IE84898B1 - A liquid bridge and system - Google Patents
A liquid bridge and systemInfo
- Publication number
- IE84898B1 IE84898B1 IE2007/0069A IE20070069A IE84898B1 IE 84898 B1 IE84898 B1 IE 84898B1 IE 2007/0069 A IE2007/0069 A IE 2007/0069A IE 20070069 A IE20070069 A IE 20070069A IE 84898 B1 IE84898 B1 IE 84898B1
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- Prior art keywords
- liquid
- bridge
- droplets
- flow
- chamber
- Prior art date
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Abstract
ABSTRACT The invention relates to control of liquids at small volumes such as at the microfluidic level.
Description
A Liquid Bridge and System
INTRODUCTION
Field of the Invention
The invention relates to control of liquids at small volumes such as at the microfluidic
level.
Prior Art Discussion
Microfluidics is a technology which in simple terms refers to the micro-scale devices
which handle small volumes of fluids — as small as micro-, nano- and pico- and
femtolitre volumes. Microfluidic devices have dimensions ranging from several
millimetres to micrometers. Typically dimensions of the device are measured in
micrometers. Given the small dimensions of microfluidic devices or components
thereof, not surprisingly microfluidic devices require construction and design which
differ from macro-scale devices. Simple sealing down in size of conventional scale
devices to microfluidic scale is not a simple option. Liquid flow in microfluidic
devices differs from that of macro-scale size devices. Liquid flow tends to be laminar,
surface flux and surface tension start to dominate and as a result effects not seen at the
macro level become significant. At the microfluidic level other differences include
faster thermal diffusion, predominately laminar flow and, surface forces are
responsible for capillary phenomena and an electric double layer (EDL).
Because microfluidics can accurately and reproducibly control and dispense minute
volumes of fluid — in particular volumes less than one ill. The application of
microfluidics can have significant cost-saving potential. The use of microfluidics
technology can potentially reduce cycle times, shorten time-to-results and increase
throughput. Furthermore incorporation of microfluidics technology can, in theory,
enhance system integration and automation.
Heretofore. handling of small quantities of liquids being transferred from one device
or conduit to another often involves pipetting, which is a time-consuming manual
procedure.
US2005/0092681 describes a device for mixing liquids by transporting them to a
confluent portion. US2005/0272144 discloses user of a mixing flow path for diffusion
and mixing of liquids.
The invention is directed towards achieving improved liquid control in microfluidic
systems.
SUMMARY OF THE INVENTION
In this specification the term “droplet” is used to mean a small quantity or plug of
liquid as it flows in an immiscible carrier liquid along a conduit.
According to the invention, there is provided a liquid bridge comprising:
at least one inlet port for delivery of a liquid A and an immiscible carrier liquid
3.
at least one outlet port,
a chamber within which the ports are located, the chamber having a capacity
such that it fills with carrier liquid B to fill the space between an inlet port and
an outlet port; and
wherein the ports are mutually located and have dimensions such that liquid A
periodically bridges to the outlet port due to fluidic instability and droplets are
periodically delivered to the outlet port;
wherein the ports have a width dimension in the range of 150nm to 400nm;
wherein the separation of an inlet port and a corresponding outlet port is in the
range of 0.2 mm and 2.0 mm.
In one embodiment, the liquid bridge comprises at least two aligned inlet and outlet
ports for droplet formation during flow of liquid from one port to the other.
In another embodiment, the ports are circular in cross-section and said dimension is
diameter.
In a further embodiment, at least one port is provided by an end of a capillary tube.
In one embodiment, said separation is approximately 1.5mm.
In another embodiment, the bridge comprises at least first and second inlet ports
arranged for delivery of inlet liquid droplets and an output port for outlet of a mixed
liquid droplet.
In a firrther embodiment, the first inlet port and the outlet port are co-axial and the
second inlet port is substantially perpendicular to their axis.
In one embodiment, the distance between the second inlet port and the axis of the co-
axial ports is in the range of 1.0mm to 2.0mm.
In another embodiment, the second inlet port has a smaller cross-sectional area than
the first inlet port.
In another aspect of the invention, there is provided a liquid bridge system comprising
a liquid bridge as claimed in any preceding claim, and a flow controller for controlling
flow of liquids A and B to the bridge and operation of the bridge according to a plot of
volumetric ratio vs. droplet slendemess, or capillary separation, or droplet volume vs.
slendemess ratio, or V * (Q *) versus A*, or ratio of capillary diameters, or volumetric
ratio vs. funicular slenderness.
In one embodiment, the controller comprises means for directing flow of carrier liquid
B at a flow rate in the range of 2/11/min and 5,ul/min.
In a further embodiment, the controller comprises means for directing pressure in the
chamber to be in the range of 0.5 bar and 1.0 bar above atmospheric.
In another aspect of the invention, there is provided a method of controlling operation
of any liquid bridge as defined above, the method comprising directing flow of liquids
A and B into the bridge with flow characteristics detennined according to the
geometry of the bridge ports and their separations and according to liquid properties,
such that liquid A periodically bridges to the outlet port due to fluidic instability and
droplets are periodically delivered to the outlet port.
In one embodiment, the flow is controlled to cause segmentation of a continuous
stream of large droplets of the liquid A in the chamber to provide a sequence of
segmented droplets derived from the inlet liquid A.
In another embodiment, different, miscible, liquids A and C are delivered to the
bridge, and flow is controlled so that droplets of liquids A and C mix within the
chamber, in which the liquids A and C are delivered to different inlet ports of the
bridge, said ports being located so that droplets of liquids A and C mix to form a
single droplet which exits via the outlet port.
In a further embodiment, different, miscible, liquids A and C are delivered to the
bridge, and flow is controlled so that droplets of liquids A and C mix within the
chamber, and the liquids A and C are delivered to the bridge in a single inlet port as
successive droplets, and flow is controlled in the inlet port and in the chamber so that
the droplets collide within the chamber to mix.
In one embodiment, the method comprises withdrawing carrier liquid B from between
said droplets before they enter the bridge so that they are caused to collide within the
chamber.
In another embodiment, the liquids A and C comprise different chemical species
contained within an aqueous phase.
In a further embodiment, the carrier liquid B has a viscosity in the range of 0.08 Pas
and 0.1 Pas.
In one embodiment, the carrier liquid B flows into the bridge at a flow rate in the
range of Zptl/min and 5/.tl/min.
In another embodiment, the carrier liquid B is density matched with the liquid A such
that a neutrally buoyant environment is created within the chamber.
In a further embodiment, the carrier liquid B has characteristics causing the droplets in
the chamber to have a spherical shape between an inlet port and an outlet port.
In one embodiment, the carrier liquid B is oil.
In another embodiment, the density of the carrier liquid B and the cross—sectional
areas of the ports are such that the carrier liquid B flows and surrounds the droplets of
liquid A in the outlet port.
In a further embodiment, the velocity profile across the outlet port is such as to cause
internal movement within droplets of liquid as they flow from an outlet port.
In one embodiment, said internal flow is internal circulation.
In another embodiment, the carrier liquid B forms a protective film which is static
very close to the internal surface of an outlet port and flows with the droplets further
from the said surface.
DETAILED DESCRIPTION OF THE INVENTION
Brief Description of the Drawings
The invention will be more clearly understood from the following description of some
embodiments thereof, given by way of example only with reference to the
accompanying drawings in which:-
Figs. 1 and 2 are schematic diagrams of a PCR preparation microfluidic system
of the invention;
Figs. 3 and 4 are above and beneath perspective views of a pumping system of
the microfluidic system, and Fig. 5 is a cross-sectional diagram illustrating a
sample well of the pumping system in more detail;
Fig. 6 is a sequence of diagrams A to G illustrating operation of a mixing
bridge incorporating a fiinicular bridge;
Fig. 7 is a diagram showing internal circulation and the protective oil film
around a micro-reactor droplet;
Fig. 8 is a sequence of diagrams A to D illustrating operation of a
segmentation bridge;
Fig. 9 is a sequence of photographs showing liquid dynamics and dimensions
at a liquid bridge;
Fig. 10 is a diagram showing a characteristic plot of volumetric ratio vs.
slendemess at a liquid bridge for segmentation;
Fig. 11 is a set of photographs of liquid bridge segmentors having different
geometries;
Fig. 12 is a characteristic plot for a liquid bridge segmentor;
Fig. 13 is a collapsed data characteristic plot for liquid bridge segmentation;
Fig. 14 is a set of photographs of three liquid bridge segmentors having
different capillary radii;
Figs. 15 and 16 are further characteristic plots for a liquid bridge segmentor;
Fig. 17 is a photograph of a funicular bridge, also showing dimension
parameters;
Fig. 18 is a funicular liquid bridge characteristic stability plot; and
Fig. 19 is a set of diagrams A to D showing operation of an alternative mixer
bridge of the invention.
Description of the Embodiments
Referring to Figs. 1 and 2 a microfluidic preparation system 1 for a Polymerase Chain
Reaction (“PCR”) system is shown at a high level. It comprises components 2 — 9 for,
respectively flow control, sample loading, sample queuing, mixing, reaction
segmentation, PCR thermocycling, RT fluorescence monitoring, and waste handling.
The system 1 is operated to find the gene expression profile of a patient sample using
the PC R technique. Standard scientific protocols are available on the extraction and
purification of mRNA and the subsequent production of cDNA. This is then mixed
with the specific primers and the general other reagents prior to amplification.
In the simple example illustrated sample streams of droplets A2, B2, C2, and D2 are
managed in the queuing network 4 so that the order in which they are delivered to the
mixer 5 is controlled as desired. For example, there may be a long sequence of
droplets of type A2, followed by equivalent streams for B2, C2, and D2. Alternatively,
there may be repeated sequences of A2, B2, C2, and D2, the droplets being
interleaved in the queuing network 4. The mixer 5 joins together a droplet of sample
type D1 and each particular droplet which passes through the mixer 5. The chemical
composition of the droplets is not the subject of this invention, suffice to say that they
are chosen according to the particular PCR experiment being run. The invention
applies particularly to the manner in which the droplets are mixed by the mixer 5 to
provide micro reactor droplets. It also applies to the manner in which a long stream of
a sample is broken up into a sequence of droplets of a particular size by the segmentor
6. These droplets are separated by an enveloping carrier liquid, in this case oil Al
delivered to the segmentor 6.
Thus, the system 1 achieves automation of the production of liquid droplets in a
carrier fluid with the sample, primers and reagents mixed. Take for example a
situation where there are N genes, each interrogated m times. To increase the
experimental certainty, there may be m>
housekeeping genes and q negative controls. The total number of drop1ets,M , may
be calculated by:
M =(mxN)+_p+q.
This may be of the order of 400 droplets.
Liquid bridges are used to mix the reagents and segment droplets in the components 5
and 6. These generate a line of droplets carrying a variety of different chemistries. The
choice of fluid properties will be described so that a liquid film is always present
between the droplet and the channel wetted surfaces. This has the dual effect of
preventing carryover contamination and surface inhibitory effects that restrict
amplification.
A typical Q-PCR reaction contains: fluorescent double-stranded binding dye, Taq
polymerase, deoxynucleotides of type A,C,G and T, magnesium chloride, forward and
reverse primers and patient cDNA, all suspended within an aqueous buffer. Reactants,
however, may be assigned into two broad groups: universal and reaction specific.
Universal reactants are those common to every Q-PCR reaction, and include:
fluorescent double-stranded binding dye, Taq polymerase, deoxynucleotides A, C, G
and T, and magnesium chloride. Reaction specific reactants include the forward and
reverse primers and patient cDNA.
In more detail, the following describes the components 2 — 8.
: A flow control system consisting of a precision pump with a motor controlled
drive that regulates the flow rates used to load and drive samples through the
system.
: Sample loading is accomplished by infusing oil into small wells. These wells
contain the universal reactants for each PCR reaction in addition to specific
primers. Bach Well contains a different primer set to quantify specific gene
expression levels. The samples exit the wells as long microfluidic plugs.
: The loading process is followed by sample queuing where the reactants flow
through a network such that they are arranged serially or alternatively as
desired in a tube.
: The reactants then flow through the mixer where they are combined with the
final PCR reactant, the patient CDNA. The production of negative reactants,
containing no patient CDNA, is also possible during this process.
: The relatively large reaction plugs are then segmented into smaller plugs or
droplets in a liquid bridge segmentation process. This is performed to reduce
experimental uncertainty.
Referring also to Figs. 3 to 5, the wells labelled Al, A2, B1, B2, etc. in Fig. 2 connect
directly to the flow control system 2, as shown in Fig. 3. The system 2 consists of
eight Teflon-tipped plungers 10 each inserted into a 686pl cylinder in a polycarbonate
infusion manifold 11 filled with silicone oil. There is a substrate microchannel 21
formed between the manifold 11 and a cradle 12. The plungers 10, when driven at a
constant velocity, pump the oil from the infusion manifold 11 through the sample
wells and into the substrate microchannels. The flow rates are maintained equal in
each well so that the queuing of droplets only depends upon path length. The locations
of the ports are shown in Fig. 4. They connect directly to opposing substrate wells.
The plungers are driven by a pusher block (not shown) on a lead-screw connected to a
stepper motor to infuse silicone oil at equal flow rates into each sample well to pump
the contents of the wells into microchannels. This method of pumping could also be
done from a reservoir, possibly with internal baffling to equalise the flow. In this case
only a single plunger would be necessary.
The sample wells may be either integrated on disposable substrates or connected via
polycarbonate sample well strips on non-disposable versions. Aligmncnt dowels 15
are used to position the wells accurately under the infusion manifold 11. Sealing
between mating components is ensured with the use of elastomer gaskets 16 on each
side of a sample well strip 19. The gaskets provide a liquid-tight seal with the
application of a constant upward force to the substrate cradle 12 and a sealing sheet
. The substrate measures 70mm x 90mm in this embodiment. When an upward
force is applied to the substrate cradle, a liquid tight seal is formed between the
infusion manifold, sample well strip and the microchannel substrate with the aid of
elastomer gaskets at mating interfaces.
As silicone oil is pumped by the plungers 10 it passes through the wells 19 into which
samples have been loaded. Thus, the flow into each microchannel 21 is a stream of
carri er oil and sample droplets for entry into the queuing network 4.
Differences in microchannel path lengths delay the arrival of sample droplets to the
outlet of the queuing network 4. It can be seen from Fig. 2, that the shortest path
length exists from well A2 to the network outlet. The next shortest path length exists
from well B2 to outlet and so on. The result of the queuing network is a linear array of
reaction droplets separated by silicone oil. This queuing network design has the
additional benefit of being scalable to array many samples.
The aqueous phase reaction droplets are combined with patient cDNA at the mixer 5.
The two-phase nature of microfluidic flow necessitates a means of combining fluids.
The fluids are combined into a single droplet in the mixer 5 by virtue of fimicular
bridge instability. Fig 6 shows funicular bridge mixing in a particular funicular bridge
of the mixer 5. The bridge 30 comprises a first inlet port 31 at the end of a
capillary, a narrower second inlet port 32 which is an end of a capillary, an outlet port
33 which is an end of a capillary, and a chamber 34 for silicone oil. Initially, the entire
system is primed with a density matched oil. The inlet and outlet ports 31 and 33 are
of diameter 200nm, more generally preferably in the range of 150nm to 400nm. The
separation of the ports 31 and 33 is c. 1mm, and the distance between the second inlet
port 32 and the axis of the ports 21 and 33 is e. 1.5mm. The chamber 34 is 5mm in
diameter and 3mm in depth. The enveloping oil provides a pressure of no more than
0.5 to 1.0 bar above atmospheric. It has a viscosity of 0.08 to 0.1 Pas, and the flow
rate is in the range of 2 to 5 pl/mn
The oil is density-matched with the reactor droplets such that a neutrally buoyant
enviromnent is created within the chamber 34. The oil within the chamber is
continuously replenished by the oil separating the reactor droplets. This causes the
droplets to assume a stable capillary-suspended spherical form upon entering the
chamber 34, steps B and C. The spherical shape grows until large enough to span the
gap between the ports, forming an axisymmetric liquid bridge.
The introduction of a second droplet from the second inlet port 32 causes the
formation of an unstable funicular bridge that quickly ruptures from the, finer, second
inlet port 32, causing all the fluid to combine at the liquid bridge 30.
The sequence of illustrations A to G in Fig. 6 show how the enveloping liquid controls
droplet formation and mixing according to surface tension. The pressure in the
chamber 34 is atmospheric. The interfacial tension within the chamber 34 is important
for effective mixing. Also, the relative viscosity between the aqueous and oil phases is
important. The internal pressure (Laplace pressure) within each droplet is inversely
proportional to the droplet radius. Thus there is a higher internal pressure within the
droplet at the second inlet port 32. Because they are of the same phase, there is little
interfacial tension between the large and small droplets, and the internal pressures
cause a joining of the droplets, akin to injection of one into the other. Also, physical
control of the locations of the droplets is achieved by the enveloping oil, which is of
course immiscible with the droplets. Further addition of a surfactant to either phase
can change the interfacial tension.
Fig. 7 shows the internal circulation that takes place within a flowing droplet 40
carried by an immiscible oil 41. The velocity profile on the right shows the velocity
distribution within the plug relative to the average velocity of the flow. This internal
circulation causes excellent mixing and enhances chemical reactions within the
droplet 40, and hence it may be regarded as a micro-reactor. The location of a
protective oil film 41 is also shown, separating the droplet 40 from the channel walls
42 in addition to separating the droplets 40 from each other. The patterns observed
may be visualised if the observer adopts a reference frame that moves at the mean
velocity of the flow. With this in mind, the flow can be imagined as a fast moving
fluid along the centre-streamline toward the leading face of the plug. Fluid is then
circulated back to the rear of the plug near the walls of the microchannel. Internal
circulation within flowing microfluidic plugs is a powerful mixing mechanism that, in
contrast to existing three dimensional serpentine micromixers, does not require
complex microchannel geometries. Homogenous mixing is known to increase reaction
kinetics and internal circulation is an important advantage of the two-phase plug flow
regime. The establishment of internal circulation and protective films is enhanced by
the use of circular polymeric microchannels, specifically F EP fluorocarbon polymeric
microchannels. This mechanism enhances mixing within a droplet downstream of the
mixer 5.
Fully mixed plugs then enter the segmentor 6 to split master reaction plugs into four
smaller droplets, containing identical chemistries that are individually monitored
during thermocycling. This step reduces experimental uncertainty. A bridge 50 of the
segmentor 6 is shown in Fig. 8. The bridge 50 comprises an aqueous inlet 51, an oil
inlet 52, an outlet 53, and a chamber 54. The chamber 54 is 5mm in diameter and
3mm in depth and the internal pressure caused by flow of silicone oil is no more than
0.5 to l.0bar above atmospheric. The diameter of the ports 51 and 53 is 200nm, and is
more generally preferably in the range of 150nm to 400;.im. The spacing between the
ports 51 and 53 is 0.5mm, and is more generally preferably in the range of 0.2 to
l.0mm.The outlet flow rate from the segmentor is Sul/min, more generally in the
range of 2 to 8 ul/min.
The liquid bridge’s geometry and the enveloping carrier liquid create a periodic
instability between the opposing ports 51 and 52 due to surface tension. A droplet 55
is initially formed at the end of the inlet port 51 (diagram A). As shown in diagram B
the droplet liquid then momentarily bridges the ports 51 and 53. The volume held in
this bridge is then steadily reduced by the action of the silicone oil inlet. This causes
the formation of an unstable liquid bridge that ruptures to release a smaller plug at the
outlet. When the inlet oil flowrate matches the aqueous droplet flowrate, smaller
segmented droplets, separated by the same volume of silicone oil, are produced by this
bridge. The segmenting mechanism reliably produces uniform aqueous plugs
separated by oil that do not rely on the shear force exerted by the carrier fluid.
Multiple dispensing bridges with N inputs 51 and N outputs 53 are also provided. In
this case multi lumen tubing may be used as a conduit to carry fluid from the
preparation system through the continuous flow PCR therrnocycler. Multi lumen
tubing contains many micro-bores such that each bore represents a fluid path through
which PCR thermocycling may occur.
Liquid bridge stability was studied as a means to predicting the geometric conditions
at which rupture occurs. Liquid bridge rupture may be defined as the complete
breakage of the liquid filament connecting one solid support to the other. The
dimensionless parameters characterising liquid bridges are used to define the stability
boundary at which rupture was observed. Fig. 9 presents images of liquid bridges at
three slendemess conditions just prior to rupture. The rupture was caused by the
withdrawal of liquid bridge fluid from one capillary tube. It was observed that low
slendemess ratio liquid bridges, an example of which is shown in Fig. 9 (A), adopt a
thimble shape at the minimum volume stability. Larger slendemess ratio liquid
bridges, such as that shown in Fig. 9 (C), possess a barrel form with a maximum
radius at the bridge mid-span. Intermediate slendemess ratios were found to have a
near cylindrical shape at the minimum volume stability limit. Images (A), (B) and (C)
of Fig. 9 show liquid bridges with slenderness ratios of 1.09, 2.45 and 6.16
respectively.
The stability of liquid bridges was examined as a function of slcndcmess, A* , which
is the ratio of tip separation, L, to the mean diameter, 2R0, of the supporting
capillaries, ie A* = L/2R0 . Stability was also investigated as a function of volumetric
ratio, V *, which is the ratio of liquid bridge volume to the volume of a cylinder with
a radius R0, the average radius of the supporting capillaries, i.e. V*= I7/(7rR02 L),.
The location of the stability boundary, or rupture point, was determined
experimentally by fixing the slendemess, establishing a stable liquid bridge between
capillary tips and withdrawing fluid from one capillary until rupture was observed. A
digital image of the liquid bridge just prior to rupture was then analysed, using an
edge detection measurement technique to determine the total volume and hence the
volumetric ratio, V*. The slenderness was then adjusted and the experiment repeated.
K * represents the ratio of the radius of the smaller disk, R1, to the radius of the larger
one, R3, that is K*=R,/R2. Figure 12 shows the approximate location of the
minimum volume stability boundary for liquid bridges with a lateral Bond number of
1.25 x l0‘4, a near weightless environment. Vertical and horizontal error bars indicate
experimental uncertainty.
At high volumetric ratios, Fig. 9 (C) for example, bridges maintain their integrity and
reach a minimum energy configuration. At low volumetric ratios, Fig. 9 (A) for
example, the bridges break before the interfacial energy is minimized. The initial dip
in the stability boundary at low slendemess ratios is caused by low-volume droplets
not fully wetting the exposed fused silica of the capillary tips. The influence of
unequal capillaries on the A*- V * stability diagram is also shown in Fig. 10. It can be
seen that the unstable region of the A * - V * plane increases as the parameterK *, the
ratio of capillary radii, decreases. The results presented in Fig. 10 confirmed that the
static stability of liquid bridge is purely geometrical at low Bond numbers. It is
notable that low slenderness ratio bridges are almost completely stable, with respect to
rupture, for all capillary radii measured. Rupture was observed only at very low
volumetric ratios with the liquid bridge assuming a thimble shape. Liquid bridge
instability when applied to fluid dispensing is particularly useful as a replacement for
microchannel shear-based dispensing systems. In more detail, Fig. 10 shows an
experimentally determined stability diagram for a de-ionized water liquid bridge in a
density matched silicone oil, Bond number: 1.25 x 104. Vertical error bars indicate the
volumetric ratio uncertainty as a result of camera frame rate. Horizontal error bars
indicate slendemess uncertainty due to capillary tip misalignment. The parameter K*
is the ratio of supporting capillary radii.
The following describes the use of liquid bridge instability as a mechanism for
dispensing sub-microlitre volumes of fluid in a continuous manner. The approach uses
the liquid bridge’s dependence on geometry to create a periodic instability between
opposing capillary tips. The dispensing mechanism provides a reliable means of
producing uniform aqueous plugs separated by silicone oil that did not rely on the
shear force exerted by the carrier fluid. The repeatability with which the method can
dispense plugs is examined. The approach uses the liquid bridge’s dependence on
geometry to create a periodic instability between opposing capillary tips. A stable
liquid bridge is first established between aqueous inlet and outlet. The volume held in
this bridge is then steadily reduced by the action of the silicone oil inlet. This causes
the formation of an unstable liquid bridge that ruptures to release a smaller plug at the
outlet. The segmenting mechanism provides a reliable means of producing uniform
aqueous plugs separated by silicone oil that does not rely on the shear force exerted by
the carrier fluid. Furthermore, a protective oil film is established between the walls of
the circular capillaries and the droplet to prevent carryover contamination.
Fig. 11 (A) - (D) presents images of liquid bridge dispensing at four different
slendemess ratios. (A) A*-=0, (B) A*=0.76, (C) A*=l.37 and (D) /\*=2.3l.
Q*=0.5, K*=0.44. Increasing the capillary tip separation, and hence the
slcndcrness ratio increases the plug volumes dispensed. Q*, the oil flowrate as a
fraction of the total flowrate, was maintained constant at 0.5. Image (A) shows
V dispensing with the dispensing capillary inserted inside the outlet capillary. This
configuration was assigned a slendemess ratio, A* , of zero. Slendemess ratios close
to zero resulted in the smallest volume plugs dispensed for this geometry. The effect
of increasing tip separation on dispensed plug volume is shown in Fig. 11 (B)-(D).
Increasing tip separation, i.e. slenderness ratio, resulted in larger volume aqueous
plugs punctuated by approximately the same volume of silicone oil. This was due to
the silicone oil inlet flowrate being maintained constant and equal to the aqueous
droplet inlet flowrate.
Fig. 12 presents a plot of V*, against slendemess ratio, A*, where V* is the
dimensionless plug volume sealed with R03 , i.e. V* = 17/ R03 . Results are presented
for three different values of the oil flowrate fraction, Q* , with the ratio of capillary tip
radii, K *, maintained constant at 0.44. The axis on the right—hand side of the plot
indicates the measured plug volume. Horizontal error bars indicate slendemess
uncertainty as a result of positional inaccuracy. Vertical error bar are a result of
uncertainty in the plug volume calculation due to image processing. The results show
the expected trend of increased plug volume with liquid bridge slenderness ratio.
Decreasing Q* resulted in a dramatic increase in dimensionless plug volume.
Altering Q* also affected the volume of silicone oil separating the aqueous plugs as
Q* is the oil flowrate as a fraction of the total flowrate. The lowest repeatable volume
measured using this particular geometry was approximately 90 nL with A*=O,
Q* = 0.75. The highest volume measured was approximately 3.9 uL with A* = 2.36,
Q* = 0.25 .
In flows where the non-wetting fluid, i.e. the aqueous phase, is displaced by wetting
fluid, i.e. oil, a thin film of the wetting fluid separates the droplets from the capillary
surface. The thickness of the film results from a balance between the oil viscosity, :7,
and the interfacial tension, 0,. . The thickness of the oil film deposited in a capillary of
radius r is given by;
h =1.34r(Ca“) .
The capillary number, Ca , is given by:
(0.1)
Cazfl
O7 ,
(0.2
where U represents the mean velocity of the flow. Equation (0.1) is obeyed if the film
is thin enough to neglect geometric forces, 11 < 0.1r, and thick enough to avoid the
influence of long range molecular attraction, h>l00 nm. Typical oil film thicknesses
for plug flow through 400 um polymeric fluorocarbon internal diameter tubing were
calculated to be of the order of 1 mn. This film thickness was too small to resolve with
any degree of accuracy from experimental images. The oil film does, however, form a
protective coating preventing aqueous reactor fluid from contacting the Teflon tubing.
This has the advantage of preventing a mechanism responsible for carryover
contamination whereby small droplets may be deposited onto the walls of
microchannels. Table 1 below presents two examples of oil-surfactant combinations
used to successfully establish protective oil films around flowing droplets. Surfactant
additives act to change the interfacial tension between droplets and the oil carrier fluid
such as to promote the establishment of a protective oil film, the thickness of which is
given by Equation 0.1.
TABLE 1
Oil Surfactant Concentration
FC40 1H, 1H,2H,2H-Perfluoro- 1- 2%W/V
decanol
AS100 Silicone Oil Triton X-100 0.1% V/V in PCR Buffer
Solution
Fig. 13 presents a dimensionless plot of the product of V* and Q* versus A*. The
data, taken from the plot shown in Fig. 12, collapsed on to the trend line within the
bounds of uncertainty. The data applies to geometries with K*=0.44.
Notwithstanding this geometric constraint, the collapsed data does yield valuable
design information. Consider a microfluidie system designer deciding on an
appropriate geometry for a segmenting device. The designer will usually know the
exact volume to dispense from the outline specification for the device. If there is a
sample frequency requirement, the designer may also know a value for Q *. Recalling
that K *= R, /R2 , where R, and R2 are the inlet and outlet diameters respectively
makes the design process relatively easy. Deciding on an arbitrary value for an outlet
diameter fixes the aqueous inlet diameter as the data shown in Fig. 15 applies to only
to geometries with K * = 0.44. With this information in hand, an appropriate value for
V *(Q*) may be calculated. The corresponding value for A* may then be read from
the design curve shown in Fig. 13. Finally, A* may be used to calculate the tip
separation between the inlet and outlet.
As mentioned previously, the data presented in Figs. 12 and 13 applies to geometries
with K *=0.44. The effect of altering K * on plug volumes dispensed was also
investigated. Images of liquid bridge dispensing at three different values for K * are
presented in Fig. 14. Images (A), (B) and (C) correspond to K * values of 0.25. 0.44
and 1.0 respectively. A K * value of 0.25 was achieved by assembling a 200 um fused
silica microcapillary at the end of a polymeric capillary tube by a reduction of internal
diameter through appropriately sized fused silica. Sealing was ensured with the
addition of cyanoacrylate glue at the sleeve interfaces.
Fig. 15 presents a dimensionless plot of V* versus A* for three different values of
K *. The dimensionless plug volume, V*, was scaled with R23 , and not R03 as
previously. This permitted a direct comparison of dimensionless plug volumes as R:
remained constant throughout the experiment. It can be seen that decreasing K *
generally lowers the plug volumes dispensed for any given value of slendemess, A * .
The minimum volume dispensed with K * = 0.25 was approximately 60 nL whilst that
of K *= 0.44 and K * :1 was approximately 110 nL. Attempts to collapse the data
shown in Fig. 15 onto a single line, similar to the plot shown in Fig. 13, were
unsuccessful. This was due to the highly non-linear relationship between K * and V *
for any given value of A *.
The repeatability with which the liquid bridge dispensing system could deliver fluid
was of particular interest. Fig. 16 plots plug volume variation over fourteen
measurements for a dispensing system with K * = 0.44. The results show mean plug
volumes of approximately 120 nL and 56 nL with maximum volumetric variations of
i4.46% and 453.53% respectively. These volumetric variations compare favourably
to commercial available micropipettes which have an uncertainty of il2% when
dispensing 200 nL. The accuracy with which one may dispense using micropipettes,
however, is thought to be largely dependant upon user skill. The automation of
dispensing systems may therefore be justified as a means of eliminating user-user
variability. The volumetric analysis presented in Fig. 16 shows liquid bridge
dispensing to be a very repeatable means of continuously dispensing sub-microlitre
volumes of fluid.
Referring again to the funicular bridge of Fig. 6 an example is presented in Fig. 17.
The bridge consists of two opposing capillaries of the same external diameter. The
second inlet part is of a finer capillary orientated at right angles to and situated half-
way between the other two capillaries. Constraints on opposing capillary radius and
the placement of the third capillary helped to simplify the dimensionless stability
study. The investigation also necessitated modifications to the dimensionless
parameters characterising axisymmetric liquid bridge geometry. The slendemess ratio,
A * , was calculated using:
N‘ = —-——, (1)
where L and S correspond to the distances indicated in Fig. 17. R0 is defined as the
mean radius, i.e. (R, +RZ)/2. K * is defined as R1/R2 . The volumetric ratio, V*, is
defined as:
I7
V* = , (2)
rrR02\/L2 +52
where I7 is the measured volume at which bridge collapse occurs. In terms of the
geometry presented in Fig. 17, a funicular bridge collapse corresponds to detachment
from the finer capillary.
Fig. 18 shows a stability diagram the approximate location of the minimum volume
stability boundary for purified water funicular liquid bridges with a lateral Bond
number of 1.25 x 104, a near weightless environment. The boundaries of stability
were found by fixing a Value for A*, establishing a stable funicular bridge and
withdrawing fluid until the bridge collapsed. The collapse was recorded via a CCD
and the frame immediately following rupture was analysed to measure the volume.
The calculation of the bridge volume was simplified by the fact that the collapsed
funicular bridge exhibited axisymrnetry with respect to the axis of the two larger
capillaries. Minimum volume stability boundaries are plotted for K *=0.25 and
K * = 0.44 . Lower K * values displayed increased instability. Volumetric data for A *
values lower than approximately 1.5 was difficult to obtain with the geometry used
and so was omitted from the stability diagram. This is, to the best of the inventors
knowledge, the first experimental study of funicular bridges for this application. The
formation of a funicular bridge deemed unstable by the graph shown in Fig. 18
ensures the injection of fluid into an aqueous plug passing through opposing
capillaries. A further advantage to using funicular bridge dispensers is based on the
speed at which the process takes place. Typical instabilities last of the order of lO0ms,
insufficient time for the host droplet fluid to diffuse to the dispensing capillary tip.
This is a further preventative measure against carryover contamination.
The two input one output, funicular bridge can be configured so that the expression
profile of many genes may be addressed. One input contains the primer and premix in
a continuous phase, the outlet then delivers them in droplet form. Firstly many input
and output capillaries, say p , can be set in planes perpendicular to that of Fig. 6. A
perpendicular arrangement allows for good optical access in the planar thermal cycler
which is connected to the output. Each arrangement of two inputs and one output can
be used to address a single primer, giving p primers. This, however, would make for
a very long device in the plane perpendicular to Fig. 6. If serially variant primers were
fed into each input, as described in Figs. 2 and 8, numbering q , this would reduce the
scale. Further, if the primers were multiplexed, to order r , in each droplet the scale
would be further reduced. The number of primers that could then be addressed would
be:
N = p x q x r .
By this means a PCR test of the whole genome of any living form, including the
human, could be addressed, which would have applications beyond diagnosis, in many
fields of pure and applied science.
In another embodiment, mixing of droplets may be achieved with only one inlet port
and two outlet ports. Inlet droplets are close together, and the delay for droplet
formation within the chamber due to a reduction in fluid flow through the main line
causes a collision and hence mixing. Such mixing may be caused by withdrawal of oil
from the chamber, or upstream of it. Referring to Fig. 19 a bridge 60 has an inlet port
61, an outlet port 62, an oil withdrawal port 63, and a chamber 64. A large leading
droplet entering the chamber fonns a droplet 65 in the chamber. As oil is withdrawn
from the chamber 64 through the oil withdrawal port 63, a smaller trailing droplet
collides with the leading droplet bubble 65 so that the mixing occurs. A larger mixed
droplet 67 leaves via the outlet port 62.
In more detail, initially, the entire system is primed with a density matched oil. The
diameter of the ports 61, 62 and 63 is 200nm, and is more generally preferably in the
range of 150nm to 400nm. The spacing between the ports 61 and 62 is c. 1mm, and is
more generally preferably in the range of 0.2 to 1.5mm. The enveloping oil is
controlled to have a pressure of no more than 0.5 to 1.0 bar above atmospheric. The
enveloping liquid is silicone oil with a viscosity of 0.08 to 0.1 Pas.
As with the lateral mixing bridge 30 and the segmentation bridge 50, droplets are
enveloped by carrier oil entering and exiting the bridge 60 via a protective oil film
around the droplets. This provides a non-contacting solid surface that prevents
carryover contamination from one droplet to the other. The oil is used as the control
fluid and is density-matched with the reactor plugs such that a neutrally buoyant
environment is created within the oil chamber 64. When two umnixed droplets arrive
at the chamber in series from the inlet port 61, the first droplet assumes a stable
capillary-suspended spherical form upon entering the chamber, step A. The spherical
shape grows until large enough to span the gap between the ports, forming an
axisymmetric liquid bridge, step B. The control outlet port 63 removes a flow of oil
from the chamber causing the first droplet to slow and remain as a spherical shape at
the outlet port 62 for a longer period. This allows time for the second droplet to form a
stable capillary-suspended spherical shape on entering the chamber. With the first
droplet fonned as a spherical shape at the outlet 62 and the second droplet formed as a
spherical shape at the inlet, the droplets can form as one and create an axisymmetric
liquid bridge, step C. The mixed droplet then exits through the outlet port 62, step D.
The flow conditions most be matched so that the flow through the inlet 61 is greater
than the flow out of the control outlet port 63. A typical flow in through the inlet is
typically 5 pl/mn and more generally in the range of 2 to 7 p.l/mn. The flow away
from the chamber through the control port 63 is typically 2.5 pl/mn and more
generally in the range of 1 to 5 pl/mn. Since there is conservation of mass flow within
the bridge, this means that the flow through the outlet port 62 will balance the bridge
to give a flow of typically 2.5 pl/mn and more generally in the range of l to 5 ul/mn.
This serial mixing bridge 60 can be used with a constant outlet flow through the
control port 63. In doing so not only will droplets be mixed but also the fluid flow
through the system can be decreased. In addition, this bridge 60 can be used in
conjunction with a sensor to time the withdrawal of fluid through the control port 63
so as to maintain the main fluid flow at a generally constant flowrate. The sensor used
can be a droplet detection sensor which comprises of a LED and photodiode. The
LED is projected directly onto the centre of the tube. A photodiode is positioned
directly opposite the LED to pick up the light refracted through the tube. As a droplet
of varying properties to that of oil flows past the LED and photodiode, the light
refracted through the liquid is altered slightly. This slight alteration is detected by the
photodiode in the form of a change in voltage. This change in voltage can be used to
time the control flow through outlet port 63. The serial mixing bridge 60 can also be
used downstream of the lateral mixing bridge 30. In doing so, the system can
compensate for droplets that have not yet mixed after flowing through the lateral
mixer 30. The reason for droplets not mixing may be that the droplets are out of phase
with each other and have not met simultaneously at the lateral mixing bridge 30.
Another system of ensuring mixing has occurred is to include a droplet detection
sensor with the serial mixing bridge 60 downstream of the lateral mixing bridge 30. If
the droplet detection senses two droplets in unusually quick succession then the
droplets have not yet mixed. The droplet detection sensor can then switch on the serial
mixer 60 and mix the two droplets.
It will be appreciated that the invention provides for particularly effective processing
of droplets for applications such as amplification of nucleic acids. It provides
particularly effective mechanisms for mixing of droplets and for segmenting long
droplets or plugs.
The invention is not limited to the embodiments described but may be varied in
construction and detail. For example, the sample liquid may be delivered continuously
to an inlet port in some embodiments, for segmentation. In this case the same basic
mechanism provides droplets of a controlled size in to the outlet port. Also, the flow
controller may merely be a passive feed system based on gravity or indeed capillary
action rather than an active pumping means such as an infusion pump.
Claims (1)
1.0 bar above atmospheric. A method of controlling operation of a liquid bridge as claimed in any of claims 1 to 9, the method comprising directing flow of liquids A and B into the bridge with flow characteristics determined according to the geometry of the bridge ports and their separations and according to liquid properties, such that liquid A periodically bridges to the outlet port due to fluidic instability and droplets are periodically delivered to the outlet port. A method as claimed in claim 13, wherein the flow is controlled to cause segmentation of a continuous stream of large droplets of the liquid A in the chamber to provide a sequence of segmented droplets derived from the inlet liquid A. A method as claimed in claim 13, wherein different, miscible, liquids A and C are delivered to the bridge, and flow is controlled so that droplets of liquids A and C mix within the chamber, in which the liquids A and C are delivered to different inlet ports of the bridge, said ports being located so that droplets of liquids A and C mix to form a single droplet which exits via the outlet port. A method as claimed in claim 13, wherein different, miscible, liquids A and C are delivered to the bridge, and flow is controlled so that droplets of liquids A and C mix within the chamber, and the liquids A and C are delivered to the bridge in a single inlet port as successive droplets, and flow is controlled in the inlet port and in the chamber so that the droplets collide within the chamber to mix. A method as claimed in claim 16, comprising withdrawing carrier liquid B from between said droplets before they enter the bridge so that they are caused to collide within the chamber. A method as claimed in any of claims 15 to 17, wherein the liquids A and C comprise different chemical species contained within an aqueous phase. A method as claimed in any of claims 13 to 18, wherein the carrier liquid B has a viscosity in the range of 0.08 Pas and 0.1 Pas. A method as claimed 11 any of claims 13 to 19, wherein the carrier liquid B flows into the bridge at a flow rate in the range of 2,ul/min and Sal/min. A method as claimed in any of claims 13 to 20, wherein the carrier liquid B is density matched with the liquid A such that a neutrally buoyant environment is created within the chamber. A method as claimed in any of claims 13 to 21, wherein the carrier liquid B has characteristics causing the droplets in the chamber to have a spherical shape between an inlet port and an outlet port. A method as claimed in any of claims 13 to 22, wherein the carrier liquid B is oil. A method as claimed in any of claims 13 to 23, wherein the density of the carrier liquid B and the cross—sectional areas of the ports are such that the carrier liquid B flows and surrounds the droplets of liquid A in the outlet port. A method as claimed in any of claims 13 to 24, wherein the velocity profile across the outlet port is such as to cause internal movement within droplets of liquid as they flow from an outlet port. A method as claimed in claim 25, wherein said internal flow is internal circulation. A method as claimed in any of claims 13 to 26, wherein the carrier liquid B forms a protective film which is static very close to the internal surface of an outlet port and flows with the droplets further from the said surface. 2 <[ Flow Control SystemJ: /1 Reaction Segmentation Liquid bridge dispensing at three different values capillary radii ratio,K *. (A) K* = 0.25 (B) [or = 0.44 and (C) K* =1.0 Capillary tip separations are indicated on the images. Fig.14 SlendernessRutio,/\" Dimensionless plot of dimensionless plug vo|ume,V *,versus slenderness ratio, whereA* was V *scaIed with Rf‘.Resu|ts are plotted forK * values of 1.0, 0.44 and 0.25. Measurement Number Plug volume variation over fourteen measurements for . K* =0.44 Horizontal lines represent the mean volume dispensed. The mean plug volumes were approximately 120 nL and 56 nL with maximum variations 454.46% of and 13.53% respectively. Fig.16 Funicular liquid bridge supported between three capillaries. The geometry used to investigate stability is shown superimposed over the original image. Fig.17 Funicu|arSendernessA* Experimentally determined stability diagram for a purified water funicular liquid bridge in a density matched silicone oil, Bond number: 1.25 x 104 .Vertica| error bars indicate the volumetric ratio uncertainty as a result of camera frame rate.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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IE2007/0069A IE84898B1 (en) | 2007-02-07 | A liquid bridge and system |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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IEIRELAND07/02/20062006/0077 | |||
IE20060077 | 2006-02-07 | ||
IE2007/0069A IE84898B1 (en) | 2007-02-07 | A liquid bridge and system |
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IE84898B1 true IE84898B1 (en) | 2008-06-11 |
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