Note: Descriptions are shown in the official language in which they were submitted.
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Description
METHODS AND APPARATUS FOR MANIPULATING DROPLETS BY
ELECTROWETTING-BASED TECHNIQUES
Related Applications
6 This application is related to and claims the benefit of U.S. Patent No.
6,911,132 filed September 24, 2002.
Government Interest
This invention was made with United States Government support under
Grant No. F30602-98-2-0140 awarded by the Defense Advanced Research
Projects Agency. The United States Government has certain rights in the
invention.
Technical Field
The present invention is generally related to the field of droplet-based
liquid handling and processing, such as droplet-based sample preparation,
mixing, and dilution on a microfluidic scale. More specifically, the present
invention relates to the manipulation of droplets by electrowetting-based
techniques.
Background Art
Microfluidic systems are presently being explored for their potential to
carry out certain processing techniques on capillary-sized continuous flows of
liquid. In particular, there is currently great interest in developing
microfluidic
devices commonly referred to as "chemistry-on-a-chip" sensors and analyzers,
which are also known as labs-on-a-chip (LoC) and micro total analysis systems
(p-TAS). The ultimate goal of research in this field is to reduce most common
(bio)chemical laboratory procedures and equipment to miniaturized, automated
chip-based formats, thereby enabling rapid, portable, inexpensive, and
reliable
(bio)chemical instrumentation. Applications include medical diagnostics,
environmental monitoring, and basic scientific research.
On-line monitoring of continuous flows is most often accomplished by
connecting the output of the continuous-flow to the input of a large analysis
instrument such as a HPLC (high pressure liquid chromatography), CE
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(capillary electrophoresis) or MS (mass spectrometry) system, with appropriate
flow control and valving for sample collection and injection. Microfluidic
systems for continuous monitoring typically employ miniaturized analyte-
specific biosensors where the continuous-flow stream passes over or through a
series of the biosensors. Because the sensors lie in a common channel,
crosstalk or contamination between sensors is often a concern. In analyses
where a reagent must be mixed with the flow, only one analyte can be
measured at a time unless the flow is divided into parallel streams with
separate means for adding the reagent, controlling and mixing the flow and
carrying out detection in each stream. Additionally, mixing in microfluidic
flows
is usually quite challenging. Sufficient time and distance must be provided
for
mixing, which places constraints on chip design and system flow rates.
In general, mixing is a fundamental process in chemical analysis and
biological applications. Mixing in microfluidic devices is a critical step in
realizing a ,uTAS (micro total analysis system) or "lab on a chip" system. In
accordance with the present invention described hereinbelow, it is posited
that
mixing in these systems could be used for pre-processing sample dilution orfor
reactions between sample and reagents in particular ratios. It is further
posited
that the ability to mix liquids rapidly while utilizing minimum chip area
would
greatly improve the throughput of such systems. The improved mixing would
rely on two principles: the ability to either create turbulent, nonreversible
flow at
such small scales or create multilaminates to enhance mixing via diffusion.
Mixers can be broadly categorized into continuous-flow and droplet-
based architectures. A common limitation among all continuous-flow systems
is that fluid transport is physically confined to permanently etched
structures,
and additional mechanisms are required to enhance mixing. The transport
mechanisms used are usually pressure-driven by external pumps or
electrokinetically-driven by high-voltage supplies. This in turn requires the
use
of valves and complex channeling, consuming valuable real estate on a chip.
These restrictions prevent the continuous-flow micro-mixer from becoming a
truly self-contained, reconfigurable lab-on-a-chip. Whereas conventional
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continuous-flow systems rely on a continuous liquid flow in a confined
channel,
droplet-based systems utilize discrete volumes of liquid. Both the continuous-
flow and droplet-based architectures can be further classified into passive
and
active mixers. In passive mixers, mixing is mediated through diffusion
passively without any external energy inputted for the process. Active mixing,
on the other hand, takes advantage of external energy, through actuation of
some sort, to create either dispersed multilaminates or turbulence. In the
microscopic world, effective mixing is a technical problem because it is
difficult
to generate turbulent flow by mechanical actuation. The inertial forces that
produce turbulence and the resulting large interfacial surface areas necessary
to promote mixing are absent. Thus, mixing that depends on diffusion through
limited interfacial areas is a limitation.
Recently, active mixing by acoustic wave (see Vivek et al., "Novel
acoustic micromixer", MEMS 2000 p. 668-73); ultrasound (see Yang et al.,
"Ultrasonic micromixer for microfluidic systems", MEMS 2000, p. 80); and a
piezoelectrically driven, valveless micropump (see Yang et al., "Micromixer
incorporated with piezoelectrically driven valveless micropump", Micro Total
Analysis System '98, p. 177-180) have been proposed, and their effectiveness
has been demonstrated. Mixing by electroosmotic flow has also been
described in U.S. Pat. No. 6,086,243 to Paul et al. Another mixing technique
has been recently presented by employing chaotic advection for mixing. See
Lee et al., "Chaotic mixing in electrically and pressure driven microflows",
The
14th IEEE workshop on MEMS 2001, p. 483-485; Liu et al., "Passive Mixing in a
Three-Dimensional Serpentine Microchannel", J. of MEMS, Vol 9 (No. 2), p.
190-197 (June 2000); and Evans et al., "Planar laminar mixer", Proc. of IEEE,
The tenth annual workshop on Micro Electro Mechanical Systems (MEMS 97),
p. 96-101 (1997). Lee et al. focus on employing dielectrophoretic forces or
pressure to generate chaotic advection, while Liu et al. rely on the geometry
of
a microchannel to induce the similar advection. Evans et al. constructed a
planar mixing chamber on the side of which an asymmetrical source and sink
generate a flow field, whereby small differences in a fluid particle's initial
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location leads to large differences in its final location. This causes chaotic
rearrangement of fluid particles, and thus the mixing two liquids. Most
recently,
a technique has been proposed that uses electrohydrodynamic convection for
active mixing. See Jin et at., "An active micro mixer using
electrohydrodynamic
(EHD) convection for microfluidic-based biochemical analysis", Technical
Digest, Solid-State Sensor and Actuator Workshop, p. 52-55).
Molecular diffusion plays an important role in small Reynolds number
liquid flow. In general, diffusion speed increases with the increase of the
contact surface between two liquids. The time required for molecular diffusion
increases in proposition to the square of the diffusion distance. A fast
diffusion
mixer consisting of a simple narrowing of a mixing channel has been
demonstrated by Veenstra et al., "Characterization method for a new diffusion
mixer applicable in micro flow injection analysis systems", J. Micromech.
Microeng., Vol. 9, pg. 199-202 (1999). The primary approach for diffusion-
based micromixing has been to increase the interfacial area and to decrease
the diffusion length by interleaving two liquids. Interleaving is done by
manipulating the structure's geometry. One approach is to inject one liquid
into
another through a micro nozzle array. See Miyake et al., "Micro mixer with
fast
diffusion", Proceedings of Micro Electro Mechanical Systems, p. 248-253
(1993). An alternative method is to stack two flow streams in one channel as
thin layers by multiple stage splitting and recombining. See Branebjerg et
al.,
"Fast mixing by lamination"', Proc. IEEE Micro Electro Mechanical Systems, p.
441 (1996); Krog et at., "Experiments and simulations on a micro-mixer
fabricated using a planar silicon/glass technology", MEMS, p. 177-182 (1998);
Schwesinger et al., "A modular microfluidic system with an integrated
micromixer", J. Micromech. Microeng., Vol 6, pg. 99-102 (1996); and
Schwesinger et at., "A static micromixer built up in silicon", Proceedings of
the
SPIE, The International Society for Optical Engineering, Micromachined
Devices and Components, Vol. 2642, p. 150-155. The characterizations of this
type of mixer are provided by Koch et at., "Two simple micromixers based on
silicon", J. Micromech. Microeng., Vol 8, p. 123-126 (1998); Koch et at.,
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"Micromachined chemical reaction system", Sensors and Actuators, Physical
(74), p. 207-210; and Koch et al., "Improved characterization technique for
micromixer, J. Micromech. Microeng, Vol 9, p. 156-158 (1999). A variation of
the lamination technique is achieved similarly by fractionation, re-
arrangement,
and subsequent reunification of liquids in sinusoidally shaped fluid channels
(see Kamper et al., "Microfluidic components for biological and chemical
microreactors", MEMS 1997, p. 338); in alternative channels of two counter
current liquids (see http://www.imm-mainz.de/Lnews/Lnews_4/mire.html); or in
a 3D pipe with a series of stationary rigid elements forming intersecting
channels inside (see Bertsch et al., "3D micromixers-downscaling large scale
industrial static mixers", MEMS 2001 14th International Conference on Micro
Electro Mechanical Systems, p. 507-510). One disadvantage of purely
diffusion-based static mixing is the requirement of a complex 3D structure in
order to provide out-of-plane fluid flow. Another disadvantage is the low
Reynolds number characterizing the flow, which results in a long mixing time.
A problem for active mixers is that energy absorption during the mixing
process makes them inapplicable to temperature-sensitive fluids. Moreover,
some active mixers rely on the charged or polarizable fluid particles to
generate
convection and local turbulence. Thus, liquids with low conductivity could not
be properly mixed. When the perturbation force comes from a mechanical
micropump, however, the presence of the valveless micropump makes the
control of flow ratios of solutions for mixing quite complex.
In continuous flow systems, the control of the mixing ratio is always a
technical problem. By varying the sample and reagent flow rates, the mixing
ratio can be obtained with proper control of the pressure at the reagent and
sample ports. However, the dependence of pressure on the properties of the
fluid and the geometry of the mixing chamber/channels makes the control very
complicated. When inlets are controlled by a micropump, the nonlinear
relationship between the operating frequency and flow rate make it a
nontrivial
task to change the flow rate freely. The discontinuous mixing of two liquids
by
integration of a mixer and an electrically actuated flapper valve has been
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demonstrated by Voldman et al., "An Integrated Liquid Mixer/Valve", Journal of
Microelectromechanical Systems", Vol. 9, No. 3 (Sep. 2000). The design
required a sophisticated pressure-flow calibration to get a range of mixing
ratios.
Droplet-based mixers have been explored by Hosokawa et al., "Droplet
based nano/picoliter mixer using hydrophobic microcapillary vent", MEMS '99,
p. 388; Hosokawa et al., "Handling of Picoliter Liquid Samples in a
Poly(dimethylsiloxane)-Based Microfluidic Device", Anal. Chem 1999, Vol. 71,
p. 4781-4785; Washizu et al., Electrostatic actuation of liquid droplets for
micro-
reactor applications, IEEE Transactions on Industry Applications, Vol. 34 (No.
4), p. 732-737 (1998); Burns et al., "An Integrated Nanoliter DNA Analysis
Device", Science, Vol. 282 (No. 5388), p. 484 (Oct. 16, 1998); Pollack et al.,
"Electrowetting-based actuation of liquid droplets for microfluidic
applications",
Appl. Phys. Lett., Vol. 77, p. 1725 (Sept. 2000); Pamula et al.,
`ivlicrofluidic
electrowetting-based droplet mixing", MEMS Conference, 2001, 8-10.; Fowler
et al., "Enhancement of Mixing by Droplet-based Microfluidics", IEEE MEMS
Proceedings, 2002, 97-100.; Pollack, "Electrowetting-based microactuation of
droplets for digital microfluidics", Ph.D. Thesis, Department of Electrical
and
Computer Engineering, Duke University; and Wu, "Design and Fabrication of
an Input Buffer for a Unit Flow Microfluidic System", Master thesis,
Department
of Electrical and Computer Engineering, Duke University.
It is believed that droplet-based mixers can be designed and constructed
to provide a number of advantages over continuous-flow-based microfluidic
devices. Discrete flow can eliminate the limitation on flow rate imposed by
continuous microfluidic devices. The design of droplet-based mixing devices
can be based on a planar structure that can be fabricated at low cost.
Actuation mechanisms based on pneumatic drive, electrostatic force, or
electrowetting do not require heaters, and thus have a minimum effect on (bio)
chemistry. By providing a proper droplet generation technique, droplet-based
mixers can provide better control of liquid volume. Finally, droplet-based
mixers can enable droplet operations such as shuttling or shaking to generate
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internal recirculation within the droplet, thereby increasing mixing
efficiency in
the diffusion-dominated scale.
In view of the foregoing, it would be advantageous to provide novel
droplet-manipulative techniques to address the problems associated with
previous analytical and mixing techniques that required continuous flows. In
particular, the present invention as described and claimed hereinbelow
developed in part from the realization that an alternative and better solution
to
the continuous flow architecture would be to design a system where the
channels and mixing chambers are not permanently etched, but rather are
virtual and can be configured and reconfigured on the fly. The present
invention enables such a system by providing means for discretizing fluids
into
droplets and means for independently controlling individual droplets, allowing
each droplet to act as a virtual mixing or reaction chamber.
Disclosure of the Invention
The present invention provides droplet-based liquid handling and
manipulation methods by implementing electrowetting-based techniques. The
droplets can be sub-microliter-sized, and can be moved freely by controlling
voltages to electrodes. Generally, the actuation mechanism of the droplet is
based upon surface tension gradients induced in the droplet by the voltage-
induced electrowetting effect. The mechanisms of the invention allow the
droplets to be transported while also acting as virtual chambers for mixing to
be
performed anywhere on a chip. The chip can include an array of electrodes
that are reconfigurable in real-time to perform desired tasks. The invention
enables several different types of handling and manipulation tasks to be
performed on independently controllable droplet samples, reagents, diluents,
and the like. Such tasks conventionally have been performed on continuous
liquid flows. These tasks include, for example, actuation or movement,
monitoring, detection, irradiation, incubation, reaction, dilution, mixing,
dialysis,
analysis, and the like. Moreover, the methods of the invention can be used to
form droplets from a continuous-flow liquid source, such as a from a
continuous
input provided at a microfluidic chip. Accordingly, the invention provides a
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method for continuous sampling by discretizing or fragmenting a continuous
flow into a desired number of uniformly sized, independently controllable
droplet units.
The partitioning of liquids into discrete, independently controlled packets
or droplets for microscopic manipulation provides several important advantages
over continuous-flow systems. For instance, the reduction of fluid
manipulation, or fluidics, to a set of basic, repeatable operations (for
example,
moving one unit of liquid one unit step) allows a hierarchical and cell-based
design approach that is analogous to digital electronics.
In addition to the advantages identified hereinabove, the present
invention utilizes electrowetting as the mechanism for droplet actuation or
manipulation for the following additional advantages:
1. Improved control of a droplet's position.
2. High parallelism capability with a dense electrode array layout.
3. Reconfigurability.
4. Mixing-ratio control using programming operations, yielding better
controllability and higher accuracy in mixing ratios.
5. High throughput capability, providing enhanced parallelism.
6. Enabling of integration with optical detection that can provide
further enhancement on asynchronous controllability and accuracy.
In particular, the present invention provides a sampling method that
enables droplet-based sample preparation and analysis. The present invention
fragments or discretizes the continuous liquid flow into a series of droplets
of
uniform size on or in a microfluidic chip or other suitable structure by
inducing
and controlling electrowetting phenomena. The liquid is subsequently
conveyed through or across the structure as a train of droplets which are
eventually recombined for continuous-flow at an output, deposited in a
collection reservoir, or diverted from the flow channel for analysis.
Alternatively, the continuous-flow stream may completely traverse the
structure,
with droplets removed or sampled from specific locations along the continuous
flow for analysis. In both cases, the sampled droplets can then be transported
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to particular areas of the structure for analysis. Thus, the analysis is
carried out
on-line, but not in-line with respect to the main flow, allowing the analysis
to be
de-coupled from the main flow.
Once removed from the main flow, a facility exists for independently
controlling the motion of each droplet. For purposes of chemical analysis, the
sample droplets can be combined and mixed with droplets containing specific
chemical reagents formed from reagent reservoirs on or adjacent to the chip or
other structure. Multiple-step reactions or dilutions might be necessary in
some
cases with portions of the chip assigned to certain functions such as mixing,
reacting or incubation of droplets. Once the sample is prepared, it can be
transported by electrowetting to another portion of the chip dedicated to
detection or measurement of the analyte. Some detection sites can, for
example, contain bound enzymes or other biomolecular recognition agents,
and be specific for particular analytes while others can consist of a general
means of detection such as an optical system for fluorescence or absorbance
based assays. The flow of droplets from the continuous flow source to the
analysis portion of the chip (the analysis flow) is controlled independently
of the
continuous flow (the input flow), allowing a great deal of flexibility in
carrying out
the analyses. Other features and advantages of the methods of the present
invention are described in more detail hereinbelow.
Methods of the present invention use means for forming microdroplets
from the continuous flow and for independently transporting, merging, mixing,
and other processing of the droplets. The preferred embodiment uses
electrical control of surface tension (i.e., electrowetting) to accomplish
these
manipulations. In one embodiment, the liquid is contained within a space
between two parallel plates. One plate contains etched drive electrodes on its
surface while the other plate contains either etched electrodes or a single,
continuous plane electrode that is grounded or set to a reference potential.
Hydrophobic insulation covers the electrodes and an electric field is
generated
between electrodes on opposing plates. This electric field creates a surface-
tension gradient that causes a droplet overlapping the energized electrode to
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move towards that electrode. Through proper arrangement and control of the
electrodes, a droplet can be transported by successively transferring it
between
adjacent electrodes. The patterned electrodes can be arranged in a two
dimensional array so as to allow transport of a droplet to any location
covered
by that array. The space surrounding the droplets maybe filled with a gas such
as air or an immiscible fluid such as oil.
In another embodiment, the structure used for ground or reference
potential is co-planar with the drive electrodes and the second plate, if
used,
merely defines the containment space. The co-planar grounding elements can
be a conductive grid superimposed on the electrode array. Alternatively, the
grounding elements can be electrodes of the array dynamically selected to
serve as ground or reference electrodes while other electrodes of the array
are
selected to serve as drive electrodes.
Droplets can be combined together by transporting them simultaneously
onto the same electrode. Droplets are subsequently mixed either passively or
actively. Droplets are mixed passively by diffusion. Droplets are mixed
actively
by moving or "shaking" the combined droplet by taking advantage of the
electrowetting phenomenon. In a preferred embodiment, droplets are mixed by
rotating them around a two-by-two array of electrodes. The actuation of the
droplet creates turbulent non-reversible flow, or creates dispersed
multilaminates to enhance mixing via diffusion. Droplets can be split off from
a
larger droplet or continuous body of liquid in the following manner: at least
two
electrodes adjacent to the edge of the liquid body are energized along with an
electrode directly beneath the liquid, and the liquid moves so as to spread
across the extent of the energized electrodes. The intermediate electrode is
then de-energized to create a hydrophobic region between two effectively
hydrophilic regions. The liquid meniscus breaks above the hydrophobic
regions, thus forming a new droplet. This process can be used to form the
droplets from a continuously flowing stream.
According to one embodiment of the present invention, an apparatus for
manipulating droplets comprises a substrate comprising a substrate surface, an
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array of electrodes disposed on the substrate surface, an array of reference
elements, a dielectric layer disposed on the substrate surface, and an
electrode
selector. The reference elements are settable to a reference potential. The
array of reference elements is disposed of in substantially co-planar relation
to
the electrode array, such that each reference element is adjacent to at least
one of the electrodes. The dielectric layer is disposed on the substrate
surface
and is patterned to cover the electrodes. The electrode selector can be
provided as a microprocessor or other suitable component for sequentially
activating and de-activating one or more selected electrodes of the array to
sequentially bias the selected electrodes to an actuation voltage. The
sequencing performed by the electrode selector enables a droplet disposed on
the substrate surface to move along a desired path that is defined by the
selected electrodes.
According to one method of the present invention, a droplet is actuated
by providing the droplet on a surface that comprises an array of electrodes
and
a substantially co-planar array of reference elements. The droplet is disposed
on a first one of the electrodes, and at least partially overlaps a second one
of
the electrodes and an intervening one of the reference elements disposed
between the first and second electrodes. The first and second electrodes are
activated to spread at least a portion of the droplet across the second
electrode. The first electrode is de-activated to move the droplet from the
first
electrode to the second electrode.
According to one aspect of this method, the second electrode is
adjacent to the first electrode along a first direction. In addition, the
electrode
array comprises one more additional electrodes adjacent to the first electrode
along one or more additional directions. The droplet at least partially
overlaps
these additional electrodes as well as the second electrode. In accordance
with this aspect of the method, the first direction that includes the first
electrode
and the second electrode is selected as a desired direction along which the
droplet is to move. The second electrode is selected for activation based on
the selection of the first direction.
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In accordance with another method of the present invention, a droplet is
split into two or more droplets. A starting droplet is provided on a surface
comprising an array of electrodes and a substantially co-planar array of
reference elements. The electrode array comprises at least three electrodes
comprising a first outer electrode, a medial electrode adjacent to the first
outer
electrode, and a second outer electrode adjacent to the medial electrode. The
starting droplet is initially disposed on at least one of these three
electrodes,
and at least partially overlaps at least one other of the three electrodes.
Each
of the three electrodes is activated to spread the starting droplet across the
three electrodes. The medial electrode is de-activated to split the starting
droplet into first and second split droplets. The first split droplet is
disposed on
the first outer electrode and the second split droplet is disposed on the
second
outer electrode.
In yet another method of the present invention, two or more droplets are
merged into one droplet. First and second droplets are provided on a surface
comprising an array of electrodes in a substantially co-planar array of
reference
elements. The electrode array comprises at least three electrodes comprising
a first outer electrode, a medial electrode adjacent to the first outer
electrode,
and a second outer electrode adjacent to the medial electrode. The first
droplet is disposed on the first outer electrode and at least partially
overlaps the
medial electrode. The second droplet is disposed on the second outer
electrode and at least partially overlaps the medial electrode. One of the
three
electrodes is selected as a destination electrode. Two or more of the three
electrodes are selected for sequential activation and de-activation, based on
the selection of the destination electrode. The electrodes selected for
sequencing are sequentially activated and de-activated to move one of the
first
and second droplets toward the other droplet, or both of the first and second
droplets toward each other. The first and second droplets merge together to
form a combined droplet on the destination electrode.
According to one aspect of this method, the first droplet comprises a first
composition, the second droplet comprises a second composition, and the
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combined droplet comprises both the first and second compositions. The
method further comprises the step of mixing the first and second compositions
together. In accordance with the present invention, the mixing step can be
passive or active. In one aspect of the invention, the mixing step comprises
moving the combined droplet on a two-by-two sub-array of four electrodes by
sequentially activating and de-activating the four electrodes to rotate the
combined droplet. At least a portion of the combined droplet remains
substantially stationary at or near an intersecting region of the four
electrodes
while the combined droplet rotates. In another aspect of the invention, the
mixing step comprises sequentially activating and de-activating a linearly
arranged set of electrodes of the electrode array to oscillate the combined
droplet back and forth along the linearly arranged electrode set a desired
number of times and at a desired frequency. Additional mixing strategies
provided in accordance with the invention are described in detail hereinbelow.
According to another embodiment of the present invention, an apparatus
for manipulating droplets comprises a substrate comprising a substrate
surface,
an array of electrodes disposed on the substrate surface, a dielectric layer
disposed on the substrate surface and covering the electrodes, and an
electrode selector. The electrode selector dynamically creates a sequence of
electrode pairs. Each electrode pair comprises a selected first one of the
electrodes biased to a first voltage, and a selected second one of the
electrodes disposed adjacent to the selected first electrode and biased to a
second voltage that is less than the first voltage. Preferably, the second
voltage is a ground voltage or some other reference voltage. A droplet
disposed on the substrate surface moves along a desired path that runs
between the electrode pairs created by the electrode selector.
According to yet another method of the present invention, a droplet is
actuated by providing the droplet on a surface comprising an array of
electrodes. The droplet is initially disposed on a first one of the electrodes
and
at least partially overlaps a second one of the electrodes that is separated
from
the first electrode by a first gap. The first electrode is biased to a first
voltage
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and the second electrode is biased to a second voltage lower than the first
voltage. In this manner, the droplet becomes centered on the first gap. A
third
one of the electrodes that is proximate to the first and second electrodes is
biased to a third voltage that is higher than the second voltage to spread the
droplet onto the third electrode. The bias on the first electrode is then
removed
to move the droplet away from the first electrode. The droplet then becomes
centered on a second gap between the second and third electrodes.
According to still another method of the present invention, a droplet is
split into two or more droplets. A starting droplet is provided on a surface
comprising an array of electrodes. The electrode array comprises at least
three
electrodes comprising a first outer electrode, a medial electrode adjacent to
the
first outer electrode, and a second outer electrode adjacent to the medial
electrode. The starting droplet is initially disposed on at least one of the
three
electrodes and at least partially overlaps at least one other of the three
electrodes. Each of the three electrodes is biased to a first voltage to
spread
the initial droplet across the three electrodes. The medial electrode is
biased to
a second voltage lower than the first voltage to split the initial droplet
into first
and second split droplets. The first split droplet is formed on the first
outer
electrode and the second split droplet is formed on the second outer
electrode.
According to a further method of the present invention, two or more
droplets are merged into one droplet. First and second droplets are provided
on a surface comprising an array of electrodes. The electrode array comprises
at least three electrodes comprising a first outer electrode, a medial
electrode
adjacent to the first outer electrode, and a second outer electrode adjacent
to
the medial electrode. The first droplet is disposed on the first outer
electrode
and at least partially overlaps the medial electrode. The second droplet is
disposed on the second outer electrode and at least partially overlaps the
medial electrode. One of the three electrodes is selected as a destination
electrode. Two or more of the three electrodes are selected for sequential
biasing based on the selection of the destination electrode. The electrodes
selected for sequencing are sequentially biased between a first voltage and a
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second voltage to move one of the first and second droplets toward the other
droplet or both of the first and second droplets toward each other. The first
and
second droplets merge together to form a combined droplet on the destination
electrode.
The present invention also provides a method for sampling a continuous
liquid flow. A liquid flow is supplied to a surface along a first flow path.
The
surface comprises an array of electrodes and a substantially co-planar array
of
reference elements. At least a portion of the liquid flow is disposed on a
first
one of the electrodes, and at least partially overlaps a second one of the
electrodes and a reference element between the first and second electrodes.
The first electrode, the second electrode, and a third one of the electrodes
adjacent to second electrode are activated to spread the liquid flow portion
across the second and third electrodes. The second electrode is de-activated
to form a droplet from the liquid flow on the third electrode. The droplet is
distinct from and in controllable independently of the liquid flow.
In accordance with another method of the present invention for sampling
a continuous liquid flow, a liquid flow is supplied to a surface along a first
flow
path. The surface comprises an array of electrodes. At least a portion of the
liquid flow is disposed on a first one of the electrodes and at least
partially
overlaps a second one of the electrodes. The first electrode, the second
electrode, and a third one of the electrodes adjacent to the second electrode
are biased to a first voltage to spread the liquid flow portion across the
second
and third electrodes. The second electrode is biased to a second voltage that
is less than the first voltage to form a droplet from the liquid flow on the
third
electrode. The droplet so formed is distinct from and controllable
independently of the liquid flow.
According to still another embodiment of the present invention, a binary
mixing apparatus comprises a first mixing unit, a second mixing unit, and an
electrode selector. The first mixing unit comprises a first surface area, an
array
of first electrodes disposed on the first surface area, and an array of first
reference elements disposed in substantially co-planar relation to the first
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electrodes. The second mixing unit comprises a second surface area, an array
of second electrodes disposed on the second surface area, an array of second
reference elements disposed in substantially co-planar relation to the second
electrodes, and a droplet outlet area communicating with the second surface
area and with the first mixing unit. The electrode selector sequentially
activates
and de-activates one or more selected first electrodes to mix together two
droplets supplied to the first surface area. The electrode selector also
sequentially activates and de-activates one or more selected second electrodes
to mix together two other droplets supplied to the second surface area.
It is therefore an object of the present invention to sample a continuous
flow liquid input source from which uniformly sized, independently
controllable
droplets are formed on a continuous and automated basis.
It is another object of the present invention to utilize electrowetting
technology to implement and control droplet-based manipulations such as
transportation, mixing, detection, analysis, and the like.
It is yet another object of the present invention to provide an architecture
suitable for efficiently performing binary mixing of droplets to obtain
desired
mixing ratios with a high degree of accuracy.
Some of the objects of the invention having been stated hereinabove,
other objects will become evident as the description proceeds when taken in
connection with the accompanying drawings as best described hereinbelow.
Brief Description of the Drawings
Figure 1 is a cross-sectional view of an electrowetting microactuator
mechanism having a two-sided electrode configuration in accordance with the
present invention;
Figure 2 is a top plan view of an array of electrode cells having
interdigitated perimeters accordance with one embodiment of the present
invention;
Figure 3 is a plot of switching rate as a function of voltage demonstrating
the performance of an electrowetting microactuator mechanism structured in
accordance with the present invention;
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Figures 4A - 4D are sequential schematic views of a droplet being
moved by the electrowetting technique of the present invention;
Figures 5A - 5C are sequential schematic views illustrating two droplets
combining into a merged droplet using the electrowetting technique of the
present invention;
Figures 6A- 6C are sequential schematic views showing a droplet being
split into two droplets by the electrowetting technique of the present
invention;
Figures 7A and 7B are sequential schematic views showing a liquid
being dispensed on an electrode array and a droplet being formed from the
liquid;
Figure 8A is a cross-sectional view illustrating an electrowetting
microactuator mechanism of the invention implementing a one-dimensional
linear droplet merging process;
Figure 8B is a top plan view of the configuration in Figure 8A with the
upper plane removed;
Figures 9A, 9B, and 9C are respective top plan views of two-, three-, and
four-electrode configurations on which one-dimensional linear mixing of
droplets can be performed in accordance with the present invention;
Figures 10A, 10B, and 10C are schematic diagrams illustrating the
examples of a mixing-in-transport process enabled by the present invention;
Figure 11 is a schematic view illustrating a two-dimensional linear mixing
process enabled by the present invention;
Figure 12A is a top plan view of an array of electrode cells on which a
two-dimensional loop mixing process is performed in accordance with the
present invention;
Figure 12B is a top plan view of a 2 X 2 array of electrode cells on which
a two-dimensional loop mixing process is performed in which a portion of the
droplet remains pinned during rotation;
Figure 13 is a plot of data characterizing the performance of active
droplet mixing using the two-, three- and four- electrode configurations
respectively illustrated in Figures 9A, 9B, and 9C;
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Figure 14 is a plot of data characterizing the performance of the 2 X 2
electrode configuration illustrated in Figure 12B;
Figure 15A is a schematic view illustrating the formation of droplets from
a continuous flow source and movement of the droplets across an electrode-
containing surface to process areas of the surface;
Figure 15B is a schematic view illustrating the formation of droplets from
a continuous flow that traverses an entire electrode-containing surface or
section thereof;
Figure 16 is a top plan view of a droplet-to-droplet mixing unit that can
be defined on an electrode array on a real-time basis;
Figure 17 is a schematic view of a binary mixing apparatus provided in
accordance with the present invention;
Figure 18A is a schematic view of the architecture of a binary mixing unit
capable of one-phase mixing according to the present invention;
Figure 18B is a schematic sectional view of the binary mixing unit
illustrated in Figure 18A, showing details of the matrix section thereof where
binary mixing operations occur;
Figures 19A-19F are sequential schematic views of an electrode array
or section thereof provided by a binary mixing unit of the present invention,
showing an exemplary process for performing binary mixing operations to
obtain droplets having a predetermined, desired mixing ratio;
Figure 20 is a schematic view illustrating the architecture for a binary
mixing unit capable of two-phase mixing in accordance with the present
invention;
Figure 21 is a plot of mixing points of a one- and two-phase mixing plan
enabled by the binary mixing architecture of the present invention; and
Figure 22 is a plot of mixing points of a one-, two- and three-phase
mixing plan enabled by the binary mixing architecture of the present
invention.
Figure 23A is a cross-sectional view of an electrowetting microactuator
mechanism having a single-sided electrode configuration in accordance with
another embodiment of the present invention;
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Figure 23B is a top plan view of a portion of the mechanism illustrated in
Figure 23A with its upper plane removed;
Figures 24A - 24D are sequential schematic views of an electrowetting
microactuator mechanism having an alternative single-sided electrode
configuration, illustrating electrowetting-based movement of a droplet
positioned on a misaligned electrode array of the mechanism; and
Figures 25A and 25B are schematic views of an alternative
electrowetting microactuator mechanism having a single-sided electrode
configuration arranged as an aligned array, respectively illustrating a
droplet
actuated in north-south and east-west directions.
Detailed Description of the Invention
For purposes of the present disclosure, the terms "layer" and "film" are
used interchangeably to denote a structure or body that is typically but not
necessarily planar or substantially planar, and is typically deposited on,
formed
on, coats, treats, or is otherwise disposed on another structure.
For purposes of the present disclosure, the term "communicate" (e.g., a
first component "communicates with" or "is in communication with" a second
component) is used herein to indicate a structural, functional, mechanical,
electrical, optical, or fluidic relationship, or any combination thereof,
between
two or more components or elements. As such, the fact that one component is
said to communicate with a second component is not intended to exclude the
possibility that additional components may be present between, and/or
operatively associated or engaged with, the first and second components.
For purposes of the present disclosure, it will be understood that when a
given component such as a layer, region or substrate is referred to herein as
being disposed or formed "on", "in", or "at" another component, that given
component can be directly on the other component or, alternatively,
intervening
components (for example, one or more buffer layers, interlayers, electrodes or
contacts) can also be present. It will be further understood that the terms
"disposed on" and "formed on" are used interchangeably to describe how a
given component is positioned or situated in relation to another component.
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Hence, the terms "disposed on" and "formed on" are not intended to introduce
any limitations relating to particular methods of material transport,
deposition,
or fabrication.
For purposes of the present disclosure, it will be understood that when a
liquid in any form (e.g., a droplet or a continuous body, whether moving or
stationary) is described as being "on", "at", or "over" an electrode, array,
matrix
or surface, such liquid could be either in direct contact with the
electrode/array/matrix/surface, or could be in contact with one or more layers
or
films that are interposed between the liquid and the
electrode/array/matrix/surface.
As used herein, the term "reagent" describes any material useful for
reacting with, diluting, solvating, suspending, emulsifying, encapsulating,
interacting with, or adding to a sample material.
The droplet-based methods and apparatus provided by the present
invention will now be described in detail, with reference being made as
necessary to the accompanying Figures 1 - 25B.
Droplet-Based Actuation by Electrowetting
Referring now to Figure 1, an electrowetting microactuator mechanism,
generally designated 10, is illustrated as a preferred embodiment for
effecting
electrowetting-based manipulations on a droplet D without the need for pumps,
valves, or fixed channels. Droplet D is electrolytic, polarizable, or
otherwise
capable of conducting current or being electrically charged. Droplet D is
sandwiched between a lower plane, generally designated 12, and an upper
plane, generally designated 14. The terms "upper" and "lower" are used in the
present context only to distinguish these two planes 12 and 14, and not as a
limitation on the orientation of planes 12 and 14 with respect to the
horizontal.
Lower plane 12 comprises an array of independently addressable control
electrodes. By way of example, a linear series of three control or drive
electrodes E (specifically El, E2, and E3) are illustrated in Figure 1. It
will be
understood, however, that control electrodes E1, E2, and E3 could be arranged
along a non-linear path such as a circle. Moreover, in the construction of
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devices benefiting from the present invention (such as a microfluidic chip),
control electrodes E1, E2, and E3 will typically be part of a larger number of
control electrodes that collectively form a two-dimensional electrode array or
grid. Figure 1 includes dashed lines between adjacent control electrodes E1,
E2, and E3 to conceptualize unit cells, generally designated C (specifically
C1,
C2 and C3). Preferably, each unit cell C1, C2, and C3 contains a single
control
electrode, E1, E2, and E3, respectively. Typically, the size of each unit cell
C or
control electrode E is between approximately 0.05 mm to approximately 2 mm.
Control electrodes E1, E2, and E3 are embedded in or formed on a
suitable lower substrate or plate 21. A thin lower layer 23 of hydrophobic
insulation is applied to lower plate 21 to cover and thereby electrically
isolate
control electrodes E1, E2, and E3. Lower hydrophobic layer 23 can be a single,
continuous layer or alternatively can be patterned to cover only the areas on
lower plate 21 where control electrodes E1, E2 and E3 reside. Upper plane 14
comprises a single continuous ground electrode G embedded in or formed on a
suitable upper substrate or plate 25. Alternatively, a plurality of ground
electrodes G could be provided in parallel with the arrangement of
corresponding control electrodes E1, E2 and E3, in which case one ground
electrode G could be associated with one corresponding control electrode E.
Preferably, a thin upper layer 27 of hydrophobic insulation is also applied to
upper plate 25 to isolate ground electrode G. One non-limiting example of a
hydrophobic material suitable for lower layer 23 and upper layer 27 is
TEFLON AF 1600 material (available from E. I. duPont deNemours and
Company, Wilmington, Delaware). The geometry of microactuator mechanism
10 and the volume of droplet D are controlled such that the footprint of
droplet
D overlaps at least two control electrodes (e.g., E1 and E3) adjacent to the
central control electrode (e.g., E2) while also making contact with upper
layer
27. Preferably, this is accomplished by specifying a gap or spacing d, which
is
defined between lower plane 12 and upper plane 14 as being less than the
diameter that droplet D would have in an unconstrained state. Typically, the
cross-sectional dimension of spacing d is between approximately 0.01 mm to
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approximately 1 mm. Preferably, a medium fills gap d and thus surrounds
droplet D. The medium can be either an inert gas such as air or an immiscible
fluid such as silicone oil to prevent evaporation of droplet D.
Ground electrode G and control electrodes E1, E2 and E3 are placed in
electrical communication with at least one suitable voltage source V, which
preferably is a DC voltage source but alternatively could be an AC voltage
source, through conventional conductive lead lines L1, L2 and L3. Each control
electrode E1, E2 and E3 is energizable independently of the other control
electrodes E1, E2 and E3. This can be accomplished by providing suitable
switches S1, S2 and S3 communicating with respective control electrodes E1, E2
and E3, or other suitable means for independently rendering each control
electrode E1, E2 and E3 either active (ON state, high voltage, or binary 1) or
inactive (OFF state, low voltage, or binary 0). In other embodiments, or in
other
areas of the electrode array, two or more control electrodes E can be
commonly connected so as to be activated together.
The structure of electrowetting microactuator mechanism 10 can
represent a portion of a microfluidic chip, on which conventional microfluidic
and/or microelectronic components can also be integrated. As examples, the
chip could also include resistive heating areas, microchannels, micropumps,
pressure sensors, optical waveguides, and/or biosensing or chemosensing
elements interfaced with MOS (metal oxide semiconductor) circuitry.
Referring now to Figure 2, an electrode array or portion thereof is
illustrated in which each structural interface between adjacent unit cells
(e.g.,
C1 and C2) associated with control electrodes (not shown) is preferably
characterized by an interdigitated region, generally designated 40, defined by
interlocking projections 42 and 43 extending outwardly from the main planar
structures of respective unit cells C1 and C2. Such interdigitated regions 40
can
be useful in rendering the transition from one unit cell (e.g., C1) to an
adjacent
unit cell (e.g., C2) more continuous, as opposed to providing straight-edged
boundaries at the cell-cell interfaces. It will be noted, however, that the
electrodes or unit cells according to any embodiment of the invention can have
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any polygonal shape that is suitable for constructing a closely-packed two-
dimensional array, such as a square or octagon.
Referring back to Figure 1, the basic electrowetting technique enabled
by the design of microactuator mechanism 10 will now be described. Initially,
all control electrodes (i.e., control electrode E2 on which droplet D is
centrally
located and adjacent control electrodes E1 and E3) are grounded or floated,
and the contact angle everywhere on droplet D is equal to the equilibrium
contact angle associated with that droplet D. When an electrical potential is
applied to control electrode E2 situated underneath droplet D, a layer of
charge
builds up at the interface between droplet D and energized control electrode
E2,
resulting in a local reduction of the interfacial energy YsL. Since the solid
insulator provided by lower hypdrophobic insulating layer 23 controls the
capacitance between droplet D and control electrode E2, the effect does not
depend on the specific space-charge effects of the electrolytic liquid phase
of
droplet D, as is the case in previously developed uninsulated electrode
implementations.
The voltage dependence of the interfacial energy reduction is described
by
(1)
YSL (V) - YSL (0) 9 d V2
where E is the permittivity of the insulator, d is the thickness of the
insulator,
and V is the applied potential. The change in YSL acts through Young's
equation to reduce the contact angle at the interface between droplet D and
energized control electrode E2. If a portion of droplet D also overlaps a
grounded electrode E1 or E3, the droplet meniscus is deformed asymmetrically
and a pressure gradient is established between the ends of droplet D, thereby
resulting in bulk flow towards the energized electrode E1 or E3. For example,
droplet D can be moved to the left (i.e., to unit cell C1) by energizing
control
electrode E1 while maintaining control electrodes E2 and E3 at the ground
state.
As another example, droplet D can be moved to the right (i.e., to unit cell
C3)
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by energizing control electrode E3 while maintaining control electrodes E1 and
E2 at the ground state.
The following EXAMPLE describes a prototypical embodiment of
electrowetting microactuator mechanism 10, with reference being generally
made to Figures 1 and 2.
EXAMPLE
A prototype device consisting of a single linear array of seven
interdigitated control electrodes E at a pitch of 1.5 mm was fabricated and
tested. Control electrodes E were formed by patterning a 2000-A thick layer of
chrome on a glass lower plate 21 using standard microfabrication techniques.
The chips were then coated with a 7000 A layer of Parylene C followed by a
layer 23 of approximately 2000 A of TEFLON AF 1600. Ground electrode G
consisted of an upper plate 25 of glass coated with a conducting layer (RS <
20
Q/square) of transparent indium-tin-oxide (ITO). A thin (- 500 A) layer 27 of
TEFLON' AF 1600 was also applied to ground electrode G. The thin
TEFLON coating on ground electrode G served to hydrophobize the surface,
but was not presumed to be insulative. After coating with TEFLON', both
surfaces had a contact angle of 104 with water.
Water droplets (0.7 - 1.0 NI) of 100 mM KCI were dispensed onto the
array using a pipette, and upper plate 25 was positioned to provide a gap d of
0.3 mm between the opposing electrodes E and G. A customized clamp with
spring-loaded contact pins (not shown) was used to make connections to the
bond pads. A computer was used to control a custom-built electronic interface
which was capable of independently switching each output between ground
and the voltage output of a 120 V DC power supply.
A droplet D was initially placed on the center of the grounded control
electrode (e.g., E2) and the potential on the adjacent electrode (e.g.,
control
electrode E1 or E3) was increased until motion was observed. Typically, a
voltage of 30 - 40 V was required to initiate movement of droplet D. Once this
threshold was exceeded, droplet movement was both rapid and repeatable. It
is believed that contact angle hysteresis is the mechanism responsible for
this
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threshold effect. By sequentially energizing four adjacent control electrodes
E
at 80 V of applied potential, droplet D was moved repeatedly back and forth
across all four control electrodes E at a switching frequency of 15 Hz.
The transit time ttr of the droplet D was defined as the time required for
droplet D to reach the far,edge of the adjacent electrode following the
application of the voltage potential. The transit time ttr thus represented
the
minimum amount of time allowed between successive transfers, and (1 /ttr) was
the maximum switching rate for continuous transfer of a droplet D. The
maximum switching rate as a function of voltage is plotted in Figure 3, where
ttr
was determined by counting recorded video frames of a moving droplet D.
Sustained droplet transport over 1000's of cycles at switching rates of up
to 1000 Hz has been demonstrated for droplets of 6nL volume. This rate
corresponds to an average droplet velocity of 10.0 cm/s, which is nearly 300
times faster than a previously reported method for electrical manipulation of
droplets. See M. Washizu, IEEE Trans. Ind. Appl. 34, 732 (1998).
Comparable velocities cannot be obtained in thermocapillary systems because
(for water) the required temperature difference between the ends of droplet D
exceeds 100 C. See Sammarco et al., AIChE J., 45, 350 (1999). These
results demonstrate the feasibility of electrowetting as an actuation
mechanism
for droplet-based microfluidic systems. This design can be extended to
arbitrarily large two-dimensional arrays to allow precise and independent
control over large numbers of droplets D and to serve as a general platform
for
microfluidic processing.
Referring now to Figures 4A - 7B, examples of some basic droplet-
manipulative operations are illustrated. As in the case of Figure 1, a linear
arrangement of three unit cells C1, C2 and C3 and associated control
electrodes
E1, E2 and E3 are illustrated, again with the understanding that these unit
cells
C1, C2 and C3 and control electrodes E1, E2 and E3 can form a section of a
larger linear series, non-linear series, or two-dimensional array of unit
cells/control electrodes. For convenience, in Figures 4B - 7B, corresponding
control electrodes and unit cells are collectively referred to as control
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electrodes E1, E2 and E3. Moreover, unit cells C1, C2, and C3 can be physical
entities, such as areas on a chip surface, or conceptual elements. In each of
Figures 4A - 7B, an active (i.e., energized) control electrode E1i E2, or E3
is
indicated by designating its associated electrical lead line L1, L2, or L3
"ON",
while an inactive (i.e., de-energized, floated, or grounded) control electrode
E1,
E2, or E3 is indicated by designating its associated electrical lead line L1,
L2, or
L3 "OFF".
Turning to Figures 4A - 4D, a basic MOVE operation is illustrated.
Figure 4A illustrates a starting position at which droplet D is centered on
control
electrode E1. Initially, all control electrodes E1, E2 and E3 are grounded so
that
droplet D is stationary and in equilibrium on control electrode E1.
Alternatively,
control electrode E1 could be energized while all adjacent control electrodes
(e.g., E2) are grounded so as to initially maintain droplet D in a "HOLD" or
"STORE" state, and thereby isolate droplet D from adjoining regions of an
array
where other manipulative operations might be occurring on other droplets. To
move droplet D in the direction indicated by the arrow in Figures 4A - 4B,
control electrode E2 is energized to attract droplet D and thereby cause
droplet
D to move and become centered on control electrode E2, as shown in Figure
4B. Subsequent activation of control electrode E3, followed by removal of the
voltage potential at control electrode E2, causes droplet D to move onto
control
electrode E3 as shown in Figure 4C. This sequencing of electrodes can be
repeated to cause droplet D to continue to move in the desired direction
indicated by the arrow. It will also be evident that the precise path through
which droplet D moves across the electrode array is easily controlled by
appropriately programming an electronic control unit (such as a conventional
microprocessor) to activate and de-activate selected electrodes of the array
according to a predetermined sequence. Thus, for example, droplet D can be
actuated to make right- and left-hand turns within the array. For instance,
after
droplet D has been moved to control electrode E2 from E1 as shown in Figure
4B, droplet D can then be moved onto control electrode E5 of another row of
electrodes E4 - Es as shown in Figure 4D. Moreover, droplet D can be cycled
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back and forth (e.g., shaken) along a desired number of unit cells and at a
desired frequency for various purposes such as agitation of droplet D, as
described in the EXAMPLE hereinabove.
Figures 5A - 5C illustrate a basic MERGE or MIX operation wherein two
droplets D1 and D2 are combined into a single droplet D3. In Figure 5A, two
droplets D1 and D2 are initially positioned at control electrodes E1 and E3
and
separated by at least one intervening control electrode E2. As shown in Figure
5B, all three control electrodes E1, E2 and E3 are then activated, thereby
drawing droplets D1 and D2 toward each other across central control electrode
E2 as indicated by the arrows in Figure 5B. Once the opposing sides of
droplets D1 and D2 encounter each other at central control electrode E2, a
single meniscus M is created that joins the two droplets D1 and D2 together.
As
shown in Figure 5C, the two outer control electrodes E1 and E3 are then
returned to the ground state, thereby increasing the hydrophobicity of the
surfaces of the unit cells associated with outer electrodes E1 and E3 and
repelling the merging droplets D1 and D2, whereas energized central control
electrode E2 increases the wettability of its proximal surface contacting
droplets
D1 and D2. As a result, droplets D1 and D2 combine into a single mixed droplet
D3 as shown in Figure 5C, which represents the lowest energy state possible
for droplet D3 under these conditions. The resulting combined droplet D3 can
be assumed to have twice the volume or mass as either of the original, non-
mixed droplets D1 and D2, since parasitic losses are negligible or zero. This
is
because evaporation of the droplet material is avoided due to the preferable
use of a filter fluid (e.g., air or an immiscible liquid such as silicone oil)
to
surround the droplets, because the surfaces contacting the droplet material
(e.g., upper and lower hydrophobic layers 27 and 23 shown in Figure 1) are
low-friction surfaces, and/or because the electrowetting mechanism employed
by the invention is non-thermal.
In the present discussion, the terms MERGE and MIX have been used
interchangeably to denote the combination of two or more droplets. This is
because the merging of droplets does not in all cases directly or immediately
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result in the complete mixing of the components of the initially separate
droplets. Whether merging results in mixing can depend on many factors.
These factors can include the respective compositions or chemistries of the
droplets to be mixed, physical properties of the droplets or their
surroundings
such as temperature and pressure, derived properties of the droplets such as
viscosity and surface tension, and the amount of time during which the
droplets
are held in a combined state prior to being moved or split back apart. As a
general matter, the mechanism by which droplets are mixed together can be
categorized as either passive or active mixing. In passive mixing, the merged
droplet remain on the final electrode throughout the mixing process. Passive
mixing can be sufficient under conditions where an acceptable degree of
diffusion within the combined droplet occurs. In active mixing, on the other
hand, the merged droplet is then moved around in some manner, adding
energy to the process to effect complete or more complete mixing. Active
mixing strategies enabled by the present invention are described hereinbelow.
It will be further noted that in the case where a distinct mixing operation
is to occur after a merging operation, these two operations can occur at
different sections or areas on the electrode array of the chip. For instance,
two
droplets can be merged at one section, and one or more of the basic MOVE
operations can be implemented to convey the merged droplet to another
section. An active mixing strategy can then be executed at this other section
or
while the merged droplet is in transit to the other section, as described
hereinbelow.
Figures 6A - 6C illustrate a basic SPLIT operation, the mechanics of
which are essentially the inverse of those of the MERGE or MIX operation just
described. Initially, as shown in Figure 6A, all three control electrodes E1,
E2
and E3 are grounded, so that a single droplet D is provided on central control
electrode E2 in its equilibrium state. As shown in Figure 6B, outer control
electrodes Ei and E3 are then energized to draw droplet D laterally outwardly
(in the direction of the arrows) onto outer control electrodes E1 and E3. This
has the effect of shrinking meniscus M of droplet D, which is characterized as
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"necking" with outer lobes being formed on both energized control electrodes
E1 and E3. Eventually, the central portion of meniscus M breaks, thereby
creating two new droplets D1 and D2 split off from the original droplet D as
shown in Figure 6C. Split droplets D1 and D2 have the same or substantially
the same volume, due in part to the symmetry of the physical components and
structure of electrowetting microactuator mechanism 10 (Figure 1), as well as
the equal voltage potentials applied to outer control electrodes E1 and E3. It
will
be noted that in many implementations of the invention, such as analytical and
assaying procedures, a SPLIT operation is executed immediately after a
MERGE or MIX operation so as to maintain uniformly-sized droplets on the
microfluidic chip or other array-containing device.
Referring now to Figures 7A and 7B, a DISCRETIZE operation can be
derived from the basic SPLIT operation. As shown in Figure 7A, a surface or
port I/O is provided either on an electrode grid or at an edge thereof
adjacent to
electrode-containing unit cells (e.g., control electrode E1), and serves as an
input and/or output for liquid. A liquid dispensing device 50 is provided, and
can be of any conventional design (e.g., a capillary tube, pipette, fluid pen,
syringe, or the like) adapted to dispense and/or aspirate a quantity of liquid
LQ.
Dispensing device 50 can be adapted to dispense metered doses (e.g.,
aliquots) of liquid LQ or to provide a continuous flow of liquid LQ, either at
port
I/O or directly at control electrode E1. As an alternative to using dispensing
device 50, a continuous flow of liquid LQ could be conducted across the
surface of a microfluidic chip, with control electrodes E1, E2, and E3 being
arranged either in the direction of the continuous flow or in a non-collinear
(e.g.,
perpendicular) direction with respect to the continuous flow. In the specific,
exemplary embodiment shown in Figure 7A, dispensing device 50 supplies
liquid LQ to control electrode E1.
To create a droplet on the electrode array, the control electrode directly
beneath the main body of liquid LQ (control electrode E1) and at least two
control electrodes adjacent to the edge of the liquid body (e.g., control
electrodes E1 and E3) are energized. This causes the dispensed body of liquid
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LQ to spread across control electrodes E1 and E2 as shown in Figure 7A. In a
manner analogous to the SPLIT operation described hereinabove with
reference to Figures 6A - 6C, the intermediate control electrode (control
electrode E2) is then de-energized to create a hydrophobic region between two
effectively hydrophilic regions. The liquid meniscus breaks above the
hydrophobic region to form or "pinch off" a new droplet D, which is centered
on
control electrode E3 as shown in Figure 7B. From this point, further
energize/de-energize sequencing of other electrodes of the array can be
effected to move droplet D in any desired row-wise and/or column-wise
direction to other areas on the electrode array. Moreover, for a continuous
input flow of liquid LQ, this dispensing process can be repeated to create a
train of droplets on the grid or array, thereby,discretizing the continuous
flow.
As described in more detail hereinbelow, the discretization process is highly
useful for implementing droplet-based processes on the array, especially when
a plurality of concurrent operations on many droplets are contemplated.
Droplet-Based Mixing Strategies
Examples of several strategies for mixing droplets in accordance with
the present invention will now be described. Referring to Figures 8A and 8B, a
configuration such as that of electrowetting microactuator mechanism 10,
described hereinabove with reference to Figure 1, can be employed to carry out
merging and mixing operations on two or more droplets, e.g., droplets D1 and
D2. In Figures 8A and 8B, droplets D1 and D2 are initially centrally
positioned
on control electrodes E2 and E.5, respectively. Droplets D1 and D2 can be
actuated by electrowetting to move toward each other and merge together on a
final electrode in the manner described previously with reference to Figures
5A
- 5C. The final electrode can be an intermediately disposed electrode such as
electrode E3 or E4. Alternatively, one droplet can move across one or more
control electrodes and merge into another stationary droplet. Thus, as
illustrated in Figures 8A and 8B, droplet D1 can be actuated to move across
intermediate electrodes E3 and E4 as indicated by the arrow and merge with
droplet D2 residing on electrode, such that the merging of droplets D1 and D2
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occurs on electrode E5. The combined droplet can then be actively mixed
according to either a one-dimensional linear, two-dimensional linear, or two-
dimensional loop mixing strategy.
As one example of a one-dimensional linear mixing strategy, multiple
droplets can be merged as just described, and the resulting combined droplet
then oscillated (or "shaken" or "switched") back and forth at a desired
frequency
over a few electrodes to cause perturbations in the contents of the combined
droplet. This mixing process is described in the EXAMPLE set forth
hereinabove and can involve any number of linearly arranged electrodes, such
as electrodes in a row or column of an array. Figures 9A, 9B and 9C illustrate
two-, three-, and four-electrode series, respectively, in which merging and
mixing by shaking can be performed. As another example of one-dimensional
linear mixing, multiple droplets are merged, and the combined droplet or
droplets are then split apart as described hereinabove. The resulting
split/merged droplets are then oscillated back and forth at a desired
frequency
over a few electrodes. The split/merged droplets can then be recombined, re-
split, and re-oscillated for a number of successive cycles until the desired
degree of mixing has been attained. Both of these one-dimensional, linear
mixing approaches produce reversible flow within the combined droplet or
droplets. It is thus possible that the mixing currents established by motion
in
one direction could be undone or reversed when the combined droplet
oscillates back the other way. Therefore, in some situations, the reversible
flow
attending one-dimensional mixing processes may require undesirably large
mixing times.
Referring now to Figures 10A - 10C, another example of one-
dimensional linear mixing referred to as "mixing-in-transport" is illustrated.
This
method entails combining two or more droplets and then continuously actuating
the combined droplet in a forward direction along a desired flow path until
mixing is complete. Referring to Figure 10A, a combined droplet D is
transported from a starting electrode E. along a programmed path of
electrodes on the array until it reaches a preselected destination electrode
Ef.
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Destination electrode Ef can be a location on the array at which a subsequent
process such as analysis, reaction, incubation, or detection is programmed to
occur. In such a case, the flow path over which combined droplet D is actively
mixed, indicated by the arrow, also serves as the analysis flow path over
which
the sample is transported from the input to the processing area on the array.
The number of electrodes comprising the selected path from starting electrode
Eo to destination electrode Ef corresponds to the number of actuations to
which
combined droplet D is subject. Hence, through the use of a sufficient number
of intermediate path electrodes, combined droplet D will be fully mixed by the
time it reaches destination electrode Ef. It will be noted that the flow path
does
not reverse as in the case of the afore-described oscillatory mixing
techniques.
The flow path can, however include one or more right-angle turns through the
x-y plane of the array as indicated by the respective arrows in Figures 1 OA -
10C. In some cases, turning the path produces unique flow patterns that
enhance the mixing effect. In Figure 1 OB, the flow path has a ladder or step
structure consisting of a number of right-angle turns. In Figure 1 OC,
destination
electrode Ef lies in the same row as starting electrode E0, but combined
droplet
D is actuated through a flow path that deviates from and subsequently returns
to that row in order to increase the number of electrodes over which combined
droplet D travels and the number of turns executed.
Referring now to Figure 11, an example of a two-dimensional linear
mixing strategy is illustrated. One electrode row EROW and one electrode
column ECOL of the array are utilized. Droplets Di and D2 are moved toward
each other along electrode row EROW and merged as described hereinabove,
forming a merged droplet D3 centered on the electrode disposed at the
intersection of electrode row EROW and electrode column EcOL. Selected
electrodes of electrode column ECOL are then sequentially energized and de-
energized in the manner described hereinabove to split merged droplet D3 into
split droplets D4 and D5. Split droplets D4 and D5 are then moved along
electrode column Econ. This continued movement of split droplets D4 and D5
enhances the mixing effect on the contents of split droplets D4 and D5.
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Referring now to Figures 12A and 12B, examples of two-dimensional
loop mixing strategies are illustrated. In Figure 12A, a combined droplet D is
circulated clockwise or counterclockwise in a circular, square or other closed
loop path along the electrodes of selected rows and columns of the array, as
indicated by the arrow. This cyclical actuation of combined droplet D is
effected through appropriate sequencing of the electrodes comprising the
selected path. Combined droplet D is cycled in this manner for a number of
times sufficient to mix its contents. The cycling of combined droplet D
produces nonreversible flow patterns that enhance the mixing effect and
reduce the time required for complete mixing. In Figure 12A, the path
circumscribes only one central electrode not used for actuation, although the
path could be made larger so as to circumscribe more central electrodes.
In Figure 12B, a sub-array of at least four adjacent electrodes E1- E4 is
utilized. Combined droplet D is large enough to overlap all four electrodes E1-
E4 of the sub-array simultaneously. The larger size of combined droplet D
could be the result of merging two smaller-sized droplets without splitting,
or
could be the result of first merging two pairs of droplets and thereafter
combining the two merged droplets. Combined droplet D is rotated around the
sub-array by sequencing electrodes E1 - E4 in the order appropriate for
effecting either clockwise or counterclockwise rotation. As compared with the
mixing strategy illustrated in Figure 12A, however, a portion of the larger-
sized
combined droplet D remains "pinned" at or near the intersection of the four
electrodes E1- E4 of the sub-array. Thus, combined droplet D in effect rotates
or spins about the intersecting region where the pinned portion is located.
This
effect gives rise to unique internal flow patterns that enhance the mixing
effect
attributed to rotating or spinning combined droplet D and that promote
nonreversible flow. Moreover, the ability to mix combined droplet D using only
four electrodes E1 - E4 enables the cyclical actuation to occur at high
frequencies and with less power requirements.
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The mixing strategy illustrated in Figure 12B can also be implemented
using other sizes of arrays. For instance, a2 x4 array has been found to work
well in accordance with the invention.
For all of the above-described mixing strategies, it will be noted the
droplets involved can be of equal size or unequal volumes. In a situation
where
an n:1 volume ratio of mixing is required, the electrode areas can be
proportionately chosen to yield a one-droplet (n) to one-droplet (1) mixing.
Figure 13 depicts graphical data illustrating the performance of the one-
dimensional linear mixing strategy. The time for complete mixing is plotted as
a
function of frequency of droplet oscillation (i.e., the switch time between
one
electrode and a neighboring electrode). Curves are respectively plotted for
the
2-electrode (see Figure 9A), 3-electrode (see Figure 9B), and 4-electrode (see
Figure 9C) mixing configurations. Mixing times were obtained for 1, 2, 4, 8,
and
16 Hz frequencies. The actuation voltage applied to each electrode was 50 V.
It was observed that increasing the frequency of switching results in faster
mixing times. Similarly, for a given frequency, increasing the number of
electrodes also results in improved mixing. It was concluded that increasing
the number of electrodes on which the oscillation of the merged droplets is
performed increases the number of multi-laminate configurations generated
within the droplet, thereby increasing the interfacial area available for
diffusion.
Figure 14 depicts graphical data illustrating the performance of the two-
dimensional loop mixing strategy in which the droplet is large enough to
overlap
the 2 x 2 electrode sub-array (see Figure 12B). Mixing times were obtained for
8, 16, 32, and 64 Hz frequencies. As in the experiment that produced the plot
of Figure 13, the actuation voltage applied to each electrode was 50 V. It was
concluded that two-dimensional mixing reduces the effect of flow reversibility
associated with one-dimensional mixing. Moreover, the fact that the droplet
rotates about a point enabled the switching frequency to be increased up to 64
Hz for an actuation voltage of 50 V. This frequency would not have been
possible in a one-dimensional linear actuation case at the same voltage. It is
further believed that the fact that the droplet overlaps all four electrodes
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simultaneously enabled droplet transport at such high frequencies and low
voltages. The time between the sequential firing of any two adjacent
electrodes of the 2 x 2 sub-array can be reduced because the droplet is in
electrical communication with both electrodes simultaneously. That is, the lag
time and distance needed for the droplet to physically move from one electrode
to another is reduced. Consequently, the velocity of the droplet can be
increased in the case of two-dimensional mixing, allowing vortices to form and
thereby promoting mixing.
Droplet-Based Sampling and Processing
Referring now to Figures 15A and 15B, a method for sampling and
subsequently processing droplets from a continuous-flow fluid input source 61
is schematically illustrated in accordance with the invention. More
particularly,
the method enables the discretization of uniformly-sized sample droplets S
from continuous-flow source 61 by means of electrowetting-based techniques
as described hereinabove, in preparation for subsequent droplet-based, on-
chip and/or off-chip procedures (e.g., mixing, reacting, incubation, analysis,
detection, monitoring, and the like). In this context, the term "continuous"
is
taken to denote a volume of liquid that has not been discretized into smaller-
volume droplets. Non-limiting examples of continuous-flow inputs include
capillary-scale streams, fingers, slugs, aliquots, and metered doses of fluids
introduced to a substrate surface or other plane from an appropriate source or
dispensing device. Sample droplets S will typically contain an analyte
substance of interest (e.g., a pharmaceutical molecule to be identified such
as
by mass spectrometry, or a known molecule whose concentration is to be
determined such as by spectroscopy). The several sample droplets S shown in
Figures 15A and 15B represent either separate sample droplets S that have
been discretized from continuous-flow source 61, or a single sample droplet S
movable to different locations on the electrode array over time and along
various analysis flow paths available in accordance with the sequencing of the
electrodes.
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The method can be characterized as digitizing analytical signals from an
analog input to facilitate the processing of such signals. It will be
understood
that the droplet-manipulative operations depicted in Figures 15A and 15B can
advantageously occur on an electrode array as described hereinabove. Such
array can be fabricated on or embedded in the surface of a microfluidic chip,
with or without other features or devices ordinarily associated with IC, MEMS,
and microfluidic technologies. Through appropriate sequencing and control of
the electrodes of the array such as through communication with an appropriate
electronic controller, sampling (including droplet formation and transport)
can
be done on a continuous and automated basis.
In Figure 15A, the liquid input flow of continuous-flow source 61 is
supplied to the electrode array at a suitable injection point. Utilizing the
electrowetting-based techniques described hereinabove, continuous liquid flow
61 is fragmented or discretized into a series or train of sample droplets S of
uniform size. One or more of these newly formed sample droplets S can then
be manipulated according to a desired protocol, which can include one or more
of the fundamental MOVE, MERGE, MIX and/or SPLIT operations described
hereinabove, as well as any operations derived from these fundamental
operations. In particular, the invention enables sample droplets S to be
diverted from continuous liquid input flow 61 for on-chip analysis or other on-
chip processing. For example, Figure 15A shows droplets being transported
along programmable analysis flow paths across the microfluidic chip to one or
more functional cells or regions situated on the surface of microfluidic chip
such
as cells 63 and 65.
Functional cells 63 and 65 can comprise, for example, mixers, reactors,
detectors, or storage areas. In the case of mixers and reactors, sample
droplets S are combined with additive droplets R1 and/or R2 that are supplied
from one or more separate reservoirs or injection sites on or adjacent to the
microfluidic chip and conveyed across the microfluidic chip according to the
electrowetting technique. In the case of mixers, additive droplets R1 and/or
R2
can be other sample substances whose compositions are different from sample
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droplets S. Alternatively, when dilution of sample droplets S is desired,
additive
droplets R1 and/or R2 can be solvents of differing types. In the case of
reactors, additive droplets R1 and/or R2 can contain chemical reagents of
differing types. For example, the electrode array or a portion thereof could
be
employed as a miniaturized version of multi-sample liquid handling/assaying
apparatus, which conventionally requires the use of such large components as
96-well microtitre plates, solvent bottles, liquid transfer tubing, syringe or
peristaltic pumps, multi-part valves, and robotic systems.
Functional cells 63 and 65 preferably comprise one or more electrode-
containing unit cells on the array. Such functional cells 63 and 65 can in
many
cases be defined by the sequencing of their corresponding control electrodes,
where the sequencing is programmed as part of the desired protocol and
controlled by an electronic control unit communicating with the microfluidic
chip.
Accordingly, functional cells 63 and 65 can be created anywhere on the
electrode array of the microfluidic chip and reconfigured on a real-time
basis.
For example, Figure 16 illustrates a mixer cell, generally designated MC, that
can be created for mixing or diluting a sample droplet S with an additive
droplet
R according to any of the mixing strategies disclosed herein. Mixer cell MC
comprises a 5 x 3 matrix of electrode-containing unit cells that could be part
of
a larger electrode array provided by the chip. Mixer cell MC is thus rendered
from five electrode/cell rows ROW1- ROW5 and three electrode/cell columns
COL1 - COL3. MERGE and SPLIT operations can occur at the centrally
located electrodes E1- E3 as described hereinabove with reference to Figures
5A - 6C. The electrodes associated with outer columns COL1 and COLS and
outer rows ROW1 and ROW5 can be used to define transport paths over which
sample droplet S and additive droplet R are conveyed from other areas of the
electrode array, such as after being discretized from continuous-flow source
61
(see Figure 15A or 15B). A 2 X 2 sub-array can be defined for implementing
two-dimensional loop mixing processes as illustrated in Figure 12B. During a
MIX, MERGE, SPLIT, or HOLD operation, some or all of the electrodes
associated with outer columns COL1 and COLS and outer rows ROW1 and
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ROW5 can be grounded to serve as gates and thus isolate mixer cell MC from
other areas on the chip. If necessary, complete or substantially complete
mixing can be accomplished by a passive mechanism such as diffusion, or by
an active mechanism such as by moving or "shaking" the combined droplet
according to electrowetting as described hereinabove.
The invention contemplates providing other types of fuhctional cells,
including functional cells that are essentially miniaturized embodiments or
emulations of traditional, macro-scale devices or instruments such as
reactors,
detectors, and other analytical or measuring instruments. For example, a
droplet could be isolated and held in a single row or column of the main
electrode array, or at a cell situated off the main array, to emulate a sample
holding cell or flow cell through which a beam of light is passed in
connection
with known optical spectroscopic techniques. A light beam of an initial
intensity
could be provided from an input optical fiber and passed through the droplet
contained by the sample cell. The attenuated light beam leaving the droplet
could then enter an output optical fiber and routed to an appropriate
detection
apparatus such as a photocell. The optical fibers could be positioned on
either
side of the sample cell, or could be provided in a miniature dip probe that is
incorporated with or inserted into the sample cell.
Referring back to Figure 15A, upon completion of a process executed at
a functional cell (e.g., cell 63 or 65), the resulting product droplets (not
shown)
can be conveyed to respective reservoirs 67 or 69 located either on or off the
microfluidic chip for the purpose of waste collection, storage, or output. In
addition, sample droplets S and/or product droplets can be recombined into a
continuous liquid output flow 71 at a suitable output site on or adjacent to
the
microfluidic chip for the purposes of collection, waste reception, or output
to a
further process. Moreover, the droplets processed by functional cell 63 or 65
can be prepared sample droplets that have been diluted and/or reacted in one
or more steps, and then transported by electrowetting to another portion of
the
chip dedicated to detection or measurement of the analyte. Some detection
sites can, for example, contain bound enzymes or other biomolecular
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recognition agents, and be specific for particular analytes. Other detection
sites can consist of a general means of detection such as an optical system
for
fluorescence- or absorbance-based assays, an example of which is given
hereinabove.
In the alternative embodiment shown in Figure 158, continuous liquid
flow 61 is supplied from an input site 61 A, and completely traverses the
surface
of the microfluidic chip to an output site 61B. In this embodiment, sample
droplets S are formed (i.e., continuous liquid input flow 61 is sampled) at
specific, selectable unit-cell locations along the length of continuous liquid
input
flow 61 such as the illustrated location 73, and subsequent electrowetting-
based manipulations are executed as described hereinabove in relation to the
embodiment of Figure 15A.
The methods described in connection with Figures 15A and 15B have
utility in many applications. Applications of on-line microfluidic analysis
can
include, for example, analysis of microdialysis or other biological perfusion
flows, environmental and water quality monitoring and monitoring of industrial
and chemical processes such as fermentation. Analysis can include the
determination of the presence, concentration or activity of any specific
substance within the flowing liquid. On-line continuous analysis is beneficial
in
any application where real-time measurement of a time-varying chemical signal
is required, a classic example being glucose monitoring of diabetic patients.
Microfluidics reduces the quantity of sample required for an analysis, thereby
allowing less invasive sampling techniques that avoid depleting the analyte
being measured, while also permitting miniaturized and portable instruments to
be realized.
The droplet-based methods of the invention provide a number of
advantages over known continuous flow-based microscale methods as well as
more conventional macroscale instrument-based methods. Referring to either
Figure 15A or 15B, the flow of sample droplets S from continuous-flow source
61 to the analysis portion of the chip (i.e., the analysis flow) is controlled
independently of the continuous flow (i.e., the input flow), thereby allowing
a
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great deal of flexibility in carrying out the analyses. The de-coupling of the
analysis flow from the continuous input flow allows each respective flow to be
separately optimized and controlled. For example, in microdialysis, the
continuous flow can be optimized to achieve a particular recovery rate while
the
analysis flow is optimized for a particular sensitivity or sampling rate.
Reagent
droplets R can be mixed with sample droplets S in the analysis flow without
affecting or contaminating the main input flow. Sample droplets S in the
analysis flow can be stored or incubated indefinitely without interrupting the
input flow. Analyses requiring different lengths of time can be carried out
simultaneously and in parallel without interrupting the input flow.
. In either embodiment depicted in Figures 15A or 15B, the analysis or
other processing of sample droplets S is carried out on-line insofar as the
analysis occurs as part of the same sequential process as the input of
continuous-flow source 61. However, the analysis is not carried out in-line
with
respect to continuous liquid input flow 61, because newly formed sample
droplets S are diverted away from continuous liquid input flow 61. This design
thus allows the analysis flow to be de-coupled from the input flow.
As another advantage, multiple analytes can be simultaneously
measured. Since continuous liquid flow 61 is fragmented into sample droplets
S, each sample droplet S can be mixed with a different reagent droplet R, or
R2
or conducted to a different test site on the chip to allow simultaneous
measurement of multiple analytes in a single sample without cross-talk or
cross-contamination. Additionally, multiple step chemical protocols are
possible, thereby allowing a wide range of types of analyses to be performed
in
a single chip.
Moreover, calibration and sample measurements can be multiplexed.
Calibrant droplets can be generated and measured between samples.
Calibration does not require cessation of the input flow, and periodic
recalibration during monitoring is possible. In addition, detection or sensing
can be multiplexed for multiple analytes. For example, a single fluorimeter or
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absorbance detector may be utilized to measure multiple analytes by
sequencing the delivery of sample droplets S to the detector site.
Another important advantage is the reconfigurability of the operation of
the chip. Sampling rates can be dynamically varied through software control.
Mixing ratios, calibration procedures, and specific tests can all be
controlled
through software, allowing flexible and reconfigurable operation of the chip.
Feedback control is possible, which allows analysis results to influence the
operation of the chip.
Droplet-Based Binary Interpolating Digital Mixing
Referring now to Figure 17, a binary mixing apparatus, generally
designated 100, is illustrated in accordance with the invention. Binary mixing
apparatus 100 is useful for implementing a droplet-based, variable dilution
binary mixing technique in one, two or more mixing phases to obtain desired
mixing ratios. The degree of precision of the resulting mixing ratio depends
on
the number of discrete binary mixing units utilized. As one example, Figure 17
schematically illustrates a first binary mixing unit 110 and a second binary
mixing unit 210. When more than one mixing unit is provided, a buffer 310 is
preferably provided in fluid communication with the mixing units to store
intermediate products and transfer intermediate products between the mixing
units as needed. A suitable electronic controller EC such as a microprocessor
capable of executing the instructions of a computer program communicates
with first binary mixing unit 110, second binary mixing unit 210, and buffer
310
through suitable communication lines 111, 211, and 311, respectively.
Binary mixing apparatus 100 can be fabricated on a microfluidic chip for
the purpose of carrying out binary interpolating digital mixing procedures in
accordance with the invention. In designing the physical layouts of the
various
droplet-handling components of binary mixing apparatus 100 (examples of
which are illustrated in Figures 18A and 20), electrode design and
transportation design (scheduling) were considered. The particular physical
layout at least in part determines the code or instruction set executed by
electronic controller EC to control the electrodes and thus the types and
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sequences of droplet-based manipulation to be performed. Preferably, the
electrode-containing droplet-handling regions of binary mixing apparatus 100
are structured as shown in the cross-sectional view of Figure 1, described
hereinabove in connection with electrowetting microactuator mechanism 10, or
according to a single-sided electrode configurations described hereinbelow.
The electrodes of each mixing unit can be sequenced to implement any of the
mixing strategies disclosed herein.
The architecture of binary mixing apparatus 100 is designed to take full
advantage of accelerated rates observed in droplet-to-droplet mixing
experiments, while allowing precisely controlled mixing ratios that can be
varied
dynamically for multi-point calibrations. As will become evident from the
description herein, binary mixing apparatus 100 can handle a wide range of
mixing ratios with certain accuracy, and enables mixing patterns that
demonstrate high parallelism in the mixing operation as well as scalability in
the
construction of mixing components in a two-dimensional array. Binary mixing
apparatus 100 can handle a wide range of droplet sizes. There is, however, a
lower limit on droplet size if sample droplets are being prepared for the
purpose
of a detection or measurement.
The architecture of binary mixing apparatus 100 is based on the
recognition that the most efficient mixing most likely occurs between two
droplets moving toward each other. This has been observed from experiments,
and could be explained by the fact that convection induced by shear movement
of fluids accelerates the mixing process much faster than pure physical
diffusion. Thus, as a general design principle, one-by-one mixing is utilized
as
much as possible. As indicated hereinabove, one-by-one mixing preferably
involves both mixing and splitting operations to maintain uniform droplet
size.
The basic MIX and SPLIT operations have been described hereinabove with
reference to Figures 5A - 6C.
Certain assumptions have been made in design of the architecture of
binary mixing apparatus 100, and include the following:
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1. Full mixing occurs in terms of chemical and/or physical processes
given adequate time.
2. Equal droplet splitting occurs in terms of physical volume and
chemical components.
3. Negligible residues are produced during droplet transportation.
4. Mixing time for large dilution ratios is a bottleneck.
5. There are tolerances on mixing ratios.
6. Transportation time is negligible compared to mixing.
Preferred design requirements and constraints were also considered,
and include the following:
1. Minimum volume of mixture output to guarantee detectability.
2. Maximum number of independent control electrodes.
3. Maximum mixing area.
4. Maximum number of actuation per electrode.
S. Reconfigurability for different mixing ratios.
Thus, one design objective was to complete the mixing process using a
minimum number of mixing-splitting operations while maintaining the accuracy
of the mixing ratio.
Moreover, some desirable attributes for an ideal mixing architecture
were considered to be as follows:
1. Accurate mixing ratio.
2. Small number of mixing cycles. Since many mixing processes will
involve more than one mixing phase, during the first phase the two binary
mixing units 110 and 210 are operated in parallel to and independent of each
other. The second mixing phase, however, can only start after the first phase
is
finished. Thus, the total mixing time of two-phase mixing should be the
maximum mixing time of first and second binary mixing units 110 and 210 in
the first phase plus the mixing time of either first binary mixing unit 110 or
second binary mixing unit 210 in the second phase. Accordingly, the mixing
cycle is defined as the total mixing time required to finish one mixing
process.
It is standardized in terms of mixing operations, which are assumed to be the
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most time consuming operations as compared to, for example, droplet
transport.
3. Small number of total mixing operations. A single binary mixing
operation that consists of mixing, splitting and/or transportation is a source
of
error. Also, more mixing operations also mean more usage of the electrodes,
which may be another cause of error due to the charge accumulation on
electrodes.
4. Simplicity of operations.
5. Scalability. The capability of the binary mixing apparatus 100 to
handle different mixing ratios and extendibility of the structure to multiple
mixing
units when large throughput is demanded.
6. Parallelism.
The architecture of binary mixing apparatus 100 implements multiple
hierarchies of binary mixing phases, with the first hierarchy providing the
approximate mixing ratio and the following ones employed as the calibration
mechanism. The concept is analogous to an interpolating Digital-to-Analog
Converter (DAC) whose architecture is divided into two parts, with the main
DAC handling the MSB (most significant bit) in a binary manner and the sub-
DAC dealing with calibration and correction down to the LSB (least significant
bit). An example of a one-phase binary mixing process carried out to produce
sixteen sample droplets diluted to a concentration of 1/32 is described
hereinbelow with reference to Figures 19A - 19F.
It is believed that mixing in a binary manner results in dilution to large
ratios in the power of two with only a few mixing operations. The accuracy of
the ratio can be calibrated by further mixing two intermediate products in a
binary manner. For example, one mixing process could produce
concentrations of 1/8, and another could produce concentrations of 1/16.
When these two mixtures further mix with 1:1, 1:3, 3:1, 1:7, and 7:1 ratios,
respectively, the final product would have concentrations of 1/10.67, 1/12.8,
1/9.14, 1/14.2, and 1/8.53, respectively. Based on this principle, any ratio
can
be obtained in a few mixing phases with acceptable tolerance. If further
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accuracy is needed, an additional mixing phase using products from the
previous phase can be used to calibrate the ratio. As indicated previously,
the
process of approaching the expected ratio to high accuracy could be
characterized as a successive approximation process that is similar to one
used in Analog to Digital converter design. It is an approach that trades off
speed with accuracy. However, the number of mixing phases required for
adequate accuracy is surprisingly small. Generally, when the required ratio is
smaller than 32, two mixing phases are often enough. Ratios larger than 32
but smaller than 64 would possibly need three mixing phases. It is also
observed that different combinations of intermediate products mixed with a
range of binary ratios would produce more interpolating points to further
increase the accuracy, thus eliminating the necessities of using extra mixing
phases.
Based on known mathematical principles, the architecture of binary
mixing apparatus 100 can be designed to have preferably two same-structured
mixing units (e.g., first binary mixing unit 110 and second binary mixing unit
210
shown in Figure 17), with each binary mixing unit 110 and 210 handling binary
mixing and generating certain volumes of mixture. Each binary mixing unit 110
and 210 can produce different mixing ratios of a power of two according to
different operations. In the first mixing phase, the sample is mixed with the
reagent with a ratio of any of the series (1:1, 1:3, 1:7 ... 1:2n"1) using two
binary
mixing units 110 and 210 in parallel. The products are two mixtures with the
same volume. The ratio of the two mixtures is determined by the required ratio
of the final product, and preferably is controlled by a computer program. In a
second phase, the two mixtures mix with a certain binary ratio in one of the
two
units. Buffer 310 is used to store some of the intermediate products when
second phase mixing is carried out in one of binary mixing units 110 or 210.
Since the volume of the intermediate product is limited (e.g., 16 droplets),
the
second mixing cannot be carried out with an arbitrarily large binary ratio.
From
the description herein of the structure and operation of binary mixing
apparatus
100, it can be demonstrated that the possible binary ratio in the following
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mixing phase is constrained to be less than or equal to 31, given that 4
columns and 16 droplets are generated from each unit. Even so, sufficient
accuracy could be obtained after a second phase. If further accuracy is
demanded, additional mixing can be carried out to generate a mixture closer to
the requirement, using the product from the second phase and another mixture
with power of two series ratio (e.g., a calibration mixture).
From the description above, it can be observed that generating powers
of two series mixtures can be a fundamental process in obtaining an expected
ratio. The exact ratio of this mixture could be decided ahead of time or
varied
dynamically. For example, during the first phase of mixing, the two ratios
could
be calculated ahead of time according to the required ratio. In the phase
following the second phase, however, the calibration mixture could be decided
dynamically, given the feedback from the quality of previous mixing. Even if
predecided, it is likely that extra time would be needed to prepare the
calibration mixture before a further phase mixing is carried out. In such a
case,
the use of only two binary mixing units 110 and 210 might be not enough, and
an extra binary mixing unit could be added to prepare the calibration mixture
in
parallel with the previous calibration mixing process.
The determination of a mixing strategy includes calculating the number
of mixing phases and the mixing ratio for each phase according to the required
ratio and its tolerance. This determination can be solved by an optimization
process with the number of mixing operations and time of the mixing as the
objective function.
Referring now to Figures 18A and 18B, an exemplary architecture for
first binary mixing unit 110 is illustrated, with the understanding that
second
binary mixing unit 210 and any other additional mixing units provided can be
similarly designed. The embodiment shown in Figure 18A is capable of one-
phase mixing, while the embodiment shown in Figure 20 (to be briefly
described hereinbelow) is capable of two-phase mixing. As shown in Figure
18A, first binary mixing unit 110 generally comprises a 7 x 7 electrode matrix
or
array, generally designated EA, consisting of 49 matrix electrodes and their
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associated cells E11, where "i" designates 1, 2, ..., 7 rows of electrodes and
designates 1, 2, . . ., 7 columns of electrodes. Figure 18B identifies matrix
electrodes E, of electrode array EA in accordance with a two-dimensional
system of rows ROW1 - ROW7 and columns COL1 - COLT. The invention,
however, is not limited to any specific number of electrodes, rows, and
columns. A larger or smaller electrode array EA could be provided as
appropriate.
Referring back to Figure 18A, a sample reservoir 113, waste reservoir
115, and reagent reservoir 117 are also provided. Depending on the position of
reservoirs 113, 115 and 117 in relation to electrode array EA, a suitable
number and arrangement of transport or path electrodes and associated cells
T1- T4 are provided for conveying droplets to and from electrode array EA. A
number of electrical leads (e.g., L) are connected to matrix electrodes E11
and
transport electrodes T1- T4 to control the movement or other manipulation of
droplets. It will be understood that electrical leads L communicate with a
suitable electronic controller such as a microprocessor (e.g., electronic
controller EC in Figure 17). Each matrix electrode E,1 could have its own
independent electrical lead connection. However, to reduce the number of
electrical leads L and hence simplify the architecture of first binary mixing
unit
110, the electrodes of each of columns COL2 - COLT (see Figure 18B) are
connected to common electrical leads L as shown in Figure 18A. These
common connections must be taken into consideration when writing the
protocol for mixing operations to be carried out by first binary mixing unit
110.
In effect, each binary mixing unit 110 and 210 of binary mixing apparatus
100 is designed to have 4 x 4 logic cells with each cell storing the sample,
reagent or intermediate mixture. This can be conceptualized by comparing the
matrix layout of Figure 18B with the 4 x 4 logic cell matrix illustrated in
Figures
19A-19F. The 4 x 4 construct accounts for the fact that droplets combine on
intermediate control electrodes from adjacent control electrodes (e.g.,
intermediate control electrode E2 and adjacent control electrodes E1 and E3 in
Figures 5A - 6C), the mixed droplet is then split, and the newly formed mixed
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droplets are then returned to the adjacent control electrodes at the
completion
of the MIX (or MIX-SPLIT) operation. Hence, certain rows of electrodes need
only be used as temporary intermediate electrodes during the actual droplet
combination event. The construct also accounts for the fact that certain
columns of electrodes need only be used for droplet transport (e.g., shifting
droplets from one column to another to make room for the addition of new
reagent droplets). In view of the foregoing, electrode rows ROW2, ROW4 and
ROW6, and columns COL2, COL4 and COLE in Figure 18B are depicted
simply as lines in Figures 19A - 19F. Also in Figures 19A - 19F, active
electrodes are indicated by shaded bars, mixing operations are indicated by
the
symbol "- - - -> - - -", and transport operations are indicated by the symbol
"- - - ->". Additionally, droplet concentrations are indicated by numbers
(e.g., 0,
1,'/2) next to rows and columns where droplets reside.
It can be seen that one-by-one mixing can occur between some of the
adjacent cells in horizontal or vertical directions (from the perspective of
the
drawing sheets containing Figures 19A - 19F), depending on whether active
electrodes exist between the two cells. In the first column, between any of
the
two adjacent row cells containing droplets, an active electrode exists that
allows
the two adjacent row cells to perform mixing operations. In other columns,
there are no active electrodes between two row cells. This is illustrated, for
example, in Figure 19A. Between any of the columns containing droplets,
electrodes exist that allow any of the cells in one column to conduct a mixing
operation with the cells of its adjacent column simultaneously. This is
illustrated, for example, in Figure 19D. By the use of the active electrodes,
the
content of a logic cell (i.e., a droplet) can move from one row to another in
the
first column, or move between columns. The employment of the 4 X 4 logic
structure is designed for the optimization of binary operations, as
demonstrated
by the following example. It will be noted that the volume output of the
present
one-mixing-unit embodiment of first binary mixing unit 110 is limited to 16
droplets, although the physical volume of the final product can be adjusted by
changing the size of each droplet.
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To demonstrate how binary mixing apparatus 100 can produce any of
the power of two ratios, Figures 19A -19F illustrate an example of a series of
mixing operations targeting a 1:31 ratio (equal to 1/32 concentration). It can
be
seen that the mixing process has two basic stages: a row mix and a column
mix. Generally, the purpose of the row mix is to approach the range of the
mixing ratio with a minimum volume of two mixing inputs. The purpose of the
column mix is to produce the required volume at the output and at the same
time obtain another four-fold increase in ratio. Thus, as indicated in Figures
19A - 19F, to obtain a 1:31 ratio, the row mix results in a 1:7 ratio or 1/8
concentration (see Figure 19D). The column mix assists in achieving the final
product ratio of 1:31 or 1/32 concentration (see Figure 19F).
Referring specifically to Figure 19A, a single row mix is performed by
combining a sample droplet S1 having a concentration of 1 (i.e., 100%) with a
reagent (or solvent) droplet R1 having a concentration of 0. This results in
two
intermediate-mixture droplets I1 and 12, each having a 1/2 concentration as
shown in Figure 19B. One of the intermediate-mixture droplets (e.g., 11) is
discarded, and a new reagent droplet R2 is moved to the logic cell adjacent to
the remaining intermediate-mixture droplet (e.g., 12). Another row mix is
performed by combining intermediate-mixture droplet 12 and reagent droplet R2.
This results two intermediate-mixture droplets 13 and 14, each having a 1/4
concentration as shown in Figure 19C. Two new reagent droplets R3 and R4
are then added and, in a double row mix operation, combined with respective
intermediate-mixture droplets 13 and 14. This results in four intermediate-
mixture
droplets 15 - 18, each having a 1/8 concentration as shown in Figure 19D.
As also shown in Figure 19D, four new reagent droplets R5 - R8 are then
moved onto the matrix adjacent to respective intermediate-mixture droplets 1,5
-
18. A column mix is then performed as between each corresponding pair of
intermediate-mixture droplets 15 - I8 and reagent droplets R5 - R8. This
produces eight intermediate-mixture droplets 19 - 116, each having a 1/16
concentration as shown in Figure 19E. As also shown in Figure 19E, each
column of four intermediate-mixture droplets, 19 -112 and 113 -116,
respectively,
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is shifted over one column to the right to enable two columns of new reagent
droplets, R9 - R12 and R13 - R16, respectively, to be loaded onto the outer
columns of the matrix. Each corresponding pair of intermediate-mixture
droplets and reagent droplets (e.g., 19 and R9, 110 and Rio, etc.) is then
combined through additional column mix operations.
As a result of these mixing operations, sixteen final-mixture product
droplets P1 - P16 are produced, each having a final concentration of 1/32
(corresponding to the target mix ratio of 1:31) as shown in Figure 19F.
Product
droplets P1 - P16 are now prepared for any subsequent operation
contemplated, such sampling, detection, analysis, and the like as described by
way of example hereinabove. Additionally, depending on the precise mix ratio
desired, product droplets P1 - P16 can be subjected to a second or even a
third
phase of mixing operations if needed as described hereinabove. Such
additional mixing phases can occur at a different area on the electrode array
of
which first binary mixing unit 110 could be a part. Alternatively, as
illustrated in
Figure 17, the final-mixture droplets can be conveyed to another binary mixing
apparatus (e.g., second binary mixing unit 210) that fluidly communicates
directly with first binary mixing unit 110 or through buffer 310.
The method of the invention can be applied to ratios less than or greater
than 31. For example, if the goal is to obtain a ratio of 1:15, the row mix
would
mix the input to a ratio of 1:3, which would require two mixing operations
instead of three for obtaining a mixing ratio of 1:7. In terms of mixing
operations, Figures 19A -19F can be used to show that the first stage for row
mix (single) and the discard operation for the second stage could be
eliminated
in such a case.
To further explain the detailed operations for completing the mixing of
1:31, a pseudo code for the example specifically illustrated in Figures 19A -
19F (and with general reference to Figure 18B) is listed as follows:
1. Load S (1,1), Load R (2,1), Row Mix 1,2
2. (Discard (1,1), Load R (3,1)), Row Mix 2,3
3. Load R (1,1) Load R (3,1), (Row Mix 1,2, Row Mix 3,4)
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4. Column Load R2, Column Mix 1,2
5. Column Move 2 to 3, Column Move 1 to 2, column Load R 1,
Column Load R4, (Column Mix 1,2 Column Mix 3,4)
6. Finish
The above pseudo code also standardizes the possible mixing
operations into one mixing process. The sequence of the operations is subject
to more potential optimization to increase the throughput of the mixing while
decreasing the number of mixing operations. This design also keeps in mind
that the number of active electrodes should be maintained as small as possible
while making sure all the mixing operations function properly. In the
preferred
embodiment, each binary mixing unit 110 and 210 (see Figure 17) is designed
to have 13 active electrodes to handle the mixing functions. The capability of
transporting the droplets into and inside the each binary mixing unit 110 and
210 is another consideration. Initially, the two outside columns of the array
could be used as transportation channels running along both sides of the mixer
to deliver droplets into the mixer simultaneously with other operations of the
mixer. The same number of electrodes can also handle these transportation
functions.
The second phase is the mixing process when the intermediate products
from two binary mixing units 110 and 210 (see Figure 17) are to be mixed. It
is
similar to the standard binary mixing process in the first phase described
hereinabove with reference to Figures 19A -19F. The only difference is that
the second-phase mixing is carried out in one of binary mixing units 110 and
210 holding the previous mixing product (e.g., product droplets P1 - P16 shown
in Figure 19F). As indicated previously, buffer 310 is used to hold some of
the
product during the process.
It can be calculated that the maximum ratio of mixing during the second
phase is limited to 31. The reason is that to obtain the maximum ratio, row
mixing should be used as much as possible. When row mixing is used to
increase the ratio, less input is lost during the discard process. Thus, when
there are finite amounts of input material, the first choice is to see how far
the
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row mixing can go until there is just enough volume left to fulfill the
requirement
for mixture output. In this way, it could be known that two mixtures with 16
droplets can only mix with the largest ratio of 1:31 when the output
requirement
is specified to no less than 16 droplets. It can also be demonstrated from
Figures 19A - 19F that to mix with a ratio of 1:31, 16 droplets of reagents
would be the minimum amount.
The physical layout for first binary mixing unit 110 illustrated in Figure
18A can be modified to better achieve two-phase mixing capability.
Accordingly, referring now to Figure 20, a two-phase mixing unit, generally
designated 410, is illustrated. The architecture of two-phase mixing unit 410
is
similar to that of first binary mixing unit 110 of Figure 18A, and thus
includes
the 7 x 7 matrix, a sample reservoir 413, a waste reservoir 415, a reagent
reservoir 417, and an appropriate number and arrangement of off-array
electrodes as needed for transport of droplets from the various reservoirs to
the
7 x 7 matrix. Two-phase mixing unit 410 additionally includes a cleaning
reservoir 419 to supply cleaning fluid between mixing processes, as well as an
outlet site 421 for transporting product droplets to other mixing units or to
buffer
310 (see Figure 17). Moreover, it can be seen that additional rows and
columns of electrodes are provided at the perimeter of the 7 x 7 matrix to
provide transport paths for droplets to and from the matrix.
Further insight into the performance of the architecture of binary mixing
apparatus 100 can be obtained by considering the TABLE set forth
hereinbelow. This TABLE was constructed to list all the possible interpolating
mixing ratios using a two-phase mixing strategy for a maximum mixing ratio of
63 (or, equivalently, a maximum concentration of 1/64). The corresponding
mixing parameters, such as the mixing ratio for mixing unit 1 and 2 (e.g.,
first
and second binary mixing units 110 and 210) in the first phase, the mixing
ratio
for the second phase, and the total mixing cycles are also recorded. The
TABLE can serve as a basis for selecting the proper mixing strategy and/or
further optimization in terms of trading off accuracy with time, improving
resource usage when multiple mixers exist, decreasing total mixing operations,
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improving parallelism, and so on. The TABLE can be provided as a look-up
table or data structure as part of the software used to control apparatus 100.
The TABLE shows that there are a total of 196 mixing strategies using
the architecture of the invention, which corresponds to 152 unique mixing
points. The 196 mixing strategies are calculated by interpolating any possible
combinations of two mixtures with power of two ratios under 63. These points
have non-linear instead of linear intervals. The smaller the ratio, the
smaller
the interval. The achievable points are plotted in Figure 21. It is evident
from
the TABLE that the number of achievable ratios is larger than traditional
linear
mixing points and the distribution is more reasonable. In addition, certain
volumes of output other than one droplet can allow more tolerance on the error
caused by one-by-one mixing. In terms of mixing cycles, the best performance
is for mixing ratios of the power of two compared to their nearby ratios. In
terms of accuracy, the larger the ratio, generally the worse the performance,
since a smaller number of interpolating points can be achieved.
It can be observed from the two-phase mixing plan plotted in Figure 21
that there are not enough points when the target ratio is larger than 36.
Figure
21 shows that there is no point around a ratio of 40. The difference between
the target and theoretical achievable ratio could amount to 3. However, by
careful examination of the achievable points around 40, an appropriate usage
of the remaining mixture from phase one to further calibrate the available
points
can result in several additional interpolating points between 36.5714 and
42.6667, where the largest error exists from the phase two mixing plan. For
instance, the mixing plan #183 in the TABLE calls for obtaining mixture 1 and
mixture 2 with ratios of 1:31 and 1:63, respectively, then mixing them with a
ratio of 3:1. It is known that there are 3/4 parts of mixture 2 left. So it is
possible to mix the mixture from phase two with a concentration of 36.517 with
mixture 2 of concentration 63 using ratio of 3:1, 7:1, etc. That leads to a
point
at 40.9, 38.5, etc. In such manner, more accuracy is possible with an
additional mixing phase, but with only a small increase in mixing cycles (two
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and three cycles, respectively, in this example), and at the expense of no
additional preparation of calibration mixture.
Figure 22 demonstrates all the achievable points by one-phase, two-
phase, and three-phase mixing plans. The total number of points is 2044. The
points achieved by phase three are obtained by using the product from phase
two and remaining products from phase one. They are calculated by
considering the volume of the remaining product from phase one after phase
two has finished and reusing them to mix with products from phase two. The
possible mixing ratios of phase three are determined by the mixing ratio of
phase two.
TABLE
Mix Plan Target Mix Mix Unit 1 Mix Unit 2 Phase 2 Total Mix
Number Ratio Mix Ratio Mix Ratio Mix Ratio Cycles
1 1.0159 1:0 1:1 31:1 6
2 1.0240 1:0 1:3 31:1 7
3 1.0281 1:0 1:7 31:1 8
4 1.0302 1:0 1:15 31:1 9
5 1.0312 1:0 1:31 31:1 10
6 1.0317 1:0 1:63 31:1 11
7 1.0323 1:0 1:1 15:1 5
8 1.0492 1:0 1:3 15:1 6
9 1.0579 1:0 1:7 15:1 7
10 1.0622 1:0 1:15 15:1 8
11 1.0644 1:0 1:31 15:1 9
12 1.0656 1:0 1:63 15:1 10
13 1.0667 1:0 1:1 7:1 4
14 1.1034 1:0 1:3 7:1 5
1.1228 1:0 1:7 7:1 6
16 1.1327 1:0 1:15 7:1 7
17 1.1378 1:0 1:31 7:1 8
18 1.1403 1:0 1:63 7:1 9
19 1.1429 1:0 1:1 3:1 3
1.2308 1:0 1:3 3:1 4
21 1.2800 1:0 1:7 3:1 5
22 1.3061 1:0 1:15 3:1 6
23 1.3196 1:0 1:31 3:1 7
24 1.3264 1:0 1:63 3:1 8
1.3333 1:0 1:1 1:1 2
26 1.6000 1:0 1:1 1:3 3
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27 1.6000 1:0 1:3 1:1 3
28 1.7778 1:0 1:1 1:7 4
29 1.7778 1:0 1:7 1:1 4
30 1.8824 1:0 1:1 1:15 5
31 1.8824 1:0 1:15 1:1 5
32 1.9394 1:0 1:1 1:31 6
33 1.9394 1:0 1:31 1:1 6
34 1.9692 1:0 1:63 1:1 7
35 2.0000 1:1 N/A N/A 1
36 2.0317 1:1 1:3 31:1 7
37 2.0480 1:1 1:7 31:1 8
38 2.0562 1:1 1:15 31:1 9
39 2.0604 1:1 1:31 31:1 10
40 2.0624 1:1 1:63 31:1 11
41 2.0645 1:1 1:3 15:1 6
42 2.0981 1:1 1:7 15:1 7
43 2.1157 1:1 1:15 15:1 8
44 2.1245 1:1 1:31 15:1 9
45 2.1289 1:1 1:63 15:1 10
46 2.1333 1:1 1:3 7:1 5
47 2.2069 1:1 1:7 7:1 6
48 2.2456 1:1 1:15 7:1 7
49 2.2655 1:1 1:31 7:1 8
50 2.2756 1:1 1:63 7:1 9
51 2.2857 1:0 1:3 1:3 4
52 2.2857 1:1 1:3 3:1 4
53 2.4615 1:1 1:7 3:1 5
54 2.5600 1:1 1:15 3:1 6
55 2.6122 1:1 1:31 3:1 7
56 2.6392 1:1 1:63 3:1 8
57 2.6667 1:1 1:3 1:1 3
58 2.9091 1:0 1:3 1:7 5
59 2.9091 1:0 1:7 1:3 5
60 3.2000 1:1 1:3 1:3 4
61 3.2000 1:1 1:7 1:1 4
62 3.3684 1:0 1:3 1:15 6
63 3.3684 1:0 1:15 1:3 6
64 3.5556 1:1 1:3 1:7 5
65 3.5556 1:1 1:15 1:1 5
66 3.6571 1:0 1:3 1:31 7
67 3.6571 1:0 1:31 1:3 7
68 3.7647 1:1 1:3 1:15 6
69 3.7647 1:1 1:31 1:1 6
70 3.8209 1:0 1:63 1:3 8
71 3.8788 1:1 1:3 1:31 7
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72 3.8788 1:1 1:63 1:1 7
73 4.0000 1:3 N/A N/A 3
74 4.0635 1:3 1:7 31:1 8
75 4.0960 1:3 1:15 31:1 9
76 4.1124 1:3 1:31 31:1 10
77 4.1207 1:3 1:63 31:1 11
78 4.1290 1:3 1:7 15:1 7
79 4.1967 1:3 1:15 15:1 8
80 4.2314 1:3 1:31 15:1 9
81 4.2490 1:3 1:63 15:1 10
82 4.2667 1:0 1:7 1:7 6
83 4.2667 1:3 1:7 7:1 6
84 4.4138 1:3 1:15 7:1 7
85 4.4912 1:3 1:31 7:1 8
86 4.5310 1:3 1:63 7:1 9
87 4.5714 1:1 1:7 1:3 5
88 4.5714 1:3 1:7 3:1 5
89 4.9231 1:3 1:15 3:1 6
90 5.1200 1:3 1:31 3:1 7
91 5.2245 1:3 1:63 3:1 8
92 5.3333 1:3 1:7 1:1 4
93 5.5652 1:0 1:7 1:15 7
94 5.5652 1:0 1:15 1:7 7
95 5.8182 1:1 1:7 1:7 6
96 5.8182 1:1 1:15 1:3 6
97 6.4000 1:3 1:7 1:3 5
98 6.4000 1:3 1:15 1:1 5
99 6.5641 1:0 1:7 1:31 8
100 6.5641 1:0 1:31 1:7 8
101 6.7368 1:1 1:7 1:15 7
102 6.7368 1:1 1:31 1:3 7
103 7.1111 1:3 1:7 1:7 6
104 7.1111 1:3 1:31 1:1 6
105 7.2113 1:0 1:63 1:7 9
106 7.3143 1:1 1:7 1:31 8
107 7.3143 1:1 1:63 1:3 8
108 7.5294 1:3 1:7 1:15 7
109 7.5294 1:3 1:63 1:1 7
110 7.7576 1:3 1:7 1:31 8
111 8.0000 1:7 N/A N/A 4
112 8.1270 1:7 1:15 31:1 9
113 8.1920 1:7 1:31 31:1 10
114 8.2249 1:7 1:63 31:1 11
115 8.2581 1:0 1:15 1:15 8
116 8.2581 1:7 1:15 15:1 8
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117 8.3934 1:7 1:31 15:1 9
118 8.4628 1:7 1:63 15:1 10
119 8.5333 1:1 1:15 1:7 7
120 8.5333 1:7 1:15 7:1 7
121 8.8276 1:7 1:31 7:1 8
122 8.9825 1:7 1:63 7:1 9
123 9.1429 1:3 1:15 1:3 6
124 9.1429 1:7 1:15 3:1 6
125 9.8462 1:7 1:31 3:1 7
126 10.2400 1:7 1:63 3:1 8
127 10.6667 1:7 1:15 1:1 5
125 10.8936 1:0 1:15 1:31 9
129 10.8936 1:0 1:31 1:15 9
130 11.1304 1:1 1:15 1:15 8
131 11.1304 1:1 1:31 1:7 8
132 11.6364 1:3 1:15 1:7 7
133 11.6364 1:3 1:31 1:3 7
134 12.8000 1:7 1:15 1:3 6
135 12.8000 1:7 1:31 1:1 6
136 12.9620 1:0 1:63 1:15 10
137 13.1282 1:1 1:15 1:31 9
138 13.1282 1:1 1:63 1:7 9
139 113.4737 1:3 1:15 1:15 8
140 13.4737 1:3 1:63 1:3 8
141 14.2222 1:7 1:15 1:7 7
142 14.2222 1:7 1:63 1:1 7
143 114.6286 1:3 1:15 1:31 9
144 15.0588 1:7 1:15 1:15 8
145 15.5152 1:7 1:15 1:31 9
146 16.0000 1:15 N/A N/A 5
147 16.2540 1:0 1:31 1:31 10
148 16.2540 1:15 1:31 31:1 10
149 16.3840 1:15 1:63 31:1 11
150 16.5161 1:1 1:31 1:15 9
151 16.5161 1:15 1:31 15:1 9
152 16.7869 1:15 1:63 15:1 10
153 17.0667 1:3 1:31 1:7 8
154 17.0667 1:15 1:31 7:1 8
155 17:6552 1:15 1:63 7:1 9
156 18.2857 1:7 1:31 1:3 7
157 18.2857 1:15 1:31 3:1 7
158 19.6923 1:15 1:63 3:1 8
159 21.3333 1:15 1:31 1:1 6
160 21.5579 1:0 1:63 1:31 11
161 21.7872 1:1 1:31 1:31 10
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162 21.7872 1:1 1:63 1:15 10
163 22.2609 1:3 1:31 1:15 9
164 22.2609 1:3 1:63 1:7 9
165 23.2727 1:7 1:31 1:7 8
166 23.2727 1:7 1:63 1:3 8
167 25.6000 1:15 1:31 1:3 7
168 25.6000 1:15 1:63 1:1 7
169 26.2564 1:3 1:31 1:31 10
170 26.9474 1:7 1:31 1:15 9
171 28.4444 1:15 1:31 1:7 8
172 29.2571 1:7 1:31 1:31 10
173 30.1176 1:15 1:31 1:15 9
174 31.0303 1:15 1:31 1:31 10
175 32.0000 1:31 N/A N/A 6
176 32.5079 1:1 1:63 1:31 11
177 32.5079 1:31 1:63 31:1 11
178 33.0323 1:3 1:63 1:15 10
179 33.0323 1:31 1:63' 15:1 10
180 34.1333 1:7 1:63 1:7 9
181 34.1333 1:31 1:63 7:1 9
182 36.5714 1:15 1:63 1:3 8
183 36.5714 1:31 1:63 3:1 8
184 42.6667 1:31 1:63 1:1 7
185 43.5745 1:3 1:63 1:31 11
186 44.5217 1:7 1:63 1:15 10
187 46.5455 1:15 1:63 1:7 9
188 51.2000 1:31 1:63 1:3 8
190 52.5128 1:7 1:63 1:31, 11
191 53.8947 1:15 1:63 1:15 10
192 56.8889 1:31 1:63 1:7 9
193 58.5143 1:15 1:63 1:31 11
194 60.2353 1:31 1:63 1:15 10
195 62.0606 1:31 1:63 1:31 11
196 64.0000 1:63 N/A N/A 7
Electrowetting-based Droplet Actuation on a Single-Sided Electrode
Array
The aspects of the invention thus far have been described in connection
with the use of a droplet actuating apparatus that has a two-sided electrode
configuration such as microactuator mechanism 10 illustrated in Figure 1. That
is, lower plane 12 contains control or drive electrodes E1- E3 and upper plane
14 contains ground electrode G. As regards microactuator mechanism 10, the
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function of upper plane 14 is to bias droplet D at the ground potential or
some
other reference potential. The grounding (or biasing to reference) of upper
plane 14 in connection with the selective biasing of drive electrodes E1- E3
of
lower plane 12 generates a potential difference that enables droplet D to be
moved by the step-wise electrowetting technique described herein. However,
in accordance with another embodiment of the invention, the design of the
apparatus employed for two-dimensional electrowetting-based droplet
manipulation can be simplified and made more flexible by eliminating the need
for a grounded upper plane 14.
Referring now to Figures 23A and 23B, a single-sided electrowetting
microactuator mechanism, generally designated 500, is illustrated.
Microactuator mechanism 500 comprises a lower plane 512 similar to that of
mechanism 10 of Figure 1, and thus includes a suitable substrate 521 on which
two-dimensional array of closely packed drive electrodes E (e.g., drive
electrodes E1 - E3 and others) are embedded such as by patterning a
conductive layer of copper, chrome, ITO, and the like. A dielectric layer 523
covers drive electrodes E. Dielectric layer 523 is hydrophobic, and/or is
treated
with a hydrophobic layer (not specifically shown). Asa primary difference from
microactuator mechanism 10 of Figure 1, a two-dimensional grid of conducting
lines G at a reference potential (e.g., conducting lines G1- G6 and others)
has
been superimposed on the electrode array of microactuator mechanism 500 of
Figures 23A and 23B, with each conducting line G running through the gaps
between adjacent drive electrodes E. The reference potential can be a ground
potential, a nominal potential, or some other potential that is lower than the
actuation potential applied to drive electrodes E. Each conducting line G can
be a wire, bar, or any other conductive structure that has a much narrower
width/length aspect ratio in relation to drive electrodes E. Each conducting
line
G could alternatively comprise a closely packed series of smaller electrodes,
but in most cases this alternative would impractical due to the increased
number of electrical connections that would be required.
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Importantly, the conducting line grid is coplanar or substantially coplanar
with the electrode array. The conducting line grid can be embedded on lower
plane 512 by means of microfabrication processes commonly used to create
conductive interconnect structures on microchips. It thus can be seen that
microactuator mechanism 500 can be constructed as a single-substrate device.
It is preferable, however, to include an upper plane 514 comprising a plate
525
having a hydrophobic surface 527, such as a suitable plastic sheet or a
hydrophobized glass plate. Unlike microactuator mechanism 10 of Figure 1,
however, upper plane 514 of microactuator mechanism 500 of Figures 23A and
23B does not function as an electrode to bias droplet D. Instead, upper plane
514 functions solely as a structural component to contain droplet D and any
filler fluid such as an inert gas or immiscible liquid.
In the use of microactuator mechanism 500 for electrowetting-based
droplet manipulations, it is still a requirement that a ground or reference
connection to droplet D be maintained essentially constantly throughout the
droplet transport event. Hence, the size or volume of droplet D is selected to
ensure that droplet D overlaps all adjacent drive electrodes E as well as all
conducting lines G surrounding the drive electrode on which droplet D resides
(e.g., electrode E2 in Figure 23B). Moreover, it is preferable that dielectric
layer
523 be patterned to cover only drive electrodes E so that conducting lines G
are exposed to droplet D or at least are not electrically isolated from
droplet D.
At the same time, however, it is preferable that conducting lines G be
hydrophobic along with drive electrodes E so as not to impair movement of
droplet D. Thus, in a preferred embodiment, after dielectric layer 523 is
patterned, both drive electrodes E and conducting lines G are coated or
otherwise treated so as render them hydrophobic. The hydrophobization of
conducting lines G is not specifically shown in Figures 23A and 23B. It will
be
understood, however, that the hydrophobic layer covering conducting lines G is
so thin that an electrical contact between droplet D and conducting lines G
can
still be maintained, due to the porosity of the hydrophobic layer.
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To operate microactuator mechanism 500, a suitable voltage source V
and electrical lead components are connected with conducting lines G and
drive electrodes E. Because conducting lines G are disposed in the same
plane as drive electrodes E, application of an electrical potential between
conducting lines G and a selected one of drive electrodes E1, E2, or E3 (with
the
selection being represented by switches S1- S3 in Figure 23A) establishes an
electric field in the region of dielectric layer 523 beneath droplet D.
Analogous
to the operation of microactuator mechanism 10 of Figure 1, the electric field
in
turn creates a surface tension gradient to cause droplet D overlapping the
energized electrode to move toward that electrode (e.g., drive electrode E3 if
movement is intended in right-hand direction in Figure 23A). The electrode
array can be sequenced in a predetermined manner according to a set of
software instructions, or in real time in response to a suitable feedback
circuit.
It will thus be noted that microactuator mechanism 500 with its single-
sided electrode configuration can be used to implement all functions and
methods described hereinabove in connection with the two-sided electrode
configuration of Figure 1, e.g., dispensing, transporting, merging, mixing,
incubating, splitting, analyzing, monitoring, reacting, detecting, disposing,
and
so on to realize a miniaturized lab-on-a-chip system. For instance, to move
droplet D shown in Figure 23B to the right, drive electrodes E2 and E3 are
activated to cause droplet D to spread onto drive electrode E3. Subsequent de-
activation of drive electrode E2 causes droplet D to relax to a more favorable
lower energy state, and droplet D becomes centered on drive electrode E3. As
another example, to split droplet D, drive electrodes E1, E2 and E3 are
activated
to cause droplet D to spread onto drive electrodes E1 and E3. Drive electrode
E2 is then de-activated to cause droplet D to break into two droplets
respectively centered on drive electrodes E1 and E3.
Referring now to Figures 24A - 24D, an alternative single-sided
electrode configuration is illustrated in accordance with the present
invention.
A base substrate containing an array of row and column biasing electrodes E;1
is again utilized as in previously described embodiments. Referring
specifically
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to Figure 24A, an array or portion of an array is shown in which three rows of
electrodes E11- E14, E21 - E25, and E31 - E34, respectively, are provided. The
rows and columns of the electrode array can be aligned as described herein for
other embodiments of the invention. Alternatively, as specifically shown in
Figure 24A, the array can be misaligned such that the electrodes in any given
row are offset from the electrodes of adjacent rows. For instance, electrodes
E11- E14 of the first row and electrodes E31 - E34 of the third row are offset
from
electrodes E21 - E25 of the intermediate second row. Whether aligned or
misaligned, the electrode array is preferably covered with insulating and
hydrophobic layers as in previously described embodiments. As in the
configuration illustrated in Figures 23A and 23B, a top plate (not shown) can
be
provided for containment but does not function as an electrode.
In operation, selected biasing electrodes E1 are dynamically assigned as
either driving electrodes or grounding (or reference) electrodes. To effect
droplet actuation, the assignment of a given electrode as a drive electrode
requires that an adjacent electrode be assigned as a ground or reference
electrode to create a circuit inclusive with droplet D and thereby enable the
application of an actuation voltage. In Figure 24A, electrode E21 is energized
and thus serves as the drive electrode, and electrode E22 is grounded or
otherwise set to a reference potential. All other electrodes E;j of the
illustrated
array, or at least those electrodes surrounding the driving/reference
electrode
pair E21/E22, remain in an electrically floating state. As shown in Figure
24A,
this activation causes droplet D overlapping both electrodes E21 and E22 to
seek
an energetically favorable state by moving so as to become centered along the
gap or interfacial region between electrodes E21 and E22.
In Figure 24B, electrode E21 is deactivated and electrode E11 from an
adjacent row is activated to serve as the next driving electrode. Electrode
E22
remains grounded or referenced. This causes droplet D to center itself
between electrodes E21 and E22 by moving in a resultant northeast direction,
as
indicated by the arrow. As shown in Figure 24C, droplet D is actuated to the
right along the gap between the first two electrode rows by deactivating
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electrode E11 and activating electrode E12. As shown in Figure 24D, electrode
E22 is disconnected from ground or reference and electrode E23 is then
grounded or referenced to cause droplet D to continue to advance to the right.
It can be seen that additional sequencing of electrodes E;j to render them
either
driving or reference electrodes can be performed to cause droplet D to move in
any direction along any desired flow path on the electrode array. It can be
further seen that, unlike previously described embodiments, the flow path of
droplet transport occurs along the gaps between electrodes E;1 as opposed to
along the centers of electrodes E;j themselves. It is also observed that the
required actuation voltage will in most cases be higher as compared with the
configuration shown in Figures 23A and 23B, because the dielectric layer
covers both the driving and reference electrodes and thus its thickness is
effectively doubled.
Referring now to Figures 25A and 25B, an electrode array with aligned
rows and columns can be used to cause droplet transport in straight lines in
either the north/south (Figure 25A) or east/west (Figure 25B) directions. The
operation is analogous to that just described with reference to Figures 24A -
24D. That is, programmable sequencing of pairs of drive and reference
electrodes causes the movement of droplet D along the intended direction. In
Figure 25A, electrodes E12, E22 and E32 of one column are selectively set to a
ground or reference potential and electrodes E13, E23 and E33 of an adjacent
column are selectively energized. In Figure 25B, electrodes E11, E12, E13 and
E14 of one row are selectively energized and electrodes E21, E22, E23 and E24
of
an adjacent row are selectively grounded or otherwise referenced.
It will be noted that a microactuator mechanism provided with the
alternative single-sided electrode configurations illustrated in Figures 24A -
24D and Figures 25A and 25B can be used to implement all functions and
methods described hereinabove in connection with the two-sided electrode
configuration of Figure 1. For instance, to split droplet D in either of the
alternative configurations, three or more adjacent electrodes are activated to
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spread droplet D and an appropriately selected intervening electrode is then
de-activated to break droplet D into two droplets.
It will be understood that various details of the invention may be
changed without departing from the scope of the invention. Furthermore, the
foregoing description is for the purpose of illustration only, and not for the
purpose of limitation,
