WO2010027894A2 - Droplet actuators, modified fluids and methods - Google Patents

Droplet actuators, modified fluids and methods Download PDF

Info

Publication number
WO2010027894A2
WO2010027894A2 PCT/US2009/055139 US2009055139W WO2010027894A2 WO 2010027894 A2 WO2010027894 A2 WO 2010027894A2 US 2009055139 W US2009055139 W US 2009055139W WO 2010027894 A2 WO2010027894 A2 WO 2010027894A2
Authority
WO
WIPO (PCT)
Prior art keywords
droplet
droplet actuator
actuator
following
hlb
Prior art date
Application number
PCT/US2009/055139
Other languages
French (fr)
Other versions
WO2010027894A3 (en
Inventor
Vijay Srinivasan
Vamsee Pamula
Ramakrishna Sista
Arjun Sudarsan
Prasanna Thwar
Original Assignee
Advanced Liquid Logic, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US9227808P priority Critical
Priority to US61/092,278 priority
Priority to US9489108P priority
Priority to US61/094,891 priority
Application filed by Advanced Liquid Logic, Inc. filed Critical Advanced Liquid Logic, Inc.
Publication of WO2010027894A2 publication Critical patent/WO2010027894A2/en
Publication of WO2010027894A3 publication Critical patent/WO2010027894A3/en
Priority claimed from US13/031,760 external-priority patent/US8658111B2/en

Links

Classifications

    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L13/00Cleaning or rinsing apparatus
    • B01L13/02Cleaning or rinsing apparatus for receptacle or instruments
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0809Geometry, shape and general structure rectangular shaped
    • B01L2300/0819Microarrays; Biochips
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/18Means for temperature control
    • B01L2300/1805Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1827Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0427Electrowetting
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/06Valves, specific forms thereof
    • B01L2400/0677Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/08Regulating or influencing the flow resistance
    • B01L2400/084Passive control of flow resistance
    • B01L2400/086Passive control of flow resistance using baffles or other fixed flow obstructions
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502746Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00Supporting devices; Holding devices
    • B01L9/52Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips

Abstract

The present invention provides droplet actuators, modified fluids and methods relating to droplet operations. An aspect includes a droplet actuator including a substrate comprising electrodes arranged for conducting droplet operations on a droplet operations surface of the substrate; a filler fluid phase in contact with the droplet operations surface at least partially surrounding a droplet phase including a droplet arranged on one or more of the electrodes, the filler fluid phase being divided by one or more physical barriers into zones; and an opening in the one or more physical barriers for transporting the droplet phase from one zone to another. Still other aspects are provided.

Description

Droplet Actuators, Modified Fluids and Methods Related Applications

In addition to the patent applications cited herein, each of which is incorporated herein by reference, this patent application is related to and claims priority to U.S. Provisional

Patent Application Nos. 61/092,278, filed on August 27, 2008, entitled "Droplet Actuators, Modified Fluids and Methods," and 61/094,891, filed on September 6, 2008, entitled "Droplet Actuators, Modified Fluids and Methods," the entire disclosures of which are incorporated herein by reference.

This patent application also relates to and incorporates by reference the entire disclosure of International Patent Application No. PCT/US08/72604, entitled "Use of Additives for Enhancing Droplet Actuation," filed on August 8, 2008; U.S. Patent Application No. 60/980,620, entitled "Use of Additives for Enhancing Droplet Actuation," filed on October 17, 2007; and U.S. Patent Application No. 60/954,587, entitled "Use of Additives for Enhancing Droplet Actuation," filed on August 8, 2007.

Field of the Invention

The present invention generally relates to the field of conducting droplet operations in a droplet actuator. In particular, the present invention is directed to droplet actuator designs and droplet actuator fluid compositions for enhancing droplet operations.

Background of the Invention

Droplet actuators are used to conduct a wide variety of droplet operations. A droplet actuator typically includes two substrates separated by a gap. The substrates include electrodes for conducting droplet operations. The space is typically filled with a filler fluid that is immiscible with the fluid that is to be manipulated on the droplet actuator, so that the droplet actuator includes a droplet phase in the form of a droplet at least partially bounded by a filler fluid phase consisting of the filler fluid. The formation and movement of the droplet phase droplets is controlled by electrodes, which can be employed to conduct a variety of droplet operations. Because different droplet phase fluids and droplet operations often require differences in filler fluid properties, and vice versa, there is a need for new droplet actuator designs and droplet actuator fluid compositions for enhancing droplet operations.

Brief Description of the Invention

The present invention is directed to droplet actuators, modified fluids and methods.

In one embodiment, a droplet actuator is provided comprising a substrate comprising electrodes arranged for conducting droplet operations on a droplet operations surface of the substrate; a filler fluid phase in contact with the droplet operations surface at least partially surrounding a droplet phase comprising a droplet arranged on one or more of the electrodes, the filler fluid phase being divided by one or more physical barriers into zones; and an opening in the one or more physical barriers for transporting the droplet phase from one zone to another.

In another embodiment, a droplet actuator is provided comprising a droplet operations substrate; an oil based filler fluid on the droplet operations substrate; and a droplet in contact with the oil based filler fluid forming an oil-droplet interface, the droplet comprising an aqueous soluble additive that has a hydrophile-lipophile balance (HLB) in the range of about 10 to about 20.

In yet another embodiment, a droplet actuator is provided comprising a droplet operations substrate; an oil based filler fluid on the droplet operations substrate; and a droplet in contact with the oil based filler fluid forming an oil-droplet interface, the droplet comprising an aqueous soluble additive and water soluble particles that do not bind to a significant quantity of a target substance.

In a further embodiment, a droplet actuator is provided comprising a droplet operations substrate; an oil based filler fluid on the droplet operations substrate comprising an oil soluble additive in the filler fluid; and a droplet in contact with the oil based filler fluid.

Definitions

As used herein, the following terms have the meanings indicated. "Adsorption" is the loss of substances from the droplet phase to solid surfaces of the droplet actuator.

"Activate" with reference to one or more electrodes means effecting a change in the electrical state of the one or more electrodes which results in a droplet operation.

"Bead," with respect to beads on a droplet actuator, means any bead or particle that is capable of interacting with a droplet on or in proximity with a droplet actuator. Beads may be any of a wide variety of shapes, such as spherical, generally spherical, egg shaped, disc shaped, cubical and other three dimensional shapes. The bead may, for example, be capable of being transported in a droplet on a droplet actuator or otherwise configured with respect to a droplet actuator in a manner which permits a droplet on the droplet actuator to be brought into contact with the bead, on the droplet actuator and/or off the droplet actuator. Beads may be manufactured using a wide variety of materials, including for example, resins, and polymers. The beads may be any suitable size, including for example, microbeads, microparticles, nanobeads and nanoparticles. In some cases, beads are magnetically responsive; in other cases beads are not significantly magnetically responsive. For magnetically responsive beads, the magnetically responsive material may constitute substantially all of a bead or one component only of a bead. The remainder of the bead may include, among other things, polymeric material, coatings, and moieties which permit attachment of an assay reagent. Examples of suitable magnetically responsive beads are described in U.S. Patent Publication No. 2005-0260686, entitled,

"Multiplex flow assays preferably with magnetic particles as solid phase," published on November 24, 2005, the entire disclosure of which is incorporated herein by reference for its teaching concerning magnetically responsive materials and beads. The fluids may include one or more magnetically responsive and/or non-magnetically responsive beads. Examples of droplet actuator techniques for immobilizing magnetic beads and/or nonmagnetic beads and/or conducting droplet operations protocols using beads are described in U.S. Patent Application No. 11/639,566, entitled "Droplet-Based Particle Sorting," filed on December 15, 2006; U.S. Patent Application No. 61/039,183, entitled "Multiplexing Bead Detection in a Single Droplet," filed on March 25, 2008; U.S. Patent Application No. 61/047,789, entitled "Droplet Actuator Devices and Droplet Operations

Using Beads," filed on April 25, 2008; U.S. Patent Application No. 61/086,183, entitled "Droplet Actuator Devices and Methods for Manipulating Beads," filed on August 5, 2008; International Patent Application No. PCT/US2008/053545, entitled "Droplet Actuator Devices and Methods Employing Magnetic Beads," filed on February 11, 2008; International Patent Application No. PCT/US2008/058018, entitled "Bead-based Multiplexed Analytical Methods and Instrumentation," filed on March 24, 2008; International Patent Application No. PCT/US2008/058047, "Bead Sorting on a Droplet Actuator," filed on March 23, 2008; and International Patent Application No.

PCT/US2006/047486, entitled "Droplet-based Biochemistry," filed on December 11, 2006; the entire disclosures of which are incorporated herein by reference.

"Carryover" occurs when substances that are lost from the droplet phase via, for example, adsorption and/or partitioning, make their way into another droplet phase (e.g., from one droplet phase droplet to another droplet phase droplet), resulting in droplet phase cross- contamination.

"Droplet" means a volume of liquid on a droplet actuator that is at least partially bounded by filler fluid. For example, a droplet may be completely surrounded by filler fluid or may be bounded by filler fluid and one or more surfaces of the droplet actuator. Droplets may, for example, be aqueous or non-aqueous or may be mixtures or emulsions including aqueous and non-aqueous components. Droplets may take a wide variety of shapes; nonlimiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more surfaces of a droplet actuator.

"Droplet Actuator" means a device for manipulating droplets. For examples of droplets, see U.S. Patent 6,911,132, entitled "Apparatus for Manipulating Droplets by Electrowetting-Based Techniques," issued on June 28, 2005 to Pamula et al.; U.S. Patent Application No. 11/343,284, entitled "Apparatuses and Methods for Manipulating Droplets on a Printed Circuit Board," filed on filed on January 30, 2006; U.S. Patents

6,773,566, entitled "Electrostatic Actuators for Microfluidics and Methods for Using Same," issued on August 10, 2004 and 6,565,727, entitled "Actuators for Microfluidics Without Moving Parts," issued on January 24, 2000, both to Shenderov et al.; Pollack et al., International Patent Application No. PCT/US2006/047486, entitled "Droplet-Based Biochemistry," filed on December 11 , 2006, the disclosures of which are incorporated herein by reference. Methods of the invention may be executed using droplet actuator systems, e.g., as described in International Patent Application No. PCT/US2007/009379, entitled "Droplet manipulation systems," filed on May 9, 2007. In various embodiments, the manipulation of droplets by a droplet actuator may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated.

"Droplet operation" means any manipulation of a droplet on a droplet actuator. A droplet operation may, for example, include: loading a droplet into the droplet actuator; dispensing one or more droplets from a source droplet; splitting, separating or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; retaining a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; condensing a droplet from a vapor; cooling a droplet; disposing of a droplet; transporting a droplet out of a droplet actuator; other droplet operations described herein; and/or any combination of the foregoing. The terms "merge," "merging," "combine," "combining" and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, "merging droplet A with droplet B," can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other. The terms "splitting," "separating" and

"dividing" are not intended to imply any particular outcome with respect to size of the resulting droplets (i.e., the size of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term "mixing" refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of "loading" droplet operations include microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. In various embodiments, the droplet operations may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated.

"Filler fluid" means a fluid associated with a droplet operations substrate of a droplet actuator, which fluid is sufficiently immiscible with a droplet phase to render the droplet phase subject to electrode-mediated droplet operations. The filler fluid may, for example, be a low- viscosity oil, such as silicone oil. Other examples of filler fluids are provided in International Patent Application No. PCT/US2006/047486, entitled, "Droplet-Based Biochemistry," filed on December 11, 2006; and in International Patent Application No. PCT/US2008/072604, entitled "Use of additives for enhancing droplet actuation," filed on August 8, 2008.

"Immobilize" with respect to magnetically responsive beads, means that the beads are substantially restrained in position in a droplet or in filler fluid on a droplet actuator. For example, in one embodiment, immobilized beads are sufficiently restrained in position to permit execution of a splitting operation on a droplet, yielding one droplet with substantially all of the beads and one droplet substantially lacking in the beads.

"Magnetically responsive" means responsive to a magnetic field at a field strength suitable for substantially immobilizing beads on a droplet actuator. "Magnetically responsive beads" include or are composed of magnetically responsive materials.

Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as Fe3O4, BaFeI2Oi9, CoO, NiO, Mn2O3, Cr2O3, and CoMnP. "Magnetically responsive" means not significantly responsive to a magnetic field at a field strength suitable for immobilizing beads on a droplet actuator.

"Partitioning" is the transfer of substances from the droplet phase to the filler fluid phase.

"Target" substances are those substances which are usefully retained in the droplet phase, e.g., because they are analytes or reagents involved in the chemical or biochemical reactions for which the droplet actuator is intended, or because they are waste products that could contaminate the filler fluid phase.

"Washing" with respect to washing a magnetically responsive bead means reducing the amount and/or concentration of one or more substances in contact with the magnetically responsive bead or exposed to the magnetically responsive bead from a droplet in contact with the magnetically responsive bead. The reduction in the amount and/or concentration of the substance may be partial, substantially complete, or even complete. The substance may be any of a wide variety of substances; examples include target substances for further analysis, and unwanted substances, such as components of a sample, contaminants, and/or excess reagent. In some embodiments, a washing operation begins with a starting droplet in contact with a magnetically responsive bead, where the droplet includes an initial amount and initial concentration of a substance. The washing operation may proceed using a variety of droplet operations. The washing operation may yield a droplet including the magnetically responsive bead, where the droplet has a total amount and/or concentration of the substance which is less than the initial amount and/or concentration of the substance. Other embodiments are described elsewhere herein, and still others will be immediately apparent in view of the present disclosure.

Except where otherwise indicated, the terms "top" and "bottom" are used throughout the description with reference to the top and bottom substrates of the droplet actuator for convenience only, since the droplet actuator is functional regardless of its position in space.

When a droplet is described as being "on" or "loaded on" a droplet actuator, it should be understood that the droplet is arranged on the droplet actuator in a manner which facilitates using the droplet actuator to conduct one or more droplet operations on the droplet, the droplet is arranged on the droplet actuator in a manner which facilitates sensing of a property of or a signal from the droplet, and/or the droplet has been subjected to a droplet operation on the droplet actuator.

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.

When a droplet is described as being "on" or "loaded on" a droplet actuator, it should be understood that the droplet is arranged on the droplet actuator in a manner which facilitates using the droplet actuator to conduct one or more droplet operations on the droplet, the droplet is arranged on the droplet actuator in a manner which facilitates sensing of a property of or a signal from the droplet, and/or the droplet has been subjected to a droplet operation on the droplet actuator. Large molecular weights are generally about 1000 mw or higher. Small molecular weights are generally less than 1000. Long chains are 50 carbons (for hydrocarbons) or longer or 50 silicons (silicone based) or longer. Short chains are generally less than 50.

Brief Description of the Drawings Figure 1 illustrates a side view of a portion of a droplet actuator, showing an oil film between the droplet and the surfaces of the droplet actuator;

Figures 2 illustrates a side view of a portion of a droplet actuator that includes layered filler fluids for assisting to maintain the stability of the oil film;

Figure 3 illustrates a side view of a portion of a droplet actuator that includes physical structures for droplet retention;

Figure 4A illustrates a side view of a portion of a droplet actuator that includes a droplet transport region that requires a certain electrowetting voltage for transporting droplets;

Figure 4B illustrates a side view of another portion of the droplet actuator of Figure 4A that includes an on-chip reservoir that requires a certain electrowetting voltage for dispensing droplets;

Figure 4C illustrates a side view of yet another portion of the droplet actuator of Figure 4A that includes an off-chip reservoir that requires yet another certain electrowetting voltage for dispensing droplets; and

Figure 5 illustrates a top view of the droplet actuator of Figures 4A, 4B, and 4C and shows the different regions therein that may require different voltages.

Detailed Description of the Invention

The invention provides modified droplet actuators, improved fluids for use on droplet actuators, droplet actuators including the improved fluids, and methods of conducting droplet operations using the improved fluids and/or modified droplet actuators. Droplet actuators typically employ a droplet phase (e.g., reagents, samples, etc.) and a filler fluid phase (e.g., filler fluids). The invention provides modified fluids for use in one or both of these phases. The modifications of the invention have a variety of improved attributes relative to existing fluids. For example, in certain embodiments, the modified fluids reduce (relative to corresponding fluids lacking the modifications described herein) or minimize or substantially eliminate loss of target substances from the hydrophilic phase due, for example, to the effects of adsorption and/or partitioning of target substances. Further, in certain embodiments, the modified fluids reduce (relative to corresponding fluids lacking the modifications described herein) or minimize or substantially eliminate carryover of target substances. The improved target substance retention is achieved without substantial reduction in the capability of the droplets to be subjected to one or more droplet operations on a droplet actuator of the invention.

In one embodiment, the invention provides droplet phase and filler fluid phase fluids including certain additives. The additives may improve retention of target substances in the droplet phase and/or improve droplet operations. Further, the invention provides droplet actuators including the modified droplet phase and/or filler fluid phase fluids of the invention. The invention also provides methods of conducting droplet operations using such modified droplet phase and/or filler fluid phase fluids of the invention, which methods exhibit improved retention of target substances in the droplet phase and/or improved droplet operations relative to corresponding fluids lacking the additives described herein.

As will be discussed in more detail in the ensuing sections, the invention exhibits advantages including, but not limited to: (1) reducing adsorption, such as by adding an additive to the droplet phase and/or filler fluid phase in order to render one or more target components less likely to adsorb to surfaces of the droplet actuator, (2) reducing partitioning, such as by adding an additive to the droplet phase and/or filler fluid phase in order to reduce the partitioning of one or more target components into the filler fluid phase, (3) reducing carryover, such as by adding an additive to the droplet phase and/or filler fluid phase in order to reduce the carryover of one or more target components from one droplet phase to another droplet phase, and (4) improve one or more droplet operations relative to droplet actuators lacking the modifications and/or improved fluids; and (5) any combinations of (1), (2), (3) and (4). The invention also provides modified droplet actuators, fluids and methods for maintaining oil film stability in a droplet actuator. The maintenance of the oil film between the droplet and the surface of the droplet actuator is an important factor in optimum operation of the droplet actuator. A stabilized oil film leads to less contamination, such as contamination due to absorption and resorption. In addition, maintenance of the oil film provides for more direct electrowetting and allows for the use of lower voltages for droplet operations.

7.1 Aqueous-soluble Additives

The invention may include providing an aqueous soluble additive in a droplet phase on a droplet actuator. In one example, the additive includes an aqueous soluble substance that has a hydrophile-lipophile balance (HLB) in the range of about 10 to about 20. In another example, the additive includes an aqueous soluble substance that has a hydrophile- lipophile balance (HLB) in the range of about 11 to about 20. In another example, the additive includes an aqueous soluble substance that has a hydrophile-lipophile balance (HLB) in the range of about 12 to about 20. In another example, the additive includes an aqueous soluble substance that has a hydrophile-lipophile balance (HLB) in the range of about 13 to about 20. In another example, the additive includes an aqueous soluble substance that has a hydrophile-lipophile balance (HLB) in the range of about 14 to about 20. In another example, the additive includes an aqueous soluble substance that has a hydrophile-lipophile balance (HLB) in the range of about 15 to about 20.

Examples of suitable additives include, but are not limited to, polysorbate 20, which is commercially available as Tween 20, and Triton X-100. Tween 20 may be supplied by, for example, Pierce Biotechnology, Inc. (Woburn, MA). Triton® X-100 may be supplied by, for example, Rohm & Haas Co (Philadelphia, PA).

The aqueous-soluble additive may selected and provided in an amount sufficient to interfere with adsorption, partitioning and/or carryover to the extent that the adsorption, partitioning and/or carryover is reduced relative to the adsorption, partitioning and/or carryover of the component in the absence of the additive. The aqueous-soluble additive may selected and provided in an amount sufficient to enhance a droplet operation relative to a corresponding droplet actuator system lacking the additive. In one embodiment when additive includes Tween® 20. The concentration of Tween® 20 in the droplet phase may, for example, be in the range of from about 001% to about 0.2% by volume, or from about 0.005% to about 0.1% by volume, or from about 0.01% to about 0.08% by volume.

In one embodiment, additive includes Triton X-100. The concentration of Triton X-100 in the droplet phase may, for example, be in the range of from about 0.001% to about 0.2% by volume, or from about 0.005% to about 0.1% by volume, or from about 0.01% to about 0.08% by volume.

In another example, the additive may be an organic solvent, such as dimethyl sulfoxide (DMSO) supplied by Gaylord Chemical Corporation (Slidell, LA). The concentration of

DMSO in the droplet phase may, for example, be in the range of from about 0.01% to about 5% by volume, or from about 0.1% to about 2% by volume, or from about 0.5% to about 1% by volume.

A variety of additives may be added to the droplet phase to improve droplet operations by increasing solubility of the target. Examples include 1,3 -propanediol; 1 ,4-butanediol;

1,5-pentanediol; 2,2,2- trifluoroethanol; 2-propanol; 3-mercaptopropionic acid; acetic acid; butyl chloride; chloroform (with ethanol, e.g., 1% ethanol); diethylene glycol; dimethyl sulfoxide; dimethylformamide; ethanol; ethylene glycol; formamide; formic acid; glycerol; isoamyl alcohol; mercaptoethanol; methanol; N,N-dimethlyformamide; N- methlyacetamide; phenol; pyridine; triethanolamine; Methylene glycol; and trifluoroacetic acid. Preferred organic solvent additives are those in which the target has a solubility which is greater than about 10 mg/mL.

Still other suitable additives include partially fluorinated surfactants, such as lH,lH,2H,2H-perfluoro-l-decanol and lH,lH,2H,2H-perfluoro-l-octanol; as well as perfluorinated surfactants, such as perfluorodecanoic acid and perfluorododecanoic acid.

An important class of additives for use in the droplet fluid phase is aqueous soluble fluorinated surfactants. A list of fluorinated surfactants is available in Chapter 1 "Fluorinated Surfactants and Repellents" By Erik Kissa, Published by CRC Press, 2001, the entire disclosure of which is incorporated herein by reference. Other suitable fluorinated surfactants are described in Michael Terrazas & Rudi Dams, "A new generation of fluorosurfactants," Speciality Chemicals Magazine, March 2004, vol 24 no 3, the entire disclosure of which is incorporated herein by reference.

Combinations of any of the foregoing surfactants may be used as filler fluid phase additives in accordance with the invention. Further, combinations of organic solvents, as well as combinations of any water miscible solvents with water may also be used in accordance with the invention. Moreover, combinations of foregoing surfactants and organic solvent additives may be used.

The invention also provides a droplet actuator, such as droplet actuator 200, having one or more aqueous droplets including one or more additives selected and provided in an amount which reduces the loss of target substances due to adsorption and/or partitioning.

The invention also includes a method of conducting a droplet operation during which operation the droplet includes one or more additives selected and provided in an amount that reduces the loss of target substances due to adsorption and/or partitioning.

In some cases, the surfactant molecules in the aqueous droplets tend to diffuse to the interface of the droplet causing a decrease in the oil-water interfacial tension over time.

In some embodiments, it may be useful to diffuse surfactant molecules through the bulk of the droplet phase or otherwise reduce accumulation of the surfactant molecules at the droplet phase/filler fluid phase interface. One solution to this issue involves including water soluble particles, such as polystyrene particles, in the droplet phase. The particles are provided in an amount that enhances mixing within the droplet or otherwise reduces accumulation of surfactant molecules at the interface. In one embodiment, beads, such as polystyrene particles, are selected which tend to migrate to the oil- water interface, thereby reducing accumulation of surfactants at the interface keeping the surfactant within the aqueous phase.

7.2 Oil Soluble Additives

In addition to, or as an alternative to, the water soluble additives described above, certain oil soluble additives may be useful in the filler fluid phase for reducing loss of target droplet phase components from the droplet phase and/or for improving droplet operations. Examples of suitable additives include nonionic low HLB (hydrophile-lipophile balance) surfactants. The HLB is preferably less than about 10 or less than about 5. Suitable examples include: Triton X- 15 (HLB=4.9); Span 85 (HLB 1.8); Span 65 (2.1); Span 83 (3.7); Span 80 (4.3); Span 60 (4.7); and fluorinated surfactants.

For example, oil-soluble filler fluid additives may include Span-85 (sorbitan trioleate) and/or Triton® X- 15. Span-85 may be supplied by, for example, Merck Schuchardt OHG (Germany). Triton X- 15 may be supplied by, for example, Rohm & Haas Co

(Philadelphia, PA).

Filler fluid additives are preferably selected and provided in an amount which (1) enables the droplet actuator to conduct or repeat more droplet operations compared to corresponding droplet actuator without the additives; and/or (2) enables one or more droplet operations on the droplet actuator that are not possible on a corresponding droplet actuator without the additives; and/or (3) makes one or more droplet operations more reliable on the droplet actuator as compared to corresponding droplet actuator without the additives; and/or (4) results in less loss of target substance from the droplet phase during droplet operations as compared to a corresponding droplet operations in the absence of the additives.

In a related example, surfactant(s) are selected and provided in an amount which makes one or more droplet operations possible or more reliable for droplets including one or more specific reagents or mixtures on the droplet actuator as compared to droplet operations for the same droplets including one or more specific reagents or mixtures on a corresponding droplet actuator without the surfactant(s). In another related example, surfactant(s) are selected and provided in an amount which makes one or more droplet operations possible or more reliable for one or more droplets including amphiphilic molecules on the droplet actuator as compared to droplet operations for the same droplets including amphiphilic molecules on a corresponding droplet actuator without the surfactant(s).

In one example, the concentration of Span-85 in the filler fluid phase is about 0.05% by volume. In yet another example, the concentration of Triton® X- 15 in the filler fluid phase is in the range of about 0.05% to about 0.1% by volume. In yet another example, the concentration of Triton® X- 15 in the filler fluid phase is about 0.2% by volume. In another embodiment when the filler fluid phase additive includes Triton X- 15. The concentration of Triton X- 15 in the filler fluid phase may, for example, be in the range of from about 0.001% to about 0.3% by volume, or from about 0.005% to about 0.2% by volume, or from about 0.05% to about 0.2% by volume.

An important class of additives for use in the filler fluid phase is oil soluble fluorinated surfactants. A comprehensive list of fluorinated surfactants is available in Chapter 1 "Fluorinated Surfactants and Repellents" By Erik Kissa, Published by CRC Press, 2001, the entire disclosure of which is incorporated herein by reference.

In other embodiment, the filler fluid phase additive includes surfactants with oleophilic & hydrophilic groups. The oleophilic groups may, for example, be hydrocarbon or silicone based. In one embodiment, the surfactant has an HLB which is less than about 5 and a small hydrophilic group. In another embodiment, the surfactant has a long hydrophobic(oleophilic) chains, e.g., polymeric surfactants, such as silicone polymeric surfactants.

In yet another embodiment, the surfactants include oleophobic, oleophilic and hydrophilic groups. For example, the oleophobic groups may include fluorinated groups. The oleophilic groups may include hydrocarbon/silicone groups. In one embodiment, the surfactant has a short or low mw hydrophilic group. In another embodiment, the surfactant has a short or low mw fluorinated group. In one embodiment, the surfactant has a short or low mw hydrophilic group and a long or high mw hydrophobic or oleophilic group. In yet another embodiment, the surfactant has a short or low mw fluorinated group and a long or high mw hydrophobic or oleophilic group. In certain embodiments, such as semifluorinated alkanes, the surfactant may lack a hydrophilic group. Further, certain surfactants suitable for use in the present invention lack a hydrophilic group and include a short fluorinated group or a short fluorinated group with a long hydrophobic group. As described herein, short fluorines have generally 20 or less, 15 or less, or 10 or less fluorinated groups (eg -CF2- or CF3-). In one embodiment, the surfactant is a fluorosilicone.

Silicone surfacants may be used as filler fluid additives in accordance with the invention. Examples include DBE-224, DBE-621 , and ABP-263, manufactured by Gelest. Hydrocarbon surfactants are also suitable additives for the filler fluid phase. Examples include Tetronic 701, Tetronic 901, Tetronic 70R2, Tetronic 150R4, Tetronic 11 ORl, Tetronic 1301, Tetronic 150Rl, Tetronix 1502, Pluronic 25Rl, Pluronic LlOl, Pluronic L61, Pluronic L81, Plurafac A-24, by BASF; IGEPAL CA-210 and IGEPAL CO-210 by GEF; and SPAN 60, SPAN 65, SPAN 80, SPAN 85, ARLACEL 60, ARLACEL 83, BRIJ

52, BRIJ 93, ATMUL 500, ARSURF 2802, by ICI.

Fluorinated surfactants are also useful as additives to the filler fluid phase, e.g., PolyFox PF-636, 6320, 656, 6520, 651, 652 by Omnova; Masurf FS-910, FS-1400, FS-1900 by Mason Chemical Company; FC-4432 by 3M; FMS-141, FMS-736, FMS- 121 (all examples of fluorosilicones) by Gelest; Zonyl 8857 and Zonyl FTS by Dupont; and fluorinated surfactants without hydrophilic groups.

7.2.1 Combinations of Surfactants

Combinations of surfactants may be used as droplet phase additives in accordance with the invention. Many droplet operations scenarios essentially have conflicting interfacial tension requirements. For example, while large-volume dispensing from a 2 mm dia opening is ideally conducted using a low interfacial tension, transport of droplets without tailing, hyperstability of droplets in the reservoir and bead handling are all best conducted using a moderate-to-large interfacial tension. Often, handling biological samples with a high protein load also imposes additional requirements on the surfactant solubility and HLB values.

In one embodiment of the invention, multiple surfactants are combined to satisfy different interfacial tension requirements for a particular application. For example, Span 85 is useful for selectively reducing the surface tension of oil, Triton Xl 5 is useful for covering the droplet phase/filler fluid interface and thus preventing proteins from accumulating at the interface. Span 85 can be combined with very small quantities of Span 80 or a polymeric surfactant that can accumulate at the droplet phase/filler fluid interface and mimic the Triton Xl 5 property, but still rendering a low oil surface tension. In one embodiment, the invention provides a filler fluid doped with a first surfactant having an HLB that is less than about 2 and a second surfactant having an HLB that is between about 2 and about 5. In another embodiment, the first surfactant forms forming the major proportion of surfactant and the second surfactant is included in trace quantities. 7.3 Changing pH to Adjust Solubility

The invention includes a droplet actuator having a droplet thereon having a target substance therein, where the droplet has a pH which has been adjusted away from the isoelectric point of the target substance in order to increase the solubility of the target substance. Similarly, the invention provides a method for preparing a fluid for conducting one of more droplet operations on a droplet actuator, where the method comprises adjusting the pH of the fluid in a direction which is away from the isoelectric point of the target substance in order to increase the solubility of the target substance. The adjustment may, for example, be achieved by combining the droplet with another droplet having a different pH. The invention further includes methods of conducting droplet operations, where the droplet operations are conducted using a droplet in which the pH has been adjusted as described here. The droplet having the adjusted pH may be wholly or partially surrounded by a filler fluid while present on the droplet actuator and/or while undergoing droplet operations.

Another aspect of the invention relates to changing the pH of a droplet in order to increase retention of a target substance in the droplet. For example, a first droplet having a target substance and a first pH may be combined with a second droplet having a second pH which is different from the fist pH. When the first droplet and second droplet are combined using one or more droplet operations, the resulting combined droplet has a pH which is adjusted relative to the pH of the first droplet. In one aspect of the invention, the pH of the second droplet is selected so that the pH of the first droplet will be adjusted in a direction which is which is away from the isoelectric point of the target substance.

7.4 Filler Fluid Zones

The invention also provides an embodiment in which a single chip includes multiple oil zones. For example, different zones may have different additives or different surfactants or surfactant concentrations. Each zone may be loaded with an appropriate filler fluid formulation (surfactant concentration, viscosity, etc) to assist with droplet operations and other functions that are to occur within that zone. The zones may be separated by physical barriers, such as strips of gasket. For example, different parts of the assay protocol occur in each zone: the filler fluid in a washing zone may be doped with a higher concentration of surfactant to assist wash buffer loading from large off-chip wells; the filler fluid in the detection zone may have a reduced amount of surfactant to assist in transport of double droplets using a single electrode. An opening in the barriers may be associated with an electrode path for transporting droplets from one barrier to another. Filler fluids may mix through the openings so long as the mixing is not sufficient to eliminate the benefits conferred by the tailored zones.

The droplet actuator layout is scalable, such that a droplet actuator may include a few as one filler fluid zone up to tens, hundreds or more filler fluid zones.

In some cases, filler fluids may be selected with appropriate properties to prevent mixing between the filler fluids in different zones. For example, a fluorinated oil may be provided in a middle zone between two non-fluorinated oils.

In one specific embodiment, the invention provides a PCR chip with base fluid that is generally used throughout the droplet actuator except in heated locations where the temperature would be unduly detrimental to droplet operations using the base fluid; and a heat stable filler fluid that is used in heated locations where the temperature would be unduly detrimental to electowetting function using the base fluid. For example, in one embodiment, 2.0 cSt Silicone oil is used as the base oil and hexadacane is used in regions that are sufficiently heated to be unduly detrimental to electowetting function with the silicone oil.

In another embodiment, the opening in a barrier between zones may be sealed with a wax plug. In operation, when sufficient heat is applied, the wax melts. The wax droplet may be immiscible with the surrounding filler fluid and may be transported away from the opening. The wax droplet may be transported into the using droplet operations and cooled to seal the opening.

7.5 Heating Elements

In general, thermal control may be provided in three ways: (1) thermal control of the entire droplet actuator; (2) thermal control of a region of a droplet actuator using a heater that is in contact with or in proximity to the controlled region; and (3) thermal control of a region of the droplet actuator or the entire droplet actuator using a heater that is integrated into the droplet actuator (e.g., in the substrate comprising the path or array of electrodes and/or in a top substrate of the droplet actuator, when present). Combinations of the foregoing approaches are also possible.

In an integrated heater approach, temperature zones can be created and controlled using thermal control systems directly integrated into the droplet actuator. Thermal control elements (heating and/or cooling) may be integrated on the bottom substrate and/or top substrate (when present) of the droplet actuator and on the bottom and/or top surface of either substrate, or integrated within the structure of either substrate, or arranged between substrates. In one embodiment, the heating element is located in the barrier between filler fluid zones.

Each filler fluid zone may include distinct heating elements and may thus serve as a distinct thermal zone within the droplet actuator. This arrangement permits multiple steps in an analysis, such as sample preparation and thermal cycling, requiring different temperatures to be performed simultaneously at different temperatures in different filler fluid zones on a droplet actuator. For example, droplets can be physically transported or shuttled between filler fluid zones of different fixed temperatures to perform thermal cycling for an amplification reaction.

In one embodiment, heaters in the filler fluid zones may be formed using thin