexington, MA)
; Link; Darren Roy; (Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Raindance Technologies, Inc. |
Billerica |
MA |
US |
|
|
Assignee: |
Raindance Technologies,
Inc.
Billerica
MA
|
Family ID: |
48135176 |
Appl. No.: |
14/173974 |
Filed: |
February 6, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13554655 |
Jul 20, 2012 |
8658430 |
|
|
14173974 |
|
|
|
|
61509837 |
Jul 20, 2011 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
239/61 |
Current CPC
Class: |
B01L 2400/0487 20130101;
B01L 3/502761 20130101; B01L 2200/0647 20130101; B01L 3/502784
20130101; B05B 7/0012 20130101; B01L 2300/0867 20130101; Y10T
436/2575 20150115; B01F 3/0807 20130101; C12Q 1/6806 20130101; B01F
15/00357 20130101; B01L 2200/141 20130101; B01L 2200/148 20130101;
B01L 2300/14 20130101; B05B 1/08 20130101; B01L 7/525 20130101;
B01F 13/0062 20130101; B01L 2200/143 20130101; B01L 2200/025
20130101; B05B 1/02 20130101; B05B 1/26 20130101 |
Class at
Publication: |
435/6.12 ;
239/61 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B05B 1/02 20060101 B05B001/02 |
Claims
1-17. (canceled)
18. A microfluidic apparatus comprising: a plurality of
microfluidic circuits, each circuit comprising: a first fluid
channel comprising a first resistance and a first fluid; a second
fluid channel, comprising a second resistance and a second fluid
that is immiscible with the first fluid; a junction where the first
fluid channel and the second fluid channel meet such that a
plurality of droplets are formed; and an outlet channel that exits
the junction and receives the droplets; wherein the first and
second resistances of the microfluidic circuits are configured such
that variation in pressure exerted on the first fluid in each
circuit results in a change in flow rate of the second fluid and a
change in a dimension of the droplets.
19. The microfluidic apparatus of claim 18, wherein the change in
flow rate of the second fluid is an inverse change to the variation
in pressure of the first fluid.
20. The microfluidic apparatus of claim 18, wherein the droplets
flow in the second fluid through the outlet channel.
21. The microfluidic apparatus of claim 18, wherein the change in
dimension of the droplets allows for control of coalescence of the
droplets from a first microfluidic circuit with droplets from a
second microfluidic circuit.
22. The microfluidic apparatus of claim 18, wherein the junction
comprises a nozzle.
23. The microfluidic apparatus of claim 18, wherein the outlet
channel from each of the microfluidic circuits connects to the same
main channel.
24. The microfluidic apparatus of claim 23, wherein the main
channel connects to one or more analysis modules.
25. The microfluidic apparatus of claim 24, wherein the analysis
modules comprise a merging module, an amplification module, a
detection module, and a sorting module.
26. The microfluidic apparatus of claim 18, wherein the first
fluids in the microfluidic circuits are different from each
other.
27. The microfluidic apparatus of claim 18, wherein the first
fluids in the microfluidic circuits are the same.
28. The microfluidic apparatus of claim 18, wherein the variation
in pressure is exerted on the first fluid in all of the
microfluidic circuits independently.
29. A microfluidic apparatus comprising: a plurality of
microfluidic circuits each configured to produce droplets; and a
regulator operably associated with each microfluidic circuit that
controls pressure exerted on a first fluid that causes a change in
flow rate of a second fluid, and adjusts a dimension of the
droplets from each microfluidic circuit.
30. The microfluidic apparatus of claim 29, wherein each
microfluidic circuit comprises: a first fluid channel comprising a
first resistance and the first fluid; a second fluid channel
comprising a second resistance and the second fluid that is
immiscible with the first fluid; a junction where the first fluid
channel and the one or more second fluid channels meet such that a
plurality of the droplets are formed; and an outlet channel that
exits the junction and receives the droplets; wherein the first and
second resistances of the microfluidic circuits are configured such
that variation in pressure exerted on the first fluid in each
circuit results in the change in flow rate of the second fluid and
a change in the dimension of the droplets.
31. The microfluidic apparatus of claim 29, wherein the pressure
regulator associated with each circuit controls pressure exerted on
the first fluid in each microfluidic circuit.
32. The microfluidic apparatus of claim 29, wherein a second
pressure regulator controls pressure exerted on the second fluid in
all of the microfluidic circuits.
33. The microfluidic apparatus of claim 29, further comprising a
pressure source that provides pressure exerted on the first fluid
in the microfluidic circuits.
34. The microfluidic apparatus of claim 33, wherein the pressure
source provides pressure exerted on the second fluid in the
microfluidic circuits.
35. The microfluidic apparatus of claim 30, wherein the junction
comprises a nozzle.
36. The microfluidic apparatus of claim 30, wherein the outlet
channel from each of the microfluidic circuits connects to the same
main channel.
37. The microfluidic apparatus of claim 36, wherein the main
channel connects to one or more analysis modules.
38. The microfluidic apparatus of claim 37, wherein the analysis
modules comprise a merging module, an amplification module, a
detection module, and a sorting module.
Description
RELATED APPLICATION
[0001] The present application claims benefit of and priority to
U.S. provisional application Ser. No. 61/509,837, filed Jul. 20,
2011, the content of which is incorporated by reference herein in
its entirety.
FIELD OF THE INVENTION
[0002] The invention generally relates to methods and systems for
manipulating fluidic droplet size.
BACKGROUND
[0003] The ability to precisely manipulate fluidic streams enhances
the use and effectiveness of microfluidic devices. Typically,
networks of small channels provide a flexible platform for
manipulation of small amounts of fluids. Certain microfluidic
devices utilize aqueous droplets in an immiscible carrier fluid.
The droplets provide a well-defined, encapsulated microenvironment
that eliminates cross contamination and changes in concentration
due to diffusion or surface interactions.
[0004] Microfluidic devices for performing biological, chemical,
and diagnostic assays generally include at least one substrate
containing one or more etched or molded channels. The channels are
generally arranged to form individual fluid circuits, each circuit
including a sample fluid channel, an immiscible carrier fluid
channel, and an outlet channel. The channels of each circuit may be
configured such that they meet at a junction so that droplets of
aqueous fluid surrounded by carrier fluid are formed at the
junction and flow into the outlet channel. In some cases, the
outlet channel of each circuit is connected to a main channel that
receives all of the droplets from the different fluidic circuits
and flows them to an analysis module. In other cases, the outlet
channels connect to exit ports to carry the droplets to a
collection vessel.
[0005] Since each fluidic circuit may have different samples, and
because different compositions (e.g., concentration and/or length
of nucleic acid) from different samples affect how droplets form,
droplets of different sizes may be produced by each circuit. A
problem with droplets of different sizes flowing through the same
channel is that the droplets travel at different velocities.
Droplets traveling at different velocities may cause unwanted
collisions or unwanted coalescence of droplets in the channel.
Thus, it is important that individual fluidic circuits produce
droplets of uniform size so that the droplets travel at the same
velocity in the channel and do not collide or coalesce in an
unwanted manner.
[0006] Droplets are typically generated one at a time at a junction
between an aqueous fluid and an immiscible carrier fluid. Droplet
volume and frequency (the number of droplet generated per unit
time) are determined by geometrical factors such as the
cross-sectional area of the channels at the junction and the
fluidic properties such as the fluid viscosities and surface
tensions as well as the infusion rates of the aqueous and carrier
fluids. To control the volume of the aqueous droplet, within a
range, droplet volume can be adjusted by tuning the oil infusion
rate through the junction. This is readily achieved with a pressure
regulator on the carrier fluid stream. In some cases it is
desirable to have multiple junctions operating as separate circuits
to generate droplets and have independent control over the oil
infusion rates through each circuit. This is readily achieved by
using separate pressure regulators for each aqueous stream and each
carrier fluid stream. A simpler and lower cost system would have a
single carrier oil source at a single pressure providing a flow of
carrier oil through each system. The problem with such a system is
that in adjusting the pressure to regulate the flow of carrier oil
in one circuit the carrier oil in all circuits would be effected
and independent control over droplet volume would be compromised.
Thus, it is important to have a means whereby at a fixed carrier
oil pressure the flow of carrier oil in each of the circuits can be
independently controlled to regulate droplet volume.
SUMMARY
[0007] The invention generally relates to methods and systems for
manipulating droplet size. The invention recognizes that in a
fluidic circuit, changing the pressure exerted on the aqueous phase
changes the flow rate of the immiscible carrier fluid. Changing the
flow rate of the immiscible fluid manipulates the size of the
droplet. Thus, adjusting pressure, which changes flow rate, adjusts
droplet size. Pressure adjustments may be made independent of one
another such that the pressure exerted on the aqueous phase in
individual fluidic circuits can be adjusted to produce droplets of
uniform size from the different fluidic circuits. In this manner,
droplets produced from different fluidic circuits travel at the
same velocity in a main channel and do not collide or coalesce in
an unwanted manner.
[0008] In certain aspects, the invention provides methods for
manipulating droplet size that involve forming droplets of aqueous
fluid surrounded by an immiscible carrier fluid, and manipulating
droplet size during the forming step by adjusting pressure exerted
on the aqueous fluid or the carrier fluid. Methods of the invention
involve forming a sample droplet. Any technique known in the art
for forming sample droplets may be used with methods of the
invention. An exemplary method involves flowing a stream of sample
fluid so that the sample stream intersects two opposing streams of
flowing carrier fluid. The carrier fluid is immiscible with the
sample fluid. Intersection of the sample fluid with the two
opposing streams of flowing carrier fluid results in partitioning
of the sample fluid into individual sample droplets. The carrier
fluid may be any fluid that is immiscible with the sample fluid. An
exemplary carrier fluid is oil. In certain embodiments, the carrier
fluid includes a surfactant, such as a fluorosurfactant.
[0009] Methods of the invention may be conducted in microfluidic
channels. As such, in certain embodiments, methods of the invention
may further involve flowing the droplet channels and under
microfluidic control. Methods of the invention further involve
measuring the size of a generated droplet. Any method known in the
art may be used to measure droplet size. Preferable methods involve
realtime image analysis of the droplets, which allows for a
feedback loop to be created so that droplet size may be adjusted in
real-time. In certain embodiments, measuring the droplet size is
accomplished by taking an image of the droplet and measuring a
midpoint of an outline of the droplet image, as opposed to
measuring an inside or an outside of the droplet.
[0010] Another aspect of the invention provides methods for forming
droplets of a target volume that include flowing an aqueous fluid
through a first channel, flowing an immiscible carrier fluid
through a second channel, forming an aqueous droplet surrounded by
the carrier fluid, and adjusting resistance in the first or second
channels during the forming step to adjust volume of the droplets,
thereby forming droplets of a target volume.
[0011] Another aspect of the invention provides methods for forming
substantially uniform droplets that involve flowing a plurality of
different aqueous fluids through a plurality of different channels,
flowing an immiscible carrier fluid through a carrier fluid
channel, forming substantially uniform droplets of the different
aqueous fluids, each droplet being surrounded by the carrier fluid,
by independently adjusting resistance in the different
channels.
[0012] Another aspect of the invention provides microfluidic chips
that include a substrate, and a plurality of channels, in which at
least two of the channels include pressure regulators, the pressure
regulators being independently controllable. Generally, the
plurality of channels include at least one aqueous fluid channel,
at least one immiscible carrier fluid channel, at least one outlet
channel, and a main channel. In certain embodiments, the channels
are configured to form microfluidic circuits, each circuit
including an aqueous fluid channel, a carrier fluid channel, and an
outlet channel. The channels of each circuit meet at a junction
such that droplets of aqueous fluid surrounded by carrier fluid are
formed at the junction and flow into the outlet channel. Each
outlet channel of each circuit is connected to the main channel.
The channels may be etched or molded into the substrate. The
channels may be open channels or enclosed channels. Droplets may be
collected in a vessel on the device or off of the device.
[0013] Another aspect of the invention provides droplet systems
that include a microfluidic chip that include a substrate, and a
plurality of channels, in which at least two of the channels
include pressure regulators, the pressure regulators being
independently controllable; and a pressure source coupled to the
chip.
[0014] Other aspects and advantages of the invention are provided
in the following description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a drawing showing a device for droplet
formation.
[0016] FIG. 2 is a drawing showing a device for droplet
formation.
[0017] FIG. 3 is a graph showing droplet size sensitivity to
changes in aqueous flow rate when using positive displacement
pumping.
[0018] FIG. 4 is a graph showing droplet size sensitivity to
changes in aqueous flow rate when using pressure driven
pumping.
[0019] FIG. 5 shows a diagram of a single fluidic circuit.
[0020] FIG. 6 is a drawing illustrating that the same volume drop
is subject to extreme changes in the lighting but the midpoint is
always the same. From left to right, the intensity of the lighting
decreases but the midpoint of the outline is always the same.
[0021] FIGS. 7A-C provides three graphs that demonstrate the
differences in the droplet measuring techniques, and the projected
area required to produce 5 pL drops when using the inside, outside
and midpoint of a droplet image.
[0022] FIG. 8 is a schematic illustrating measurement of droplet
size using the midpoint technique described herein.
[0023] FIG. 9 is a schematic diagram showing a microfluidic
interconnect as described in the Specification, containing a
plurality of aqueous fluid ports and an immiscible fluid port for
use in methods of the invention.
[0024] FIG. 10 is a schematic diagram showing an apparatus as
described in the Specification showing the microfluidic
interconnect shown in FIG. 9 with a manifold overlay and immiscible
fluid storage.
[0025] FIG. 11 is a schematic diagram showing the relationship
between the microfluidic interconnect of FIG. 9 with a microfluidic
chip for use in methods of the invention.
DETAILED DESCRIPTION
[0026] The invention generally relates to methods and systems for
manipulating droplet size. In certain aspects, the invention
provides methods for manipulating droplet size that involve forming
droplets of aqueous fluid surrounded by an immiscible carrier
fluid, and manipulating droplet size during the forming step by
adjusting pressure exerted on the aqueous fluid or the carrier
fluid.
Droplet Formation
[0027] Methods of the invention involve forming sample droplets. In
certain embodiments, the droplets include nucleic acid from
different samples. In particular embodiments, each droplet includes
a single nucleic acid template, a single protein molecule or single
cell. The droplets are aqueous droplets that are surrounded by an
immiscible carrier fluid. Methods of forming such droplets are
shown for example in Link et al. (U.S. patent application numbers
2008/0014589, 2008/0003142, and 2010/0137163), Stone et al. (U.S.
Pat. No. 7,708,949 and U.S. patent application number
2010/0172803), Anderson et al. (U.S. Pat. No. 7,041,481 and which
reissued as U.S. Pat. No. RE41,780) and European publication number
EP2047910 to Raindance Technologies Inc. The content of each of
which is incorporated by reference herein in its entirety.
[0028] FIG. 1 shows an exemplary embodiment of a device 100 for
droplet formation. Device 100 includes an inlet channel 101, and
outlet channel 102, and two carrier fluid channels 103 and 104.
Channels 101, 102, 103, and 104 meet at a junction 105. Inlet
channel 101 flows sample fluid to the junction 105. Carrier fluid
channels 103 and 104 flow a carrier fluid that is immiscible with
the sample fluid to the junction 105. Inlet channel 101 narrows at
its distal portion wherein it connects to junction 105 (See FIG.
2). Inlet channel 101 is oriented to be perpendicular to carrier
fluid channels 103 and 104. Droplets are formed as sample fluid
flows from inlet channel 101 to junction 105, where the sample
fluid interacts with flowing carrier fluid provided to the junction
105 by carrier fluid channels 103 and 104. Outlet channel 102
receives the droplets of sample fluid surrounded by carrier
fluid.
[0029] The sample fluid is typically an aqueous buffer solution,
such as ultrapure water (e.g., 18 mega-ohm resistivity, obtained,
for example by column chromatography), 10 mM Tris HCl and 1 mM EDTA
(TE) buffer, phosphate buffer saline (PBS) or acetate buffer. Any
liquid or buffer that is physiologically compatible with enzymes
can be used. The carrier fluid is one that is immiscible with the
sample fluid. The carrier fluid can be a non-polar solvent, decane
(e.g., tetradecane or hexadecane), fluorocarbon oil, silicone oil
or another oil (for example, mineral oil).
[0030] In certain embodiments, the carrier fluid contains one or
more additives, such as agents which reduce surface tensions
(surfactants). Surfactants can include Tween, Span,
fluorosurfactants, and other agents that are soluble in oil
relative to water. In some applications, performance is improved by
adding a second surfactant to the sample fluid. Surfactants can aid
in controlling or optimizing droplet size, flow and uniformity, for
example by reducing the shear force needed to extrude or inject
droplets into an intersecting channel. This can affect droplet
volume and periodicity, or the rate or frequency at which droplets
break off into an intersecting channel. Furthermore, the surfactant
can serve to stabilize aqueous emulsions in fluorinated oils from
coalescing.
[0031] In certain embodiments, the droplets may be coated with a
surfactant. Preferred surfactants that may be added to the carrier
fluid include, but are not limited to, surfactants such as
sorbitan-based carboxylic acid esters (e.g., the "Span"
surfactants, Fluka Chemika), including sorbitan monolaurate (Span
20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span
60) and sorbitan monooleate (Span 80), and perfluorinated
polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/or FSH). Other
non-limiting examples of non-ionic surfactants which may be used
include polyoxyethylenated alkylphenols (for example, nonyl-,
p-dodecyl-, and dinonylphenols), polyoxyethylenated straight chain
alcohols, polyoxyethylenated polyoxypropylene glycols,
polyoxyethylenated mercaptans, long chain carboxylic acid esters
(for example, glyceryl and polyglycerl esters of natural fatty
acids, propylene glycol, sorbitol, polyoxyethylenated sorbitol
esters, polyoxyethylene glycol esters, etc.) and alkanolamines
(e.g., diethanolamine-fatty acid condensates and
isopropanolamine-fatty acid condensates).
[0032] In certain embodiments, the carrier fluid may be caused to
flow through the outlet channel so that the surfactant in the
carrier fluid coats the channel walls. In one embodiment, the
fluorosurfactant can be prepared by reacting the perflourinated
polyether DuPont Krytox 157 FSL, FSM, or FSH with aqueous ammonium
hydroxide in a volatile fluorinated solvent. The solvent and
residual water and ammonia can be removed with a rotary evaporator.
The surfactant can then be dissolved (e.g., 2.5 wt %) in a
fluorinated oil (e.g., Flourinert (3M)), which then serves as the
carrier fluid.
Manipulating Droplet Size
[0033] The invention recognizes that in a fluidic circuit, changing
the pressure exerted on the aqueous phase changes the flow rate of
the immiscible carrier fluid. Changing the flow rate of the
immiscible fluid manipulates the size of the droplet. Thus,
adjusting pressure, which changes flow rate, adjusts droplet size.
Pressure adjustments may be made independently of each other such
that the pressure exerted on the aqueous phase in individual
fluidic circuits can be adjusted to produce droplets of uniform
size from the different fluidic circuits. In this manner, droplets
produced from different fluidic circuits travel at the same
velocity in a main channel and do not collide or coalesce in an
unwanted manner. When the pressure is the variable parameter used
for control, there is coupling between the aqueous and immiscible
carrier fluid (e.g., oil) channels in an individual circuit.
Therefore, any change to the aqueous pressure has an impact on the
pressure at the nozzle and in turn affects the flow rate of the
immiscible carrier fluid (IMF). For instance, increasing P.sub.Aq,
decreases Q.sub.IMF and vice-versa. Proper design of the
resistances in both the aqueous and immiscible carrier fluid
channels controls the degree of coupling that can be expected when
making a change to one or more of the input pressures. This in turn
controls the sensitivity of the change in drop volume as a function
of P.sub.A.
[0034] For comparison, the sensitivity of drop size to a change in
flow rate is compared using both a positive displacement pump and a
pressure driven system. FIG. 3 is a graph showing droplet size
sensitivity to changes in aqueous flow rate when using positive
displacement pumping. FIG. 4 is a graph showing droplet size
sensitivity to changes in aqueous flow rate when using pressure
driven pumping. Oil was used as the immiscible fluid for these
comparisons. Using a similar chip with a similar circuit, a
positive displacement pump yields a 10% change in drop volume when
changing the flow rate by a factor of two. The pressure driven
system yields a 2% change in drop volume for every psi of change in
P.sub.A. If the pressure was doubled, a 60% change in drop size
could be expected when using the pressure driven system. Using a
similar circuit, pressure gives 6.times. better control over the
droplet volume when the aqueous channel is adjusted.
[0035] In certain embodiments, multiple fluidic circuits are used
to produce droplets that all flow into a main channel. Proper
design of the fluidic circuits, specifically by adjusting the
fluidic resistance in both the aqueous and oil channels, controls
the degree of influence that adjustments to the aqueous pressure
has on each of the circuits, resulting in all of the circuits
producing droplets of the same size. Changes in droplet size as a
result of changes in pressure and flow rate can be modeled using
the below calculations.
[0036] FIG. 5 shows a diagram of a single fluidic circuit for
calculation purposes. One of skill in the art will recognize that
the calculations shown herein may be applied to multiple fluidic
circuits. (A) represents an immiscible carrier fluid channel, (B)
represents an aqueous channel, (C) represents a junction of
channels (A) and (B) where aqueous phase and immiscible carrier
fluid phase meet to form droplets of the aqueous phase surrounded
by the immiscible carrier fluid, and (D) represents outlet channel
that receives the droplets. P.sub.A represents the pressure of the
immiscible carrier fluid in the immiscible carrier fluid channel,
P.sub.B represents the pressure of the aqueous fluid in the aqueous
fluid channel, P.sub.C represents the pressure at the junction of
channels (A) and (B). P.sub.A, P.sub.B, and P.sub.C are all greater
than 0, and PD is equal to 0 because channel (D) is open to the
atmosphere. Q.sub.AC represents the flow rate of the immiscible
fluid, Q.sub.BC represents the flow rate of the aqueous fluid, and
Q.sub.CD represents the flow rate of droplets in channel (D).
R.sub.AC represents the fluidic resistance in the immiscible
carrier fluid channel, R.sub.BC represents the fluidic resistance
in the aqueous channel, and R.sub.CD represents the fluidic
resistance in the (D) channel. Equations and expressions for
Q.sub.AC and Q.sub.BC are as follows:
PA-PC=QAC(RAC) Equation 1;
PB-PC=QBC(RBC) Equation 2; and
PC=QCD(RCD)=(QAC+QBC)RCD Equation 3.
Assuming that PA, PB, RAC, RBC, and RCD are known, then the three
unknowns are PC, QAC, and QBC. QAC and QBC can be solved for as
follows:
QAC = PA ( RBC ) + ( PA - PB ) RCD RAC ( RBC ) + RCD ( RAC + RBC )
and Equation 3 QBC = PB ( RAC + ( PA - PB ) RCD RAC ( RBC ) + RCD (
RAC + RBC ) . Equation 4 ##EQU00001##
[0037] The sensitivities of the follow rates (Q) to changes in
pressure (P) are determined by obtaining partial derivatives of QAC
and QBC with respect to PA and PB, which yields:
.delta. QA .delta. PA = ( RBC + RCD ) RAC ( RBC ) + RCD ( RAC + RBC
) ; Equation 5 .delta. QA .delta. PB = - RCD RAC ( RBC ) + RCD (
RAC + RBC ) ; Equation 6 .delta. QB .delta. PB = ( RAC + RCD ) RAC
( RBC ) + RCD ( RAC + RBC ) ; and Equation 7 .delta. QB .delta. PA
= - RCD RAC ( RBC ) + RCD ( RAC + RBC ) = .delta. QA .delta. PB .
Equation 8 ##EQU00002##
Assuming that P'A=PA+.delta.PA then:
Q ' A C QAC = 1 + ( RBC + RCD ) .delta. PA PA ( RBC ) + ( PA - PB )
RCD ; and Equation 9 Q ' BC QBC = 1 - ( RCD ) .delta. PA PB ( RAC )
- ( PA - PB ) RCD . Equation 10 ##EQU00003##
Similarly, assuming that P''B=PB+.delta.PB then:
Q '' A C QAC = 1 - ( RCD ) .delta. PB PA ( RBC ) + ( PA - PB ) RCD
; and Equation 11 Q '' BC QBC = 1 + ( RAC + RCD ) .delta. PB PB (
RAC ) - ( PA - PB ) RCD . Equation 12 ##EQU00004##
Substituting chip dPCR 1.3 specifics into the above and assuming
PA.apprxeq.PB, thus neglecting PA-PB containing terms yields:
Q ' A QA = 1 + 1.4 .delta. PA PA ; Equation 13 Q ' B QB = - 0.45
.delta. PA PA ; Equation 14 Q '' A QA = 1 - 0.36 .delta. PB PB ;
and Equation 15 Q '' B QB = 1 + 3 .delta. PB PB . Equation 16
##EQU00005##
[0038] The results in FIG. 4 show that changing PA from 28 psi to
30 psi results in Q.sub.BC going from 577 .mu.L/hr to 558 .mu.L/hr,
-3.3% change. The above model predicts a -3.1% change in QB.sub.1
which is in agreement with the actually results data.
[0039] In certain embodiments, the system may be configured such
that the circuits produce droplets of different size to allow for
controlled droplet coalescence in the main channel. The fluidic
circuits are arranged and controlled to produce an interdigitation
of droplets of different sizes flowing through a channel. Such an
arrangement is described for example in Link et al. (U.S. patent
application numbers 2008/0014589, 2008/0003142, and 2010/0137163)
and European publication number EP2047910 to Raindance Technologies
Inc. Due to size variance, the smaller droplet will travel at a
greater velocity than the larger droplet and will ultimately
collide with and coalesce with the larger droplet to form a mixed
droplet.
[0040] Another benefit of the added resistance in both channels
next to the nozzle occurs during priming. Simultaneous arrival of
both the aqueous and carrier liquids is difficult to produce
reliably. If the carrier fluid enters the aqueous channel and
travels all the way back into the filter elements, the aqueous and
carrier liquids begin to mix and emulsify before the nozzle. This
mixing interference causes significant variability in the size of
the generated droplets. The added resistance next to the nozzle
eliminates the mixing interference by creating a path of relatively
high resistance without emulsifying features that are in the
filter. Therefore, if the carrier fluid arrives at the nozzle first
it will travel both into the aqueous resistor and towards the
outlet of the chip. The outlet of the chip has a resistance that is
much smaller than the aqueous resistor and therefore the majority
of the carrier fluid will flow in that direction. This gives the
aqueous liquid time to reach the nozzle before the carrier fluid
enters the filter feature.
Droplet Measurement
[0041] The volume of an individual droplet is measured using
real-time image analysis. This in turn is fed back into a control
loop where a known projected area is targeted and equal to a given
droplet volume. Microfluidic chips are calibrated using a 3 point
reference emulsion of know volumes to generate calibration curves
for each channel. The idea is that the midpoint of the outline of a
projected droplet image is always the same regardless of the
lighting. This demonstrated in FIG. 6, which is a drawing
illustrating that the same volume drop is subject to extreme
changes in the lighting but the midpoint is always the same. From
left to right, the intensity of the lighting decreases but the
midpoint of the outline is always the same. In contrast to
determining the projected area of the inside of the drop, which is
difficult due to chip and lighting imperfections and variability,
or the outside of the drop, which is also quite sensitive to
lighting and chip imperfections, methods of the invention use the
midpoint of the outline of a projected droplet image, which is
always the same regardless of the lighting and chip imperfections.
Using the midpoint "flattens out" the imperfections and is
significantly less sensitive to outside influences on projected
drop size. FIGS. 7A-C provides three graphs that demonstrate the
differences in the droplet measuring techniques, and the projected
area required to produce 5 pL drops when using the inside, outside
and midpoint of a droplet image. Finding both the outside and
inside projected area allows you calculate the outside and inside
diameters. Calculating the average of the outside and inside
diameters gives you the midpoint diameter. From there an estimated
projected area is calculated from the midpoint diameter (See FIG.
8).
Nucleic Acid Target Molecules
[0042] One of skill in the art will recognize that methods and
systems of the invention are not limited to any particular type of
sample, and methods and systems of the invention may be used with
any type of organic, inorganic, or biological molecule. In
particular embodiments the droplets include nucleic acids. Nucleic
acid molecules include deoxyribonucleic acid (DNA) and/or
ribonucleic acid (RNA). Nucleic acid molecules can be synthetic or
derived from naturally occurring sources. In one embodiment,
nucleic acid molecules are isolated from a biological sample
containing a variety of other components, such as proteins, lipids
and nontemplate nucleic acids. Nucleic acid template molecules can
be obtained from any cellular material, obtained from an animal,
plant, bacterium, fungus, or any other cellular organism. In
certain embodiments, the nucleic acid molecules are obtained from a
single cell. Biological samples for use in the present invention
include viral particles or preparations. Nucleic acid molecules can
be obtained directly from an organism or from a biological sample
obtained from an organism, e.g., from blood, urine, cerebrospinal
fluid, seminal fluid, saliva, sputum, stool and tissue. Any tissue
or body fluid specimen may be used as a source for nucleic acid for
use in the invention. Nucleic acid molecules can also be isolated
from cultured cells, such as a primary cell culture or a cell line.
The cells or tissues from which template nucleic acids are obtained
can be infected with a virus or other intracellular pathogen. A
sample can also be total RNA extracted from a biological specimen,
a cDNA library, viral, or genomic DNA.
[0043] Generally, nucleic acid can be extracted from a biological
sample by a variety of techniques such as those described by
Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., pp. 280-281 (1982). Nucleic acid molecules may
be single-stranded, double-stranded, or double-stranded with
single-stranded regions (for example, stem- and
loopstructures).
Target Amplification
[0044] Methods of the invention further involve amplifying a target
nucleic acid(s) in a droplet. Amplification refers to production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction or other technologies
well known in the art (e.g., Dieffenbach and Dveksler, PCR Primer,
a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.
[1995]). The amplification reaction may be any amplification
reaction known in the art that amplifies nucleic acid molecules,
such as polymerase chain reaction, nested polymerase chain
reaction, polymerase chain reaction-single strand conformation
polymorphism, ligase chain reaction (Barany F. (1991) PNAS
88:189-193; Barany F. (1991) PCR Methods and Applications 1:5-16),
ligase detection reaction (Barany F. (1991) PNAS 88:189-193),
strand displacement amplification and restriction fragments length
polymorphism, transcription based amplification system, nucleic
acid sequence-based amplification, rolling circle amplification,
and hyper-branched rolling circle amplification.
[0045] In certain embodiments, the amplification reaction is the
polymerase chain reaction. Polymerase chain reaction (PCR) refers
to methods by K. B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202,
hereby incorporated by reference) for increasing concentration of a
segment of a target sequence in a mixture of genomic DNA without
cloning or purification.
[0046] The process for amplifying the target sequence includes
introducing an excess of oligonucleotide primers to a DNA mixture
containing a desired target sequence, followed by a precise
sequence of thermal cycling in the presence of a DNA polymerase.
The primers are complementary to their respective strands of the
double stranded target sequence.
[0047] To effect amplification, primers are annealed to their
complementary sequence within the target molecule. Following
annealing, the primers are extended with a polymerase so as to form
a new pair of complementary strands. The steps of denaturation,
primer annealing and polymerase extension can be repeated many
times (i.e., denaturation, annealing and extension constitute one
cycle; there can be numerous cycles) to obtain a high concentration
of an amplified segment of a desired target sequence. The length of
the amplified segment of the desired target sequence is determined
by relative positions of the primers with respect to each other,
and therefore, this length is a controllable parameter.
[0048] Methods for performing PCR in droplets are shown for example
in Link et al. (U.S. patent application numbers 2008/0014589,
2008/0003142, and 2010/0137163), Anderson et al. (U.S. Pat. No.
7,041,481 and which reissued as RE41,780) and European publication
number EP2047910 to Raindance Technologies Inc. The content of each
of which is incorporated by reference herein in its entirety.
[0049] The sample droplet may be pre-mixed with a primer or primers
and other reagents for an amplification reaction, or the primer or
primers and other reagents for an amplification reaction may be
added to the droplet. In some embodiments, fluidic circuits are
controlled to produce droplets of different sizes to result in
controlled merging of droplets. In those embodiments, sample
droplets are created by segmenting the starting sample and merging
that droplet with a second set of droplets including one or more
primers for the target nucleic acid in order to produce final
droplets. The merging of droplets can be accomplished using, for
example, one or more droplet merging techniques described for
example in Link et al. (U.S. patent application numbers
2008/0014589, 2008/0003142, and 2010/0137163) and European
publication number EP2047910 to Raindance Technologies Inc.
[0050] In embodiments involving merging of droplets, two droplet
formation modules are used. A first droplet formation module
produces the sample droplets that on average contain a single
target nucleic acid. A second droplet formation module produces
droplets that contain reagents for a PCR reaction. Such droplets
generally include Taq polymerase, deoxynucleotides of type A, C, G
and T, magnesium chloride, and forward and reverse primers, all
suspended within an aqueous buffer. The second droplet also
includes detectably labeled probes for detection of the amplified
target nucleic acid, the details of which are discussed below. In
embodiments that start with a pre-mix of sample and reagents for a
PCR reaction, the pre-mix includes all of the above described
components.
[0051] The droplet formation modules are arranged and controlled to
produce an interdigitation of sample droplets and PCR reagent
droplets flowing through a channel. Such an arrangement is
described for example in Link et al. (U.S. patent application
numbers 2008/0014589, 2008/0003142, and 2010/0137163) and European
publication number EP2047910 to Raindance Technologies Inc.
[0052] A sample droplet is then caused to merge with a PCR reagent
droplet, producing a droplet that includes Taq polymerase,
deoxynucleotides of type A, C, G and T, magnesium chloride, forward
and reverse primers, detectably labeled probes, and the target
nucleic acid. Droplets may be merged for example by: producing
dielectrophoretic forces on the droplets using electric field
gradients and then controlling the forces to cause the droplets to
merge; producing droplets of different sizes that thus travel at
different velocities, which causes the droplets to merge; and
producing droplets having different viscosities that thus travel at
different velocities, which causes the droplets to merge with each
other. Each of those techniques is further described in Link et al.
(U.S. patent application numbers 2008/0014589, 2008/0003142, and
2010/0137163) and European publication number EP2047910 to
Raindance Technologies Inc. Further description of producing and
controlling dielectrophoretic forces on droplets to cause the
droplets to merge is described in Link et al. (U.S. patent
application number 2007/0003442) and European Patent Number
EP2004316 to Raindance Technologies Inc.
[0053] Primers can be prepared by a variety of methods including
but not limited to cloning of appropriate sequences and direct
chemical synthesis using methods well known in the art (Narang et
al., Methods Enzymol., 68:90 (1979); Brown et al., Methods
Enzymol., 68:109 (1979)). Primers can also be obtained from
commercial sources such as Operon Technologies, Amersham Pharmacia
Biotech, Sigma, and Life Technologies. The primers can have an
identical melting temperature. The lengths of the primers can be
extended or shortened at the 5' end or the 3' end to produce
primers with desired melting temperatures. Also, the annealing
position of each primer pair can be designed such that the sequence
and, length of the primer pairs yield the desired melting
temperature. The simplest equation for determining the melting
temperature of primers smaller than base pairs is the Wallace Rule
(Td=2(A+T)+4(G+C)). Computer programs can also be used to design
primers, including but not limited to Array Designer Software
(Arrayit Inc.), Oligonucleotide Probe Sequence Design Software for
Genetic Analysis (Olympus Optical Co.), NetPrimer, and DNAsis from
Hitachi Software Engineering. The TM (melting or annealing
temperature) of each primer is calculated using software programs
such as Oligo Design, available from Invitrogen Corp.
[0054] Once final droplets have been produced, the droplets are
thermal cycled, resulting in amplification of the target nucleic
acid in each droplet. In certain embodiments, the droplets are
flowed through a channel in a serpentine path between heating and
cooling lines to amplify the nucleic acid in the droplet. The width
and depth of the channel may be adjusted to set the residence time
at each temperature, which can be controlled to anywhere between
less than a second and minutes.
[0055] In certain embodiments, the three temperature zones are used
for the amplification reaction. The three temperature zones are
controlled to result in denaturation of double stranded nucleic
acid (high temperature zone), annealing of primers (low temperature
zones), and amplification of single stranded nucleic acid to
produce double stranded nucleic acids (intermediate temperature
zones). The temperatures within these zones fall within ranges well
known in the art for conducting PCR reactions. See for example,
Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 2001).
[0056] In certain embodiments, the three temperature zones are
controlled to have temperatures as follows: 95.degree. C.
(T.sub.H), 55.degree. C. (T.sub.L), 72.degree. C. (T.sub.M). The
prepared sample droplets flow through the channel at a controlled
rate. The sample droplets first pass the initial denaturation zone
(T.sub.H) before thermal cycling. The initial preheat is an
extended zone to ensure that nucleic acids within the sample
droplet have denatured successfully before thermal cycling. The
requirement for a preheat zone and the length of denaturation time
required is dependent on the chemistry being used in the reaction.
The samples pass into the high temperature zone, of approximately
95.degree. C., where the sample is first separated into single
stranded DNA in a process called denaturation. The sample then
flows to the low temperature, of approximately 55.degree. C., where
the hybridization process takes place, during which the primers
anneal to the complementary sequences of the sample. Finally, as
the sample flows through the third medium temperature, of
approximately 72.degree. C., the polymerase process occurs when the
primers are extended along the single strand of DNA with a
thermostable enzyme.
[0057] The nucleic acids undergo the same thermal cycling and
chemical reaction as the droplets passes through each thermal cycle
as they flow through the channel. The total number of cycles in the
device is easily altered by an extension of thermal zones. The
sample undergoes the same thermal cycling and chemical reaction as
it passes through N amplification cycles of the complete thermal
device.
[0058] In other embodiments, the temperature zones are controlled
to achieve two individual temperature zones for a PCR reaction. In
certain embodiments, the two temperature zones are controlled to
have temperatures as follows: 95.degree. C. (T.sub.H) and
60.degree.10 C (T.sub.L). The sample droplet optionally flows
through an initial preheat zone before entering thermal cycling.
The preheat zone may be important for some chemistry for activation
and also to ensure that double stranded nucleic acid in the
droplets are fully denatured before the thermal cycling reaction
begins. In an exemplary embodiment, the preheat dwell length
results in approximately 10 minutes preheat of the droplets at the
higher temperature.
[0059] The sample droplet continues into the high temperature zone,
of approximately 95.degree. C., where the sample is first separated
into single stranded DNA in a process called denaturation. The
sample then flows through the device to the low temperature zone,
of approximately 60.degree. C., where the hybridization process
takes place, during which the primers anneal to the complementary
sequences of the sample. Finally the polymerase process occurs when
the primers are extended along the single strand of DNA with a
thermostable enzyme. The sample undergoes the same thermal cycling
and chemical reaction as it passes through each thermal cycle of
the complete device. The total number of cycles in the device is
easily altered by an extension of block length and tubing.
Target Detection
[0060] After amplification, droplets are flowed to a detection
module for detection of amplification products. The droplets may be
individually analyzed and detected using any methods known in the
art, such as detecting for the presence or amount of a reporter.
Generally, the detection module is in communication with one or
more detection apparatuses. The detection apparatuses can be
optical or electrical detectors or combinations thereof. Examples
of suitable detection apparatuses include optical waveguides,
microscopes, diodes, light stimulating devices, (e.g., lasers),
photo multiplier tubes, and processors (e.g., computers and
software), and combinations thereof, which cooperate to detect a
signal representative of a characteristic, marker, or reporter, and
to determine and direct the measurement or the sorting action at a
sorting module. Further description of detection modules and
methods of detecting amplification products in droplets are shown
in Link et al. (U.S. patent application numbers 2008/0014589,
2008/0003142, and 2010/0137163) and European publication number
EP2047910 to Raindance Technologies Inc.
[0061] In certain embodiments, amplified target are detected using
detectably labeled probes. In particular embodiments, the
detectably labeled probes are optically labeled probes, such as
fluorescently labeled probes. Examples of fluorescent labels
include, but are not limited to, Atto dyes,
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine
and derivatives: acridine, acridine isothiocyanate;
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;
Brilliant Yellow; coumarin and derivatives; coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI);
5'5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives; eosin, eosin isothiocyanate,
erythrosin and derivatives; erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein and derivatives;
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein,
fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;
B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives:
pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum
dots; Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative
of sulforhodamine 101 (Texas Red);
N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5;
Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and
naphthalo cyanine. Preferred fluorescent labels are cyanine-3 and
cyanine-5. Labels other than fluorescent labels are contemplated by
the invention, including other optically-detectable labels.
[0062] During amplification, fluorescent signal is generated in a
TaqMan assay by the enzymatic degradation of the fluorescently
labeled probe. The probe contains a dye and quencher that are
maintained in close proximity to one another by being attached to
the same probe. When in close proximity, the dye is quenched by
fluorescence resonance energy transfer to the quencher.
[0063] Certain probes are designed that hybridize to the wild-type
of the target, and other probes are designed that hybridize to a
variant of the wild-type of the target. Probes that hybridize to
the wild-type of the target have a different fluorophore attached
than probes that hybridize to a variant of the wild-type of the
target. The probes that hybridize to a variant of the wild-type of
the target are designed to specifically hybridize to a region in a
PCR product that contains or is suspected to contain a single
nucleotide polymorphism or small insertion or deletion.
[0064] During the PCR amplification, the amplicon is denatured
allowing the probe and PCR primers to hybridize. The PCR primer is
extended by Taq polymerase replicating the alternative strand.
During the replication process the Taq polymerase encounters the
probe which is also hybridized to the same strand and degrades it.
This releases the dye and quencher from the probe which are then
allowed to move away from each other. This eliminates the FRET
between the two, allowing the dye to release its fluorescence.
Through each cycle of cycling more fluorescence is released. The
amount of fluorescence released depends on the efficiency of the
PCR reaction and also the kinetics of the probe hybridization. If
there is a single mismatch between the probe and the target
sequence the probe will not hybridize as efficiently and thus a
fewer number of probes are degraded during each round of PCR and
thus less fluorescent signal is generated. This difference in
fluorescence per droplet can be detected and counted. The
efficiency of hybridization can be affected by such things as probe
concentration, probe ratios between competing probes, and the
number of mismatches present in the probe.
Droplet Sorting
[0065] Methods of the invention may further include sorting the
droplets. A sorting module may be a junction of a channel where the
flow of droplets can change direction to enter one or more other
channels, e.g., a branch channel, depending on a signal received in
connection with a droplet interrogation in the detection module.
Typically, a sorting module is monitored and/or under the control
of the detection module, and therefore a sorting module may
correspond to the detection module. The sorting region is in
communication with and is influenced by one or more sorting
apparatuses.
[0066] A sorting apparatus includes techniques or control systems,
e.g., dielectric, electric, electro-osmotic, (micro-) valve, etc. A
control system can employ a variety of sorting techniques to change
or direct the flow of molecules, cells, small molecules or
particles into a predetermined branch channel. A branch channel is
a channel that is in communication with a sorting region and a main
channel. The main channel can communicate with two or more branch
channels at the sorting module or branch point, forming, for
example, a T-shape or a Y-shape. Other shapes and channel
geometries may be used as desired. Typically, a branch channel
receives droplets of interest as detected by the detection module
and sorted at the sorting module. A branch channel can have an
outlet module and/or terminate with a well or reservoir to allow
collection or disposal (collection module or waste module,
respectively) of the molecules, cells, small molecules or
particles. Alternatively, a branch channel may be in communication
with other channels to permit additional sorting.
[0067] A characteristic of a fluidic droplet may be sensed and/or
determined in some fashion, for example, as described herein (e.g.,
fluorescence of the fluidic droplet may be determined), and, in
response, an electric field may be applied or removed from the
fluidic droplet to direct the fluidic droplet to a particular
region (e.g. a channel). In certain embodiments, a fluidic droplet
is sorted or steered by inducing a dipole in the uncharged fluidic
droplet (which may be initially charged or uncharged), and sorting
or steering the droplet using an applied electric field. The
electric field may be an AC field, a DC field, etc. For example, a
channel containing fluidic droplets and carrier fluid, divides into
first and second channels at a branch point. Generally, the fluidic
droplet is uncharged. After the branch point, a first electrode is
positioned near the first channel, and a second electrode is
positioned near the second channel. A third electrode is positioned
near the branch point of the first and second channels. A dipole is
then induced in the fluidic droplet using a combination of the
electrodes. The combination of electrodes used determines which
channel will receive the flowing droplet. Thus, by applying the
proper electric field, the droplets can be directed to either the
first or second channel as desired. Further description of droplet
sorting is shown for example in Link et al. (U.S. patent
application numbers 2008/0014589, 2008/0003142, and 2010/0137163)
and European publication number EP2047910 to Raindance Technologies
Inc.
Release from Droplets
[0068] Methods of the invention may further involve releasing the
enzymes from the droplets for further analysis. Methods of
releasing contents from the droplets are shown for example in Link
et al. (U.S. patent application numbers 2008/0014589, 2008/0003142,
and 2010/0137163) and European publication number EP2047910 to
Raindance Technologies Inc.
[0069] In certain embodiments, sample droplets are allowed to cream
to the top of the carrier fluid. By way of non-limiting example,
the carrier fluid can include a perfluorocarbon oil that can have
one or more stabilizing surfactants. The droplet rises to the top
or separates from the carrier fluid by virtue of the density of the
carrier fluid being greater than that of the aqueous phase that
makes up the droplet. For example, the perfluorocarbon oil used in
one embodiment of the methods of the invention is 1.8, compared to
the density of the aqueous phase of the droplet, which is 1.0.
[0070] The creamed liquids are then placed onto a second carrier
fluid which contains a destabilizing surfactant, such as a
perfluorinated alcohol (e.g. 1H,1H,2H,2H-Perfluoro-1-octanol). The
second carrier fluid can also be a perfluorocarbon oil. Upon
mixing, the aqueous droplets begins to coalesce, and coalescence is
completed by brief centrifugation at low speed (e.g., 1 minute at
2000 rpm in a microcentrifuge). The coalesced aqueous phase can now
be removed and the further analyzed.
Microfluidic Chips
[0071] Microfluidic chips for performing biological, chemical, and
diagnostic assays are described in U.S. Published Patent
Application No. US2008/0003142 and US2008/0014589, the content of
each of which is incorporated by reference herein in its entirety.
Such microfluidic devices generally include at least one substrate
having one or more microfluidic channels etched or molded into the
substrate, and one or more interconnects (fluid interface). The one
or more interconnects contain inlet modules that lead directly into
the microfluidic channels, and serve to connect the microfluidic
channel to a means for introducing a sample fluid to the channel.
The one or more interconnects also serve to form a seal between the
microfluidic substrate and the means for introducing a sample. The
one or more interconnects can be molded directly into the
microfluidic substrate, as one or more individual pieces, or as a
single, monolithic self-aligning piece. The interconnect may also
be a separate piece and the entire assembly (the manifold,
microfluidic chip, and interconnect) can be modular as well. An
exemplary interconnect is shown in FIG. 9, which shows the
interconnect with immiscible fluid port 901 and aqueous fluid port
902. FIG. 10 shows the interconnect integrated with a manifold
having oil reservoir 1003 and a microfluidic chip thereunder. FIG.
11 shows the interconnect 1104 integrated with a microfluidic chip
1105 with the manifold (not shown) removed.
[0072] Microfluidic chips according to the invention include a
substrate defining at least one internal channel and at least one
port in fluid communication with the channels. In one particular
embodiment, a chip of the invention includes a top plate adhered to
a bottom plate to form the substrate with the channel(s) and
port(s). The top plate can include the port(s), and the bottom
plate can include the channel(s), such that when these two plates
are brought together and adhered to each other the combination
forms the substrate with the channel(s) and the port(s). The
microfluidic chip can be injection molded from a variety of
materials. Preferably the microfluidic chip is injection molded
using a cyclic olefin copolymer (COC) or cyclic olefin polymer
(COP) or blend of COC and COP.
[0073] Chips of the invention include one or more fluidic circuits.
Each circuit including a sample fluid channel, an immiscible
carrier fluid channel, and an outlet channel. The channels of each
circuit are configured such that they meet at a junction so that
droplets of aqueous fluid surrounded by carrier fluid are formed at
the junction an flow into the outlet channel. The outlet channel of
each circuit is connected to a main channel that receives all of
the droplets from the different fluidic circuits and flows the
droplets to different modules in the chip for analysis. In certain
embodiments, each fluid circuit carries a different aqueous sample
fluid in order to produce different sample droplets. In other
embodiments, the fluidic circuits all carry the same aqueous sample
fluid, and thus produce the same sample droplets.
[0074] A pressure source, optionally coupled to electronic pressure
regulators, is used to pump fluids through multiple microfluidic
channels in parallel. Multiple pressure regulators control the
aqueous inputs. The immiscible carrier fluid input is under gain
control for all channels simultaneously. In this configuration,
there is independent control of individual circuits to adjust
projected area to obtain a target droplet volume. Droplet volume is
measured either relatively or absolutely (depending on the
application) via real-time image analysis. Proper design of the
microfluidic circuits is required to obtain sensitive and precise
control of the droplet volume in all channels.
[0075] Pressure driven flow allows for the replacement of expensive
mechanical parts with inexpensive pneumatic control products.
Pressure driven flow is instantaneous and pulse-free. Taking
advantage of circuits in parallel, constant pressure driven flow
instantly adjusts to changes in resistance in any and all channels
without affecting any of the other channels.
[0076] Any pressure sources known in the art may be used with chips
of the invention. In certain embodiments, the pressure source is
coupled to electronic regulators. When coupled to an electronic
regulator, the pressure source may be an external compressor with a
reservoir for pumping compressed nitrogen, argon or air. In
embodiments that do not used electronic regulators, an internal air
cylinder with a linear actuator is applied.
[0077] The regulators should be of a type capable of regulating gas
pressure from about 0 to about 5 atm in 100 evenly spaced
increments (0-10 V, step=0.1 V). Each aqueous input is
independently driven and controlled by a separate pressure
regulator. The immiscible fluid lines are controlled in a gain
control fashion, where one regulator is used to drive and control
the flow of immiscible fluid through the entire system.
INCORPORATION BY REFERENCE
[0078] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in
their entirety for all purposes.
EQUIVALENTS
[0079] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein.
* * * * *