Patent Description:
Electrowetting on dielectric (EWOD) is a well-known technique for manipulating droplets of fluid by the application of an electric field. Active Matrix EWOD (AM-EWOD) refers to implementation of EWOD in an active matrix array incorporating transistors, for example by using thin film transistors (TFTs). It is thus a candidate technology for digital microfluidics for lab-on-a-chip technology. An introduction to the basic principles of the technology can be found in "<NPL>).

Example configurations and operation of EWOD devices are described in the following. <CIT>) discloses a two-dimensional EWOD array to control the position and movement of droplets in two dimensions. <CIT>) further discloses methods for other droplet operations including the splitting and merging of droplets, and the mixing together of droplets of different materials. <CIT>) describes how TFT based thin film electronics may be used to control the addressing of voltage pulses to an EWOD array by using circuit arrangements very similar to those employed in AM display technologies.

The approach of <CIT> may be termed "Active Matrix Electrowetting on Dielectric" (AM-EWOD). There are several advantages in using TFT based thin film electronics to control an EWOD array, namely:.

AM-EWOD droplet manipulation devices are a highly desirable platform for automation of chemical and biochemical reactions. Such devices may carry out chemical/biochemical reactions or reaction sequences in multiple steps requiring different droplet manipulations, such as for example dispensing droplets from a reservoir, moving droplets along the array, splitting droplets, mixing droplets, and the like. Accordingly, it is significant that droplet positioning is precisely maintained and controlled, so as to prevent droplet contamination by adjacent droplets and with droplet contact only occurring in a controlled manner in accordance with a given reaction protocol.

There have been attempts to isolate fluid constituents for purposes of performing chemical reactions, but such principles largely have not been applied to AM-EWOD type devices. For example, <CIT>) pertains to a non-EWOD system using immiscible fluid media to isolate microfluidic structures (pens) in a microfluidic device. In a microfluidic device that incorporates fluidically connected microfluidic structures (storage pens), the pens and channels are filled with a first fluid medium, such as oil, and then a second fluid medium, such as gas or an aqueous medium, is used to obstruct the pen openings to reduce the diffusion of micro-objects or soluble components. <CIT>) is another non-EWOD system, which relies on diffusion of chemical species from a porous membrane into an aqueous droplet, and the droplet is then analyzed. Such a system treats droplets in oil that is somewhat permeable to affect diffusion. As referenced above, such principles largely have not been applied to AM-EWOD type devices, as droplet manipulation is controlled principally by the electrowetting forces, without a recognized need for additional droplet manipulation mechanisms.

<CIT> proposes certain novel approaches to reducing or eliminating the movement of contaminants from one droplet to another on a droplet actuator via liquid filler fluid. In one application, droplet actuators are used to conduct genetic analysis using polymerase chain reaction (PCR) techniques. The disclosure addresses the need for improved methods of performing PCR on a droplet actuator that provide for optimum amplification and detection of a sample target.

<CIT> proposes droplet actuators and droplet actuator techniques. Among other things, the droplet actuators and methods are useful for manipulating beads on a droplet actuator, such as conducting droplet operations using bead-containing droplets on a droplet actuator. For example, beads may be manipulated on a droplet actuator in the context of executing a sample preparation protocol and/or an assay protocol. An output of the methods of the invention may be beads prepared for execution of an assay protocol. Another output of the methods of the invention may be results of an assay protocol executed using beads.

<CIT> proposes an array element circuit with an integrated impedance sensor is provided. The array element circuit includes an array element which is controlled by application of a drive voltage by a drive element; writing circuitry for writing the drive voltage to the drive element; and sense circuitry for sensing an impedance presented at the drive element.

Typically, in reaction protocols using AM-EWOD devices, it has been presumed that in the absence of electrowetting forces, droplets remain largely fixed in position, with any migration or leakage of droplet constituents being considered negligible. Accordingly, barrier structures that are used in other analytical fluidic systems, such as pens, channels, or the like, have not been employed on EWOD devices, and in any event would not be suitable for EWOD devices given the size and number of droplets that may be dispensed, and the nature of the droplet manipulation operations. The inventors have found, however, that the presumption of negligible droplet migration or leakage of constituents does not hold true in many significant circumstances. Accordingly, there is a need in the art, that previously has gone unrecognized, for the capability to employ barriers against droplet content migration in an AM-EWOD device.

More particularly, the inventors have found that when an aqueous droplet on an AM-EWOD device is used to store a species for extended periods, under some circumstances there is a tendency for the stored species to migrate into the surrounding medium, and potentially into nearby droplets resulting in droplet contamination. This problem is particularly pronounced when the concentration of the stored species in the original droplet is high. For example, a droplet containing <NUM>:<NUM> v/v formic acid / water, surrounded by oil, has been shown to leak formic acid through the oil and into nearby droplets, lowering the pH of the contaminated droplets in a way that might compromise an ongoing chemical reaction.

To overcome such deficiencies, in accordance with embodiments of the present invention, in an AM-EWOD device electrowetting forces are applied to a droplet to form, as referred to herein, one or more "barrier droplets" to form a barrier droplet configuration that acts as a barrier to trap, obstruct, or otherwise prevent migration of a dissolved or suspended species in a source droplet through the surrounding oil medium. The barrier droplet configuration completely surrounds the source droplet. For example, the barrier droplet configuration may be positioned between a droplet containing an acid (the source droplet) and a second droplet whose pH must be kept high. Without the barrier droplet configuration, acid is able to migrate from the source droplet, through the oil, and into the high-pH second droplet. The barrier droplet configuration acts to restrict the migration of acid (or whatever constituent species the source droplet contains) so that high concentrations may be stored on-chip in the presence of, for example, species-sensitive components or other reaction areas on the device array.

In exemplary embodiments, the barrier droplet configuration operates by exploiting a difference in preference of the migrating species for the polar or aqueous environment of the barrier droplet versus the non-aqueous environment of the non-polar fluid (oil). That is, the migrating species (constituent) either exhibits a preference for the polar/ aqueous environment of the barrier over the non-polar environment of the oil, or exhibits a preference for the non-polar environment of the oil over the polar/aqueous environment of the barrier. On encountering an oil/water boundary, the migrating species partitions itself between the oil and the water as a result of the preference for the polar/ aqueous environment of the barrier or for the non-polar environment of the oil. Consider an example whereby a source droplet containing a high concentration of the migrating species is near an area that must be protected from the migrating species. This protected area may be where a chemical reaction takes place that may be sensitive to the presence of the migrating species, an electronic component that could be damaged by the migrating species, or another droplet or fluid reservoir that stores a reagent of another composition that could be contaminated by the migrating species. At the boundary between the source droplet and the oil, a portion of the migrating species will escape the source droplet and enter the oil. In the absence of a barrier droplet configuration, the migrating species may then move across the oil until the species reaches the area to be protected. If the system is closed, the system may move towards a state of dynamic equilibrium in which the concentration of the migrating species in the area to be protected is unacceptably high.

To prevent contamination of the protected area by the migrating species, a barrier droplet configuration is formed by electrowetting forces in an area of the device array between the source droplet and the protected area. The barrier droplet configuration provides an additional set of oil/water interfaces at which the migrating species population may become partitioned; in the event that the migrating species has a preference for polar or aqueous environments, the barrier will also act as a thermodynamic sink for the migrating species. The barrier droplet thus obstructs a portion of the potential pathways of migration, thereby reducing the rate of migration of the species to the protected area. By reducing the rate at which the migrating species enters the protected area, the useful lifetime of the reaction system on the AM-EWOD device is extended.

To be effective, the barrier droplet configuration should obstruct a significant portion of the potential pathways of migration to the protected area. The obstruction may be performed using a single barrier droplet or an ensemble of a plurality of barrier droplets. To achieve this requisite obstruction without the barrier droplet becoming unmanageably large, electrowetting forces applied by the AM-EWOD device are applied to control the shape of the barrier droplet(s). An AM-EWOD device can be used to prepare a barrier droplet configuration that completely surrounds the source droplet, or otherwise maintains a high aspect ratio in the barrier droplet configuration such that surface tension otherwise renders the barrier droplets unstable in the absence of the electrowetting forces.

For effective obstruction of migration pathways, a concentration of the migrating species in the barrier droplet(s) of the barrier droplet configuration is lower than a concentration of the migrating species in the source droplet. Under such conditions, the rate of migration out of a side of the barrier droplet opposite from the source droplet is lowered simply by dilution effects in accordance with Fick's diffusion laws. Additionally, the effectiveness of the barrier droplet configuration may be enhanced by the changing barrier droplet contents or makeup. Because of the makeup of the migrating species, at the oil/water interface the migrating species may have a preference for one environment over the other. In the case of a preference for non-polar environments, the aqueous barrier droplet will act as a barrier. In the case of a greater preference for the aqueous environment, the barrier droplet will act as a sink, absorbing the migrating species. The oil/water interface on the far side of the barrier droplet from the source droplet will also act as a barrier to migration. Accordingly, an additive to the barrier droplet that increases the strength of a migrating species's existing preference for either aqueous or non-polar media would help to slow its migration through the oil across the EWOD device. In one example, the additive includes a capturing agent, such as for example an adsorbent nanoparticle, that is able to capture the migrating species and physically prevent species migration, or that reacts with the migrating species to convert the species into something whose presence can be tolerated in the region to be protected.

An aspect of the invention, therefore, is a method of operating an electrowetting on dielectric (EWOD) device that includes an EWOD device array that applies electrowetting forces and contains a non-polar fluid, whereby a barrier droplet configuration is formed using electrowetting forces to obstruct migration of a species from a first area of the EWOD device array to a protected area of the EWOD device array. In exemplary embodiments, the method of operating includes the steps of: dispensing a source droplet into a first area of the EWOD device array, the source droplet containing a migrating species, wherein the EWOD device array includes a second area to be protected from the migrating species; and forming a barrier droplet configuration positioned between the first area and the second area of the EWOD device array that obstructs a migration pathway of the migrating species between the first area and the second area. The barrier droplet configuration includes at least one aqueous or polar barrier droplet, and the migrating species exhibits a preference for either the polar or aqueous environment of the barrier or the non-polar environment of the oil. For example, the concentration of a migrating species in the barrier droplet may be lower than the concentration of a migrating species in the source droplet. The barrier droplet may include an additive that increases the preference of the migrating species for the environment of the barrier droplet relative to the non-polar fluid, such as for example a capturing agent that reacts with or binds to the migrating species, or converts the migrating species into another form. The barrier droplet configuration may include a variety of numbers, shapes, and positionings of one or more barrier droplets, and in exemplary embodiments the barrier droplet(s) may be combined with one or more additional barrier elements that are not barrier droplets formed using the electrowetting forces.

The EWOD device may be operated by: dispensing a first droplet into a first area of the EWOD device array; dispensing a second droplet into a second area of the EWOD device array; forming a barrier droplet configuration; wherein the barrier droplet configuration comprises a first portion that separates the first area from the second area, and a second portion that separates both the first and second areas from a third area of the EWOD device array; maintaining the first portion of the barrier droplet configuration during a first stage of a reaction protocol, thereby obstructing a migration pathway between the first area and the second area during said first stage; and retracting the first portion of the barrier droplet configuration during a second stage of the reaction protocol, thereby opening the migration pathway between the first area and the second area to permit interaction of the first droplet and the second droplet during said second stage. The method further may include applying electrowetting voltages to the EWOD device array during the second stage of the reaction protocol to mix the first droplet and the second droplet.

According to another aspect of the invention, a microfluidic system includes an electro-wetting on dielectric (EWOD) device comprising an element array configured to receive one or more liquid droplets, the element array comprising a plurality of individual array elements; and a control system configured to control actuation voltages applied to the element array to perform manipulation operations as to the liquid droplets to perform the methods of operating the EWOD device that include forming a barrier droplet configuration. The microfluidic system further may include integrated impedance sensing circuitry that is integrated into the array elements of the EWOD device and is configured to determine an impedance of source and barrier droplets dispensed onto the array elements. The configuration and/or position of source and barrier droplets dispensed onto the array elements may be determined based on an impedance sensed by the impedance sensing circuitry. Another aspect of the invention is a non-transitory computer-readable medium storing program code which is executed by a processing device for controlling actuation voltages applied to array elements of an element array of an electro-wetting on dielectric (EWOD) device of the above aspect for performing droplet manipulations on droplets on the element array, the program code being executable by the processing device to perform steps of the methods of operating the EWOD device that include forming a barrier droplet configuration.

These and further features of the present invention will be apparent with reference to the following description and attached drawings. In the description and drawings, particular embodiments of the invention have been disclosed in detail as being indicative of some of the ways in which the principles of the invention may be employed, but it is understood that the invention is not limited correspondingly in scope. Rather, the invention includes all changes and modifications coming within the scope of the claims appended hereto.

The present invention pertains to a microfluidic system including in an AM-EWOD device by which electrowetting forces are applied to a liquid reservoir to form a barrier droplet configuration to act as a barrier to trap, obstruct, or otherwise prevent migration of a dissolved or suspended species in a source droplet through the surrounding oil medium. <FIG> is a drawing depicting an exemplary EWOD based microfluidic system that may be used to perform methods according to embodiments of the present invention. In the example of <FIG>, the measurement system includes a reader <NUM> and a cartridge <NUM>. The cartridge <NUM> may contain a microfluidic device, such as an EWOD or AM-EWOD device <NUM>, as well as (not shown) fluid input ports into the device and an electrical connection as are conventional. The fluid input ports may perform the function of inputting fluid into the AM-EWOD device <NUM> and generating droplets within the device, for example by dispensing from input reservoirs as controlled by electrowetting. As further detailed below, the microfluidic device includes an electrode array configured to receive the inputted fluid droplets.

The microfluidic system further may include a control system configured to control actuation voltages applied to the electrode array of the microfluidic device to perform manipulation operations to the fluid droplets. For example, the reader <NUM> may contain such a control system configured as control electronics <NUM> and a storage device <NUM> that may store any application software any data associated with the system. The control electronics <NUM> may include suitable circuitry and/or processing devices that are configured to carry out various control operations relating to control of the AM-EWOD device <NUM>, such as a CPU, microcontroller or microprocessor.

Among their functions, to implement the features of the present invention, the control electronics may comprise a part of the overall control system that may execute program code embodied as a control application within the storage device <NUM>. It will be apparent to a person having ordinary skill in the art of computer programming, and specifically in application programming for electronic control devices, how to program the control system to operate and carry out logical functions associated with the stored control application. Accordingly, details as to specific programming code have been left out for the sake of brevity. The storage device <NUM> may be configured as a non-transitory computer readable medium, such as random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), or any other suitable medium. Also, while the code may be executed by control electronics <NUM> in accordance with an exemplary embodiment, such control system functionality could also be carried out via dedicated hardware, firmware, software, or combinations thereof, without departing from the scope of the invention.

The control system may be configured to perform some or all of the following functions:.

In the example of <FIG>, an external sensor module <NUM> may be provided for sensing droplet properties. For example, optical sensors as are known in the art may be employed as external sensors for sensing droplet properties. Suitable optical sensors include camera devices, light sensors, charged coupled devices (CCDs) and image similar image sensors, and the like. A sensor alternatively may be configured as internal sensor circuitry incorporated as part of the drive circuitry in each array element. Such sensor circuitry may sense droplet properties by the detection of an electrical property at the array element, such as impedance or capacitance.

The control system, such as via the control electronics <NUM>, may supply and control the actuation voltages applied to the electrode array of the microfluidics device <NUM>, such as required voltage and timing signals to perform droplet manipulation operations and sense liquid droplets on the AM-EWOD device <NUM>. The control electronics further may execute the application software to generate and output control voltages for droplet sensing and performing sensing operations. The reader <NUM> and cartridge <NUM> may be electrically connected together while in use, for example by a cable of connecting wires <NUM>, although various other methods (e.g. wireless connection) of providing electrical communication may be used as are known to those of ordinary skill in the art.

<FIG> is a drawing depicting additional details of the exemplary AM-EWOD device <NUM> in schematic perspective. The AM-EWOD device <NUM> has a lower substrate <NUM> with thin film electronics <NUM> disposed upon the lower substrate <NUM>. The thin film electronics <NUM> are arranged to drive array element electrodes <NUM>. A plurality of array element electrodes <NUM> are arranged in an electrode or element array <NUM>, having X by Y array elements where X and Y may be any integer. A liquid droplet <NUM> which may include any polar liquid and which typically may be aqueous, is enclosed between the lower substrate <NUM> and a top substrate <NUM> separated by a spacer <NUM>, although it will be appreciated that multiple liquid droplets <NUM> can be present.

<FIG> is a drawing depicting a cross section through some of the array elements of the exemplary AM-EWOD <NUM> device of <FIG>. In the portion of the AM-EWOD device depicted in <FIG>, the device includes a pair of the array element electrodes 48A and 48B that are shown in cross section that may be utilized in the electrode or element array <NUM> of the AM-EWOD device <NUM> of <FIG>. The device configuration is similar to the conventional configuration shown in <FIG>, with the AM-EWOD device <NUM> further incorporating the thin-film electronics <NUM> disposed on the lower substrate <NUM>, which is separated from the upper substrate <NUM> by the spacer <NUM>. The uppermost layer of the lower substrate <NUM> (which may be considered a part of the thin film electronics layer <NUM>) is patterned so that a plurality of the array element electrodes <NUM> (e.g. specific examples of array element electrodes are 48A and 48B in <FIG>) are realized. The term element electrode <NUM> may be taken in what follows to refer both to the physical electrode structure <NUM> associated with a particular array element, and also to the node of an electrical circuit directly connected to this physical structure. A reference electrode <NUM> is shown in <FIG> disposed upon the top substrate <NUM>, but the reference electrode alternatively may be disposed upon the lower substrate <NUM> to realize an in-plane reference electrode geometry. The term reference electrode <NUM> may also be taken in what follows to refer to both or either of the physical electrode structure and also to the node of an electrical circuit directly connected to this physical structure.

In the AM-EWOD device <NUM>, a non-polar fluid <NUM> (e.g. oil) may be used to occupy the volume not occupied by the liquid droplet <NUM>. An insulator layer <NUM> may be disposed upon the lower substrate <NUM> that separates the conductive element electrodes 48A and 48B from a first hydrophobic coating <NUM> upon which the liquid droplet <NUM> sits with a contact angle <NUM> represented by θ. The hydrophobic coating is formed from a hydrophobic material (commonly, but not necessarily, a fluoropolymer). On the top substrate <NUM> is a second hydrophobic coating <NUM> with which the liquid droplet <NUM> may come into contact. The reference electrode <NUM> is interposed between the top substrate <NUM> and the second hydrophobic coating <NUM>.

<FIG> shows a circuit representation of the electrical load 70A between the element electrode <NUM> and the reference electrode <NUM> in the case where a liquid droplet <NUM> is present. The liquid droplet <NUM> can usually be modeled as a resistor and capacitor in parallel. Typically, the resistance of the droplet will be relatively low (e.g. if the droplet contains ions) and the capacitance of the droplet will be relatively high (e.g. because the relative permittivity of polar liquids is relatively high, e.g. ~<NUM> if the liquid droplet is aqueous). In many situations the droplet resistance is relatively small, such that at the frequencies of interest for electro-wetting, the liquid droplet <NUM> may function effectively as an electrical short circuit. The hydrophobic coatings <NUM> and <NUM> have electrical characteristics that may be modelled as capacitors, and the insulator <NUM> may also be modelled as a capacitor. The overall impedance between the element electrode <NUM> and the reference electrode <NUM> may be approximated by a capacitor whose value is typically dominated by the contribution of the insulator <NUM> and hydrophobic coatings <NUM> and <NUM> contributions, and which for typical layer thicknesses and materials may be on the order of a pico-Farad in value.

<FIG> shows a circuit representation of the electrical load 70B between the element electrode <NUM> and the reference electrode <NUM> in the case where no liquid droplet is present. In this case the liquid droplet components are replaced by a capacitor representing the capacitance of the non-polar fluid <NUM> which occupies the space between the top and lower substrates. In this case the overall impedance between the element electrode <NUM> and the reference electrode <NUM> may be approximated by a capacitor whose value is dominated by the capacitance of the non-polar fluid and which is typically small, of the order of femto-Farads.

For the purposes of driving and sensing the array elements, the electrical load 70A/70B overall functions in effect as a capacitor, whose value depends on whether a liquid droplet <NUM> is present or not at a given element electrode <NUM>. In the case where a droplet is present, the capacitance is relatively high (typically of order pico-Farads), whereas if there is no liquid droplet present the capacitance is low (typically of order femto-Farads). If a droplet partially covers a given electrode <NUM> then the capacitance may approximately represent the extent of coverage of the element electrode <NUM> by the liquid droplet <NUM>.

<FIG> is a drawing depicting an exemplary arrangement of thin film electronics <NUM> in the exemplary AM-EWOD device <NUM> of <FIG> in accordance with embodiments of the present invention. The thin film electronics <NUM> is located upon the lower substrate <NUM>. Each array element <NUM> of the array of elements <NUM> contains an array element circuit <NUM> for controlling the electrode potential of a corresponding element electrode <NUM>. Integrated row driver <NUM> and column driver <NUM> circuits are also implemented in thin film electronics <NUM> to supply control signals to the array element circuit <NUM>. The array element circuit <NUM> may also contain a sensing capability for detecting the presence or absence of a liquid droplet in the location of the array element. Integrated sensor row addressing <NUM> and column detection circuits <NUM> may further be implemented in thin film electronics for the addressing and readout of the sensor circuitry in each array element.

A serial interface <NUM> may also be provided to process a serial input data stream and facilitate the programming of the required voltages to the element electrodes <NUM> in the array <NUM>. A voltage supply interface <NUM> provides the corresponding supply voltages, top substrate drive voltages, and other requisite voltage inputs as further described herein. A number of connecting wires <NUM> between the lower substrate <NUM> and external control electronics, power supplies and any other components can be made relatively few, even for large array sizes. Optionally, the serial data input may be partially parallelized. For example, if two data input lines are used the first may supply data for columns <NUM> to X/<NUM>, and the second for columns (<NUM>+X/<NUM>) to M with minor modifications to the column driver circuits <NUM>. In this way the rate at which data can be programmed to the array is increased, which is a standard technique used in Liquid Crystal Display driving circuitry.

Generally, an exemplary AM-EWOD device <NUM> that includes thin film electronics <NUM> may be configured as follows. The AM-EWOD device <NUM> includes the reference electrode <NUM> mentioned above (which, optionally, could be an in-plane reference electrode) and a plurality of individual array elements <NUM> on the array of elements <NUM>, each array element <NUM> including an array element electrode <NUM> and array element circuitry <NUM>. Relatedly, the AM-EWOD device <NUM> may be configured to perform a method of actuating the array elements to manipulate liquid droplets on the array by controlling an electro-wetting voltage to be applied to a plurality of array elements. The applied voltages may be provided by operation of the control system described as to <FIG>, including the control electronics <NUM> and applications and data stored on the storage device <NUM>. The electro-wetting voltage at each array element <NUM> is defined by a potential difference between the array element electrode <NUM> and the reference electrode <NUM>. The method of controlling the electro-wetting voltage at a given array element typically includes the steps of supplying a voltage to the array element electrode <NUM>, and supplying a voltage to the reference electrode <NUM>, by operation of the control system.

<FIG> is a drawing depicting an exemplary arrangement of the array element circuit <NUM> present in each array element <NUM>, which may be used as part of the thin film electronics of <FIG>. The array element circuit <NUM> may contain an actuation circuit <NUM>, having inputs ENABLE, DATA and ACTUATE, and an output which is connected to an element electrode <NUM>. The array element circuit <NUM> also may contain a droplet sensing circuit <NUM>, which may be in electrical communication with the element electrode <NUM>. Typically, the read-out of the droplet sensing circuit <NUM> may be controlled by one or more addressing lines (e.g. RW) that may be common to elements in the same row of the array, and may also have one or more outputs, e.g. OUT, which may be common to all elements in the same column of the array.

The array element circuit <NUM> may typically perform the functions of:.

Exemplary configurations of array element circuits <NUM> including integrated impedance sensor circuitry are known in the art, and for example are described in detail in <CIT> referenced in the background art section, and commonly assigned <CIT> (application No. <CIT>). These patent documents include descriptions of how the droplet may be actuated (by means of electro-wetting) and how the droplet may be sensed by integrated capacitive or impedance sensing circuitry. Typically, capacitive and impedance sensing may be analogue and may be performed simultaneously, or near simultaneously, at every element in the array. By processing the returned information from such a sensor (for example in the application software in the storage device <NUM> of the reader <NUM>), the control system described above can determine in real-time, or almost real-time the position, size or volume, centroid and perimeter of each liquid droplet present in the array of elements <NUM>. As referenced in connection with <FIG>, an alternative to sensor circuitry is to provide an external sensor (e.g., sensor <NUM>), such as an optical sensor that can be used to sense droplet properties.

As referenced above, the present invention pertains to a microfluidic system including in an AM-EWOD device by which electrowetting forces are applied to a fluid reservoir to form a barrier droplet configuration to act as a barrier to trap, obstruct, or otherwise prevent migration of a dissolved or suspended species in a source droplet through the surrounding non-polar fluid (oil) medium. The following terms are used herein.

A "migrating species" is any dissolved chemical species, ion, molecule, or suspended particle, that is able to migrate by any means, including diffusion, out of a source droplet in which the species is originally located, through the surrounding non-polar fluid (oil), and thereby potentially into other droplets, structures, or other protected areas on the EWOD device that could be contaminated or damaged by the migrating species. The migrating species may have a preference for the polar or aqueous environment of the barrier droplet, or instead for the non-polar environment of the non-polar fluid (oil). The preference of the migrating species may be defined as having a partition coefficient between the two phases that is not equal to one.

A "barrier droplet" is a polar or aqueous droplet dispensed on an EWOD device from a fluid reservoir that obstructs a large proportion of potential pathways of migration of the migrating species. In exemplary embodiments, the migrating species has a high affinity for residence in the barrier droplet as compared to the surrounding oil, or for residence in the surrounding oil as compared to the barrier droplet, and this affinity may be further enhanced by the presence in the barrier droplet of one or more additives such as capturing agents. The barrier droplet's shape or position may be held constant by the selective actuation of corresponding array elements of the EWOD device. The EWOD device also may be used to reposition or reshape the barrier droplet, or to open and shut passages to allow or prevent the movement of the migrating species, or to merge with other droplets including other barrier droplets. The barrier droplet may be formed and manipulated in such manners as a single barrier droplet or as part of an ensemble of a plurality of barrier droplets.

A "barrier droplet configuration" is an arrangement that includes one or more barrier droplets, and optionally further may include additional barrier elements that are not barrier droplets.

A "capturing agent" is any chemical species, suspended particle or surface coating that may be located within the barrier droplet, that is able to restrict or prevent migration of the migrating species by reacting with, binding to, or removing the migrating species, or by converting the migrating species to another form. Examples of capturing agents may include magnetic beads, chemical scavengers, chelating agents, nanoparticles, ion exchange resins, supramolecular cages, pH buffers, microorganisms or the like.

As referenced above, AM-EWOD device electrowetting forces are applied to a fluid reservoir to form a barrier droplet configuration to act as a barrier to trap, obstruct, or otherwise prevent migration of a dissolved or suspended species in a source droplet through the surrounding oil medium. For example, the barrier droplet configuration may be positioned between a source droplet containing an acid and a second droplet whose pH must be kept high. Without the barrier droplet configuration, acid is able to migrate from the source droplet, through the oil, and into the high-pH second droplet. The barrier droplet configuration acts to restrict the migration of acid (or whatever migrating species the source droplet contains) so that high concentrations of the migrating species may be stored on-chip in the presence of, for example, species-sensitive components or reaction areas on the device array.

The barrier droplet configuration operates by exploiting a difference in preference of the migrating species for the polar or aqueous environment of the barrier droplet versus the non-aqueous environment of the non-polar fluid (oil). On encountering an oil/water boundary at an aqueous barrier droplet, the migrating species partitions itself between the oil and the water. Consider an example whereby a source droplet containing a high concentration of the migrating species is near an area that must be protected from the migrating species. This protected area may be where a chemical reaction takes place that may be sensitive to the presence of the migrating species, an electronic component that could be damaged by the migrating species, or another droplet or fluid reservoir that stores a reagent or another composition that may be contaminated by the migrating species. At the boundary between the source droplet and the oil, a portion of the migrating species will escape the source droplet and enter the oil. In the absence of a barrier droplet configuration, the migrating species may then move across the oil until the species reaches the area to be protected. If the system is closed, over time a state of dynamic equilibrium may be reached in which the concentration of the migrating species in the area to be protected is unacceptably high.

To prevent contamination of the protected area by the migrating species, a barrier droplet configuration is formed by electrowetting forces and positioned between the source droplet and the protected area. For effective obstruction of migration pathways, a concentration of the migrating species in the barrier droplet(s) of the barrier droplet configuration is lower than a concentration of the migrating species in the source droplet. Under such condition, the rate of migration out of a side of the barrier droplet opposite from the source droplet is lowered simply by dilution effects in accordance with Fick's diffusion laws. The barrier droplet configuration thus provides an additional set of oil/water interfaces, and may act as an additional thermodynamic sink for the migrating species, and obstructs a portion of the potential pathways of migration, thereby reducing the rate of migration of the species to the protected area. By reducing the rate at which the migrating species enters the protected area, the useful lifetime of the reaction system on the AM-EWOD device is extended.

To be effective, the barrier droplet configuration should obstruct a significant portion of the potential pathways of migration to the protected area. The obstruction may be performed using a single barrier droplet or an ensemble of a plurality of barrier droplets. To achieve this requisite obstruction without the barrier droplet(s) becoming unmanageably large, electrowetting forces applied by the AM-EWOD device are applied to control the shape and position of the barrier droplet(s) that form the barrier droplet configuration. An AM-EWOD device can be used to prepare a barrier droplet configuration that completely surrounds the source droplet, or otherwise maintains a high aspect ratio in the barrier droplet configuration such that surface tension otherwise renders the barrier droplet(s) unstable in the absence of the electrowetting forces. The barrier droplet configuration may be formed using structures and devices described with respect to <FIG>, including for example any control electronics and circuitry, sensing capabilities, and control systems including any processing device that executes computer application code stored on a non-transitory computer readable medium. The following figures illustrate various methods and configurations of forming and manipulating barrier droplet configurations. It will be appreciated that the following examples are not intended to be exhaustive, and other barrier droplet configurations may be employed.

<FIG> is a drawing depicting a first barrier droplet configuration, as illustrated with respect to a generalized depiction of an EWOD device array <NUM>. For simplicity, details of the structure the EWOD device array <NUM> are omitted, but again, the EWOD device array may be configured and controlled as described above with respect to <FIG>. <FIG> further illustrates a first droplet <NUM> and a barrier droplet <NUM> that is configured to restrict migration of a migrating species. In this example, the first droplet <NUM> is isolated from the rest of the EWOD device array <NUM> by a barrier droplet <NUM> that completely surrounds the first droplet <NUM>, thereby forming a first area <NUM> on the EWOD device array and a second area <NUM> on the EWOD device array that are separated from each other by the barrier droplet <NUM>. The configuration of the barrier droplet in this and subsequent embodiments, including shape and position, is formed using the electrowetting forces that are generated by the EWOD device array <NUM>. Such configuration generally is unstable, and the barrier droplet will therefore revert to a native state (essentially ellipsoid) when the electrowetting force is removed. In addition, electrowetting forces may be applied to reconfigure the shape and positioning of the barrier droplet as needed to protect other areas of the EWOD device array, and/or to remove the barrier effect without affecting other droplets as warranted.

The first droplet <NUM> may be a source droplet that contains a high concentration of a migrating species, or a group of such source droplets. In such case, the second area <NUM> is a protected area that is protected from the migrating species that otherwise can migrate from the first droplet <NUM> through the first area <NUM>. Conversely, the first area <NUM> may be the protected area, whereby the first droplet <NUM> may be a droplet in said protective area that must be separated by the barrier droplet from a migrating species that migrates from the second area <NUM>. In this embodiment, because the barrier droplet <NUM> completely surrounds the first droplet <NUM>, the barrier droplet obstructs all possible migration pathways between the first and second areas <NUM>, <NUM> of the EWOD device array <NUM>. Accordingly, when the first droplet <NUM> is the source droplet, the second area <NUM> of the EWOD device array <NUM> is protected against migration out of the source droplet <NUM>. The barrier droplet and its contents may be static, or may be moved either separately or in unison. Electrowetting forces may be used to maintain the shape and/or position of the barrier droplet. By use of electrowetting patterns, it further is possible to open and close the barrier droplet <NUM>, which allows the migration to be turned on and off or channeled in a particular manner as between different areas of the EWOD device array.

<FIG> is a drawing depicting a second barrier droplet configuration. In this example configuration, the first droplet <NUM> is located in the second area <NUM> of the EWOD device. The barrier droplet <NUM> thus completely surrounds the first area <NUM>, such that in the event the droplet <NUM> constitutes a source droplet having a migrating species, the first area <NUM> is a protected area relative to the second area <NUM> containing the source droplet. As a protected area, the first area <NUM> is fully enclosed by the barrier droplet <NUM> and may contain another droplet, or a structure or component of the EWOD device, to be protected from the migrating species. The source droplet <NUM> may be any droplet or combination of droplets containing one or more migrating species that is able to migrate through the oil. Similarly as in the previous embodiment, because the barrier droplet <NUM> completely surrounds the first area <NUM>, the barrier droplet obstructs all possible migration pathways between the first and second areas <NUM>, <NUM> of the EWOD device array <NUM>. Also as in the previous embodiment, and in all subsequent embodiments, the barrier droplet and its contents may be static, or may be moved either separately or in unison. Electrowetting forces further may be used to maintain the shape and/or position of the barrier droplet. By use of electrowetting patterns, it further is possible to open and close the barrier droplet, which allows the migration to be turned on and off or channeled in a particular manner as between different areas of the EWOD device array.

As referenced above, a concentration of the migrating species in the barrier droplet(s) of a barrier droplet configuration is lower than a concentration of the migrating species in the source droplet. Under such condition, the rate of migration out of a side of the barrier droplet opposite from the source droplet is lowered simply by dilution effects in accordance with Fick's diffusion laws. Additionally, the effectiveness of the barrier droplet may be enhanced by changing barrier droplet contents or makeup. Because of the make-up of the migrating species, at the oil/water interface of the aqueous barrier droplet, the migrating species will partition itself between the barrier droplet and the surrounding oil. Accordingly, an additive that enhances the preference for an aqueous medium relative to the non-polar fluid (oil), or enhances the preference for an oil medium over a polar or aqueous one, aids to slow migration through the oil across the EWOD device. For example:.

By merging the barrier droplet with other droplets containing a capturing agent or other additive, a supply of the additive or capturing agent in the barrier droplet may be replenished, as the supply may become depleted over time as the concentration of migrating species in the barrier droplet increases. This strategy allows a reduction in the concentration of the additive or capturing agent that must be stored in the barrier droplet, which may be beneficial if the additive or capturing agent itself poses a migration risk.

In accordance with such features, <FIG> is a drawing depicting a third barrier droplet configuration that is comparable to the embodiment of <FIG>, in which a barrier droplet <NUM> further includes a capturing agent or other additive <NUM>. In this embodiment, the first droplet <NUM> located in the first area <NUM> of the EWOD device is isolated from the rest of the EWOD device (second area <NUM>) by a barrier droplet <NUM> that completely surrounds the first droplet <NUM>. In this example, the barrier droplet <NUM> contains one or more capturing agents <NUM> that react with or bind to a migrating species, or otherwise convert the migrating species into another form. Again in this embodiment, because the barrier droplet <NUM> completely surrounds the first droplet <NUM>, the barrier droplet obstructs all possible migration pathways between the first and second areas <NUM>, <NUM> of the EWOD device array <NUM>. The first droplet <NUM> may be a source droplet that contains a high concentration of a migrating species, or a group of such source droplets. In such case, the second area <NUM> is a protected area that is protected from migrating species that can migrate from the first droplet <NUM> through the first area <NUM>. Conversely, the first area <NUM> may be the protected area, whereby the first droplet <NUM> may be a droplet in said protective area that must be separated by the barrier droplet from a migrating species that migrates from the second area <NUM>. In addition, although this embodiment has been described principally in connection with the use of a capturing agent, the substance <NUM> may be any additive that adjusts the preference for an aqueous or non-aqueous media that would help to slow migration through the oil and across the EWOD device.

<FIG> is a drawing depicting a fourth barrier droplet configuration that employs an ensemble of a plurality of barrier droplets, including in this example a first barrier droplet <NUM> and a second barrier droplet <NUM>. The use of multiple barrier droplets provides enhanced obstruction of the migration pathways. For this configuration, the first barrier droplet <NUM> and the second barrier droplet <NUM> are concentric barrier droplets to isolate the first area <NUM> from the second area <NUM> of the EWOD device. As in previous embodiments, the first droplet <NUM> may be a source droplet containing a high concentration of a migrating species, or a group of source droplets, or be another droplet or a device structure that must be protected from migrating species from outside the barrier droplets. The makeup or contents of the individual barrier droplets may be the same or different from one another. In addition, one or more of the barrier droplets may contain one or more additives or capturing agents as described above. In this embodiment, because the barrier droplets form a closed concentric configuration around the source droplet <NUM>, the barrier droplets obstruct all possible migration pathways between the first and second areas <NUM>, <NUM> of the EWOD device array <NUM>. The barrier droplets further may be moved or reconfigured individually or in unison, including to open and close pathways to turn migration on and off.

<FIG> is a drawing depicting a fifth barrier droplet configuration, which also employs an ensemble of a plurality of barrier droplets, including in this example a first barrier droplet <NUM> and a second barrier droplet <NUM>. For this configuration, a first droplet <NUM> is enclosed by the first barrier droplet <NUM>, and a second droplet <NUM> is enclosed by the second barrier droplet <NUM>. In this manner, the two droplets <NUM> and <NUM> are isolated from each other, as well as from the remainder of the EWOD device array. One of the droplets <NUM> or <NUM> may be a source droplet containing a high concentration of a migrating species, or a group of source droplets. The other of the droplets <NUM> or <NUM> may be a droplet to be protected from the migrating species, or an area, structure, or component of the EWOD device that needs to be protected from the migrating species. Similarly, both droplets may be source droplets containing different migrating species such that each droplet is to be protected from the migrating species of the other droplet. The makeup or contents of the individual barrier droplets <NUM> and <NUM> may be the same or different from one another, and in particular may depend on the contents of the droplets <NUM> and <NUM> to properly either permit or restrict migration of a given migrating species. Each of the barrier droplets further many include a capturing agent or other additive as described above. In addition, because the barrier droplets form closed configurations around the two droplets <NUM> and <NUM>, the barrier droplets obstruct all possible migration pathways between the droplets and other areas of the EWOD device array <NUM>. The barrier droplets further may be moved or reconfigured individually or in unison, including to open and close pathways to turn migration on and off.

<FIG> is a drawing depicting a sixth barrier droplet configuration, which also employs an ensemble of a plurality of barrier droplets, which in essence combines the configurations of <FIG> and <FIG>. As to the first droplet <NUM>, the barrier configuration employs a concentric, dual barrier droplet configuration of barrier droplets <NUM> and <NUM> similarly as in <FIG>. As to the second droplet <NUM>, the barrier droplet configuration employs a single closed barrier droplet <NUM>, comparably as in <FIG>. The embodiment of <FIG> otherwise is comparable to the previous embodiments with respect to such optional features as the use of capturing agents or additives, and the manner by which the barrier droplets may be moved or reconfigured individually or in unison, including to open and close pathways to turn migration on and off.

In previous embodiments, the barrier droplets are manipulated by the electrowetting forces to form closed barrier structures, thereby obstructing essentially all migration pathways between different areas of the EWOD device array. In certain circumstances, however, it may be sufficient to obstruct only a portion of migration pathways, such as for example when other droplets, structures, or device areas to be protected are located only at certain positions relative to the source droplet. Accordingly, a fully closed barrier configuration may not be warranted. In this regard, <FIG> is a drawing depicting a seventh barrier droplet configuration, which employs a barrier droplet <NUM> that only partially surrounds a first droplet <NUM>. The constituents of the barrier droplet <NUM> may be comparable as in previous embodiments, including the use of any capturing agents or other additives. With the barrier droplet <NUM> only partially surrounding the first droplet <NUM>, the first area <NUM> of the EWOD device array <NUM> is only partially isolated from the second area <NUM>. In this embodiment, a large proportion of possible migration pathways between the device areas <NUM> and <NUM> remain obstructed, although a migration pathway remains where the barrier droplet <NUM> is open to the second area <NUM>. Based upon a particular position of an object to be protected within the device area <NUM>, such partial obstruction of migration pathways may be sufficient.

The barrier droplet may be reconfigured using the electrowetting forces to alter permitted migration pathways versus obstructed migration pathways as may be suitable for a given reaction protocol. For example, the location of reagents or given reaction steps may be at different areas on the EWOD device array, Accordingly, the barrier droplet may be reconfigured as necessary to change the migration pathways in correspondence with performing different steps of a reaction protocol.

<FIG> is a drawing depicting an eighth barrier droplet configuration, in which a barrier droplet is used in combination with an additional non-droplet, barrier element to determine the obstructed migration pathways. The configuration of <FIG> is comparable to that of <FIG>, with the additional use of a barrier element <NUM> that is not formed by manipulating a droplet with electrowetting forces. The barrier element <NUM> may be an actual physical barrier that is a part of the EWOD device, such as a solid structure or a channel wall, or the barrier element may be a hydrogel or any other structure that may impede migration by a different mechanism from a barrier droplet that is manipulated by electrowetting forces. The combination of a barrier droplet <NUM> and additional barrier element <NUM> may wholly or partially separate device areas <NUM> and <NUM> as described in accordance with previous embodiments. In this embodiment, the barrier droplet may be moved and otherwise reconfigured to adjust the migration pathways, whereas the barrier element <NUM> is largely fixed or would have to be manipulated by more manual means that are independent of the electrowetting operations of the EWOD device.

<FIG> is a drawing depicting a ninth barrier droplet configuration that is a variation of the embodiment of <FIG>. In this embodiment, the additional barrier element is an edge <NUM> of the EWOD device array <NUM>. In this example, with the barrier droplet <NUM> otherwise enclosing the first droplet <NUM>, the barrier droplet <NUM> and the device edge <NUM> combine to form a barrier droplet configuration that obstructs all migration pathways between device areas <NUM> and <NUM>, i.e., to form a closed barrier configuration. <FIG> is a drawing depicting a tenth barrier droplet configuration that is a further variation of the embodiment of <FIG>, in which the device edge <NUM> that constitutes the barrier element is a corner edge. Although <FIG> and <FIG> illustrate a fully obstructive barrier configuration, the barrier droplet <NUM> alternatively may be configured to combine with the device edge <NUM> to only partially obstruct the migration pathways.

In previous embodiments, the barrier configuration is formed using a single barrier droplet. In other embodiments, the barrier configuration may be formed using an ensemble of a plurality of barrier droplets. Using electrowetting forces, it can be complex to devise and implement an EWOD driving scheme to form droplet shapes having numerous bends or turns, whereas it is simpler to form barrier droplets of more simple shapes. As an alternative, therefore, instead of shaping a single droplet into the barrier droplet configuration, multiple barrier droplets can be combined in a manner that each individual barrier droplet constitutes a segment or portion of the broader barrier droplet configuration.

In accordance with such principles, <FIG> is a drawing depicting an eleventh barrier droplet configuration, in which a plurality of barrier droplets is combined into the barrier droplet configuration. In this example, four barrier droplets <NUM> are combined to form the barrier droplet configuration that separates the first area <NUM> and the second area <NUM> of the EWOD device array <NUM>. The barrier droplets are combined into a barrier droplet configuration that surrounds the first droplet <NUM>. In this example, there are gaps in the barrier droplet configuration at adjacent barrier droplets, although these gaps can be eliminated by joining the individual barrier droplets with the electrowetting forces. Even with such gaps, a substantial portion of potential migration pathways are obstructed by the illustrated barrier droplet configuration. In addition, although four barrier droplets are depicted in this example, any suitable number of individual barrier droplets may be employed to form an ensemble of any suitable shape as warranted for a particular application. The individual barrier droplets further may have the same composition or different compositions, and may include capturing agents or other additives as in previous embodiments. The barrier droplets also may be manipulated as in previous embodiments using electrowetting forces either individually or in unison to modify the migration pathways between areas of the EWOD device array.

Multiple barrier droplets may be combined in numerous different arrangements to provide different barrier droplet configurations. For example, <FIG> is a drawing depicting a twelfth barrier droplet configuration having an alternative arrangement of a plurality of barrier droplets that are combined into the barrier droplet configuration. In this example, multiple barrier droplets <NUM> are formed, whereby each barrier droplet individually only partially encompasses or partially surrounds a portion of the first area <NUM> containing the first droplet <NUM>. The barrier droplets are layered and positioned so that gaps between an inner layer of the barrier droplets are obstructed by an outer layer of the barrier droplets, thereby essentially forming a barrier configuration that completely surrounds the first droplet <NUM>. In actual positioning, there remain continuous pathways between the first area <NUM> and the second area <NUM> that run between the layers of barrier droplets. In practice, however, the convoluted pathways between the first area <NUM> and the second area <NUM> would take significantly longer for a migrating species to traverse. Accordingly, the barrier configuration of <FIG> effectively provides full separation of device areas <NUM> and <NUM>, comparably as closed barrier configurations of previous embodiments.

In previous embodiments, barrier droplet configurations are formed that surround or encompass, at least partially, a first area of the EWOD device array, or otherwise extend across a substantial portion of the EWOD device array, to separate the first area from the second area of the EWOD device array. In alternative embodiments, a linear elongated, high aspect ratio barrier droplet can be formed to separate areas of the EWOD device array. A linear elongated, high aspect ratio barrier droplet is a barrier droplet configuration in which the barrier droplet spans a larger number of array elements in a first dimension than in a second dimension. For example, <FIG> is a drawing depicting a thirteenth barrier droplet configuration having a linear elongated barrier droplet <NUM>, which separates the first area <NUM> of the EWOD device from the second area <NUM>. The high aspect ratio is implemented with the barrier droplet <NUM> having a rectangular shape, with the length dimension being a substantial multiple of the width dimension. For example, the length dimension may be approximately an order of magnitude larger than the width dimension. The first droplet <NUM> is located within the first area <NUM>. Similarly as in previous embodiments, the first droplet <NUM> may be a source droplet containing a migrating species, or may be another droplet, device structure, or device area that is to be protected from a migrating species from the second area <NUM>. The linear elongated barrier droplet <NUM> may have a composition comparably as in other embodiments, including having a capturing agent or other additive that aids in obstruction of the migrating species. The linear elongated barrier droplet <NUM> further may be manipulated or reconfigured comparably as in previous embodiments, including in ways that alter the migration pathways to turn migration on and off. In the configuration of <FIG>, migration pathways may remain around the edges of the barrier droplet <NUM>, but such migration pathways may be considered negligible provided the expanse of the barrier droplet <NUM> extends over a substantial portion of a dimension (e.g., length or width) of the EWOD device array. <FIG> has an advantage that a single, elongated droplet tends to be easier to form with electrowetting operations than a non-standard shaped droplet having multiple bends and turns.

In another embodiment, an ensemble of a plurality of linear elongated barrier droplets may be employed to enhance the obstruction of the migration pathways between the first area <NUM> and the second area <NUM> of the EWOD device array. For example, <FIG> is a drawing depicting a fourteenth barrier droplet configuration having a plurality of linear elongated barrier droplets. In this example, the first linear elongated barrier droplet <NUM> is combined with a second linear elongated barrier droplet <NUM> to enhance obstruction of the migration pathways, although any suitable number of linear elongated barrier droplets may be employed. As in previous embodiments including a plurality of barrier droplets, the composition of each individual barrier droplet may be the same or different as may be suitable for any particular application. In particular, one or more of the linear elongated barrier droplets may include a capturing agent or other additive that enhances obstruction of the migration pathways. For example, <FIG> is a drawing depicting a fifteenth barrier droplet configuration having a plurality of linear elongated barrier droplets, wherein the first linear elongated barrier droplet <NUM> includes an additive <NUM>, such as a capturing agent. It will be appreciated that in such an ensemble of linear elongated barrier droplets, any one or other number, up to all, of the barrier droplets may include a capturing agent or other additive, which may be the same or different in the different barrier droplets.

In the embodiments of <FIG>, the droplet barrier configuration is formed as one or more linear elongated barrier droplets. As an alternative embodiment, <FIG> is a drawing depicting a sixteenth barrier droplet configuration, in which the barrier configuration is formed as an ensemble of a plurality of individual droplets that are linearly arranged. In this example, a plurality of individual droplets <NUM> are linearly arranged to separate the first area <NUM> from the second area <NUM> of the EWOD device array. This arrangement has a comparable performance as the embodiment of <FIG> having a single linear elongated barrier droplet. The embodiment of <FIG> may be advantageous in that there is better obstruction of the migration pathways, insofar as gaps are present between the individual droplets <NUM> in the embodiment of <FIG>. In contrast, the embodiment of <FIG> may be advantageous in that the EWOD driving scheme is simpler to generate and position relatively smaller individual droplets of more standard or native shape that the droplet has in absence of electrowetting forces (e.g., ellipsoid), as compared to generating relatively larger droplets of more non-standard elongated shapes that extend over a substantial portion of the EWOD device array.

Similarly as with the embodiment of <FIG>, multiple layers of droplets positioned in a linear arrangement may be employed to enhance the obstruction of the migration pathways. For example, <FIG> is a drawing depicting a seventeenth barrier droplet configuration having a plurality of layers of individual droplets that are linearly arranged. In this example, the first layer of individual droplets <NUM> is combined with a second layer of individual droplets <NUM> that also are linearly arranged. The droplets <NUM> and the droplets <NUM> may be offset relative to each other so as to enhance the obstruction of the migration pathways. With such arrangement, the droplets <NUM> are positioned at the gaps between the droplets <NUM> (and inherently vice versa), such that the resultant migration pathways require directional changes in the migration to move between the device areas <NUM> and <NUM>. As referenced above, migration along such multi-directional pathways tends not to occur given the nature of the migration of typical migrating species. Accordingly, the barrier configuration of <FIG> effectively provides comparable separation of areas <NUM> and <NUM> as the configuration of <FIG>, but with the more easily generated standard or natively shaped ellipsoid individual droplets rather than the linear elongated droplets.

As referenced above, it may be advantageous to form droplet configurations using patterns of smaller individual ellipsoid or natively shaped droplets rather than larger non-standard shaped droplets, in that the EWOD driving scheme is simpler to generate and position smaller individual droplets of more standard shape (e.g., ellipsoid), as compared to generating larger droplets of more non-standard shapes that singularly span a large portion of the EWOD device array. Accordingly, previous embodiments employing larger barrier droplets of non-standard shape may be modified to form comparable barrier configurations that include an arrangement of individual smaller droplets of standard or native (ellipsoid) shape. In such embodiments, any suitable number of individual droplets may be employed to form an ensemble of any suitable overall shape as warranted for a particular application. The individual barrier droplets further may have the same composition or different compositions, and may include capturing agents or other additives as in previous embodiments. The barrier droplets also may be manipulated as in previous embodiments using electrowetting forces either individually or in unison to modify the migration pathways between areas of the EWOD device array.

For example, <FIG> is a drawing depicting an eighteenth barrier droplet configuration that forms a barrier configuration comparable to that of <FIG>, but modified whereby a plurality of individual droplets <NUM> are arranged to completely surround the first area <NUM> of the EWOD device array. <FIG> is a drawing depicting a nineteenth barrier droplet configuration that forms a barrier configuration comparable to that of <FIG>, but modified whereby a plurality of individual droplets <NUM> are arranged to partially surround the first area <NUM> of the EWOD device array. <FIG> is a drawing depicting a twentieth barrier droplet configuration that forms a barrier configuration comparable to that of <FIG>, but modified whereby a plurality of individual droplets <NUM> are arranged to partially surround the first area <NUM> of the EWOD device array, and further combining with an edge <NUM> of the EWOD device array acting as a barrier element. <FIG> is a drawing depicting a twenty-first barrier droplet configuration that forms a barrier configuration comparable to that of <FIG>, but modified whereby a plurality of individual droplets <NUM> are arranged to partially surround the first area <NUM> of the EWOD device array, and further combining with a corner edge <NUM> of the EWOD device array acting as a barrier element. <FIG> is a drawing depicting a twenty-second barrier droplet configuration that forms a barrier configuration comparable to that of <FIG>, but modified whereby a plurality of individual droplets <NUM> are arranged to partially surround the first area <NUM> of the EWOD device array, and further combining with an additional barrier element <NUM>, such as a physical wall or channel, hydrogel, or any other structure that may impede migration by a different mechanism from a barrier droplet that is manipulated by electrowetting forces. <FIG> is a drawing depicting a twenty-third barrier droplet configuration that forms a barrier configuration comparable to that of <FIG>, but using two layers of individual barrier droplets 152a and 152b arranged to partially surround the first area <NUM>. <FIG> is a drawing depicting a twenty-fourth barrier droplet configuration that forms a barrier configuration comparable to that of <FIG>, but using two layers of individual barrier droplets 152a and 152b arranged to partially surround the first area <NUM> in combination with the additional barrier element <NUM>.

In accordance with the above, a myriad of barrier droplet configurations may be devised using any suitable combinations of barrier droplet arrangements, shapes, and compositions (including any capturing agents or other additives), along with any additional barrier elements that are not barrier droplets. The following illustrates potential examples, although again it will be appreciated that such examples are not intended to be exhaustive. Like references numerals are used to identify analogous components.

<FIG> is a drawing depicting a twenty-fifth barrier droplet configuration, in which two offset layers of individual barrier droplets <NUM> partially surround the device area <NUM>, in combination with a corner edge <NUM> of the EWOD device array and an additional barrier element <NUM> that aid in obstruction of migration pathways. <FIG> is a drawing depicting a twenty-sixth barrier droplet configuration, in which a single linear elongated barrier droplet <NUM> partially surrounds the device area <NUM>, in combination with a corner edge <NUM> of the EWOD device array and an additional barrier element <NUM> that aid in obstruction of migration pathways. <FIG> is a drawing depicting a twenty-seventh barrier droplet configuration, in which two linear elongated barrier droplets <NUM> are opposingly positioned to partially surround the device area <NUM>, in combination with additional opposing barrier elements <NUM>, resulting in nearly complete surrounding of the device area <NUM>. <FIG> is a drawing depicting a twenty-eighth barrier droplet configuration, in which double offset layers of individual barrier droplets 152a and 152b are opposingly positioned to partially surround the device area <NUM>, in combination with additional opposing barrier elements <NUM>, resulting in nearly complete surrounding of the device area <NUM>. <FIG> is a drawing depicting a twenty-ninth barrier droplet configuration, in which an array of offset individual barrier droplets <NUM> are positioned to partially surround the device area <NUM>, in combination with an additional barrier element <NUM>, with the barrier element <NUM> being shaped to form a receptacle having an opening at which the barrier droplets <NUM> are positioned to obstruct migration pathways between the device area <NUM> located within the receptacle and the device area <NUM> external from the receptacle. <FIG> is a drawing depicting a thirtieth barrier droplet configuration comparable to that of <FIG>, with the barrier configuration including a single linear elongated barrier droplet <NUM> rather than an array of individual barrier droplets.

In certain reaction protocols, interaction between droplets may be undesirable at certain reaction stages or steps, but desirable or required at other reaction stages or steps. Under such circumstances, barrier droplet configurations may be formed to obstruct migration pathways during certain portions of the reaction protocol, and then are reconfigured by electrowetting forces to permit droplet mixing or other interaction during other portions of the reaction protocol. For example, <FIG> is a drawing depicting a thirty-first barrier droplet configuration, by which a barrier droplet configuration <NUM> has been formed. The barrier droplet <NUM> encloses a first droplet <NUM> located within a device area <NUM>, and a second droplet <NUM> located within a device area <NUM>. The barrier droplet configuration <NUM> includes an outer barrier portion <NUM> that obstructs migration pathways between the devices areas <NUM> and <NUM> and the device area <NUM>, thereby obstructing migration pathways between the droplets <NUM> and <NUM> and the remainder of the EWOD device array. In addition, the barrier droplet configuration <NUM> includes an inner barrier portion <NUM> that obstructs migration pathways between the device areas <NUM> and <NUM> themselves, thereby obstructing migration pathways between the first droplet <NUM> and the second droplet <NUM>. Accordingly, in the state depicted in <FIG>, mixing of constituents of the droplets <NUM> and <NUM> is obstructed by the presence of the inner barrier portion <NUM>. During a reaction protocol, there may come a reaction stage or phase at which it becomes desirable for the droplets <NUM> and <NUM> to mix or otherwise interact. As such stage, electrowetting forces may be applied to retract the inner barrier portion <NUM>, such as by moving the droplet material <NUM> into the outer barrier portion <NUM>, thereby opening the areas <NUM> and <NUM> relative to each other. Obstruction of migration between the two droplets is thus removed, and electrowetting forces then may be applied to droplet <NUM> and/or droplet <NUM> to perform a suitable mixing or interaction of the two droplets, or the droplets may interact by ordinary diffusion.

In the example of <FIG>, the barrier droplet configuration <NUM> is configured as a single barrier droplet that is configured into the desired shape. Other barrier droplet arrangements may be employed, such as described with respect to previous embodiments. For example, <FIG> is a drawing depicting a thirty-second barrier droplet configuration, in which the inner barrier portion <NUM> is configured as a double layer of offset individual barrier droplets. As another example, <FIG> is a drawing depicting a thirty-third barrier droplet configuration, in which the inner barrier portion <NUM> is configured as single linear elongated barrier droplet, which is formed separately from the outer barrier portion <NUM>. The configuration of <FIG> and <FIG> can be formed using simpler EWOD driving schemes as compared to the configuration of <FIG>.

In the examples of <FIG>, the barrier droplet configuration is formed so as to separate two droplets during certain reaction stages, with a retractable portion that is retracted to permit droplet interaction at other reaction stages. Comparable principles may be expanded to apply to any number of droplets as may be suitable for a given reaction protocol. For example, <FIG> is a drawing depicting a thirty-fourth barrier droplet configuration, by which a barrier droplet configuration <NUM> has been formed that can control interaction among three droplets. Accordingly, a third droplet <NUM> is located within a device area <NUM>. In addition, the barrier droplet configuration <NUM> further includes a second inner barrier portion <NUM> in addition to the first inner barrier portion <NUM> of the previous embodiment, whereby the second barrier portion <NUM> obstructs migration pathways between the second droplet <NUM> and the third droplet <NUM>. The different portions of the barrier droplet configuration <NUM> may be configured in accordance with any of the embodiments. In addition, the different portions, including particularly the inner barrier portions <NUM> and <NUM>, may be retracted or otherwise reconfigured independently of each other as a particular reaction protocol may warrant.

An aspect of the invention, therefore, is a method of operating an electrowetting on dielectric (EWOD) device that includes an EWOD device array that applies electrowetting forces and contains a non-polar fluid, whereby a barrier droplet configuration is formed using electrowetting forces to obstruct migration of a species from a first area of the EWOD device array to a protected area of the EWOD device array. The method of operating includes the steps of: dispensing a source droplet into a first area of the EWOD device array, the source droplet containing a migrating species (or constituent), wherein the EWOD device array includes a second area to be protected from the migrating species; and forming a barrier droplet configuration positioned between the first area and the second area of the EWOD device array that obstructs a migration pathway of the migrating species between the first area and the second area. The barrier droplet configuration includes at least one polar or aqueous barrier droplet, and the barrier droplet inhibits diffusion of the migrating species by the migrating species exhibiting a preference for either the polar environment of the barrier droplet or for the non-aqueous environment of the non-polar fluid, wherein the barrier droplet configuration completely surrounds the first area of the EWOD device array. The method of operating an EWOD device may include one or more of the following features, either individually or in combination.

In an exemplary embodiment of the method of operating an EWOD device, a concentration of the migrating species in the barrier droplet is lower than a concentration of the migrating species in the source droplet.

In an exemplary embodiment of the method of operating an EWOD device, the barrier droplet includes an additive that adjusts the preference of the migrating species for either the polar environment of the barrier droplet or the non-polar fluid.

In an exemplary embodiment of the method of operating an EWOD device, the additive comprises a capturing agent that reacts with or binds to the migrating species, or converts the migrating species into another form.

In an exemplary embodiment of the method of operating an EWOD device, the method further includes replenishing the additive of the barrier droplet.

In an exemplary embodiment of the method of operating an EWOD device, the barrier droplet configuration partially surrounds the second area of the EWOD device array that does not contain the source droplet.

In an exemplary embodiment of the method of operating an EWOD device, the barrier droplet configuration includes a plurality of barrier droplets.

In an exemplary embodiment of the method of operating an EWOD device, the barrier droplet configuration includes multiple concentric barrier droplets.

In an exemplary embodiment of the method of operating an EWOD device, the barrier droplet configuration includes multiple barrier droplets that in combination surround the first area of the EWOD device array.

In an exemplary embodiment of the method of operating an EWOD device, the barrier droplet configuration includes a first barrier droplet that surrounds the source droplet and a second barrier droplet that surrounds an area of the EWOD device array that may contain a second droplet.

In an exemplary embodiment of the method of operating an EWOD device, the second droplet includes a second migrating species that is different from the migrating species of the source droplet.

In an exemplary embodiment of the method of operating an EWOD device, the at least one barrier droplet comprises a barrier droplet that is linearly elongated to have a high aspect ratio in which the barrier droplet spans a first dimension that is an order of magnitude longer than a second dimension.

In an exemplary embodiment of the method of operating an EWOD device, the barrier droplet configuration comprises a plurality of individual droplets that are natively shaped and are arranged to obstruct the migration pathway.

In an exemplary embodiment of the method of operating an EWOD device, the plurality of individual droplets includes a first layer and a second layer, wherein individual droplets of the first layer are offset relative to individual droplets of the second layer.

In an exemplary embodiment of the method of operating an EWOD device, the barrier droplet configuration further comprises an additional barrier element that obstructs the migration pathway other than by forming a barrier droplet with the electrowetting forces.

In an exemplary embodiment of the method of operating an EWOD device, the additional barrier element comprises a physical barrier or hydrogel located on the EWOD device array.

In an exemplary embodiment of the method of operating an EWOD device, the additional barrier element comprises an edge of the EWOD device array.

In another exemplary embodiment of the method of operating an EWOD device, the method includes dispensing a first droplet into a first area of the EWOD device array; dispensing a second droplet into a second area of the EWOD device array; forming a barrier droplet configuration, wherein the barrier droplet configuration includes at least one polar or aqueous barrier droplet, and constituents of the first and second droplets exhibit a preference for either a polar environment of the barrier droplet or a non-aqueous environment of the non-polar fluid; wherein the barrier droplet configuration comprises a first portion that separates the first area from the second area, and a second portion that separates both the first and second areas from a third area of the EWOD device array, the method further comprising: maintaining the first portion of the barrier droplet configuration during a first stage of a reaction protocol, thereby obstructing a migration pathway between the first area and the second area during said first stage; and retracting the first portion of the barrier droplet configuration during a second stage of the reaction protocol, thereby opening the migration pathway between the first area and the second area to permit interaction of the first droplet and the second droplet during said second stage.

In an exemplary embodiment of the method of operating an EWOD device, the method further includes applying electrowetting voltages to the EWOD device array during the second stage of the reaction protocol to mix the first droplet and the second droplet.

According to another aspect of the invention, a microfluidic system includes an electro-wetting on dielectric (EWOD) device comprising an element array configured to receive one or more liquid droplets, the element array comprising a plurality of individual array elements; and a control system configured to control actuation voltages applied to the element array to perform manipulation operations as to the liquid droplets to perform the methods of operating the EWOD device that include forming a barrier droplet configuration. The microfluidic system further may include integrated impedance sensing circuitry that is integrated into the array elements of the EWOD device, and a configuration and position of source and barrier droplets dispensed onto the array elements is determined based on an impedance sensed by the impedance sensing circuitry.

Another aspect of the invention is a computer-readable medium storing program code which is executed by a processing device for controlling actuation voltages applied to array elements of an element array of an electro-wetting on dielectric (EWOD) device for performing droplet manipulations on droplets on the element array, the program code being executable by the processing device to perform steps of the methods of operating the EWOD device that include forming a barrier droplet configuration.

Although the invention has been shown and described with respect to a certain embodiment or embodiments and modifications may occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In particular regard to the various functions performed by the above described elements (components, assemblies, devices, compositions, etc.), the terms (including a reference to a "means") used to describe such elements are intended to correspond, unless otherwise indicated, to any element which performs the specified function of the described element (i.e., that is functionally equivalent), even though not structurally equivalent to the disclosed structure which performs the function in the herein exemplary embodiment or embodiments of the invention. In addition, while a particular feature of the invention may have been described above with respect to only one or more of several embodiments, such feature may be combined with one or more other features of the other embodiments, as may be desired and advantageous for any given or particular application.

Claim 1:
A method of operating an electrowetting on dielectric (EWOD) device that includes an EWOD device array that applies electrowetting forces and contains a non-polar fluid, the method of operating comprising the steps of:
dispensing a first droplet (<NUM>,<NUM>) into a first area (<NUM>) of the EWOD device array, the first droplet containing a constituent; and
forming a barrier droplet configuration positioned between the first area (<NUM>) of the EWOD device array and a second area (<NUM>) of the EWOD device array that obstructs a migration pathway of the constituent between the first area and the second area;
wherein the barrier droplet configuration includes at least one polar or aqueous barrier droplet (<NUM>, <NUM>, <NUM>,<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>), and the barrier droplet inhibits diffusion of the constituent by the constituent exhibiting a preference for either the polar environment of the barrier droplet or for the non-aqueous environment of the non-polar fluid;
wherein the barrier droplet configuration completely surrounds the first area (<NUM>) of the EWOD device array.