CA2840949A1

CA2840949A1 - Reagent storage on a droplet actuator

Info

Publication number: CA2840949A1
Application number: CA2840949A
Authority: CA
Inventors: Jennifer Foley; Stefan Burde
Original assignee: Advanced Liquid Logic Inc
Current assignee: Advanced Liquid Logic Inc
Priority date: 2011-07-06
Filing date: 2012-06-26
Publication date: 2013-01-10
Also published as: WO2013006312A2; JP2014524741A; AU2012279420A1; CN103733059B; JP5894272B2; EP2729792A2; US9631244B2; KR20140064771A; CN103733059A; WO2013006312A3; BR112014000257A2; EP2729792A4; US20140141409A1

Abstract

A method of providing a droplet comprising one or more reagents, the method comprising, depositing a first aqueous droplet comprising the one or more reagents on a surface; drying the droplet to yield a dried composition on the surface comprising the one or more reagents; covering the dried composition with oil; and causing a second aqueous droplet in the oil to contact the dried composition and thereby resuspend one or more reagents.

Description

Docket No. 212PCT
Reagent Storage on a Droplet Actuator 1 Government Interest This invention was made with government support under HG004354 awarded by the National Institutes of Health of the United States. The United States Government has certain rights in the invention.
2 Background A droplet actuator typically includes one or more substrates configured to form a surface or gap for conducting droplet operations. The one or more substrates establish a droplet operations surface or gap for conducting droplet operations and may also include electrodes arranged to conduct the droplet operations. The droplet operations substrate or the gap between the substrates may be coated or filled with a filler fluid that is immiscible with the liquid that forms the droplets.
Droplet actuators are used in a variety of applications, including molecular diagnostic testing, such as point-of-care testing in which the flexibility and breadth of digital microfluidics provide a rapid and sensitive multifunctional testing device. For point-of-care testing, diagnostic devices are typically provided pre-loaded with the required assay reagents for a diagnostic test.
Consequently, there is a need for methods for storing assay reagents on a droplet actuator.

3 Definitions As used herein, the following terms have the meanings indicated.
"Activate," with reference to one or more electrodes, means affecting a change in the electrical state of the one or more electrodes which, in the presence of a droplet, results in a droplet operation. Activation of an electrode can be accomplished using alternating or direct current.
Any suitable voltage may be used. For example, an electrode may be activated using a voltage which is greater than about 150 V, or greater than about 200 V, or greater than about 250 V, or from about 275 V to about 375 V, or about 300 V. Where alternating current is used, any suitable frequency may be employed. For example, an electrode may be activated using alternating current having a frequency from about 1 Hz to about 100 Hz, or from about 10 Hz to about 60 Hz, or from about 20 Hz to about 40 Hz, or about 30 Hz.

Docket No. 212PCT
"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, amorphous and other three dimensional shapes. The bead may, for example, be capable of being subjected to a droplet operation 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 provided in a droplet, in a droplet operations gap, or on a droplet operations surface.
Beads may be provided in a reservoir that is external to a droplet operations gap or situated apart from a droplet operations surface, and the reservoir may be associated with a fluid path that permits a droplet including the beads to be brought into a droplet operations gap or into contact with a droplet operations surface. 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, a portion of a bead, or only one component 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 beads include flow cytometry microbeads, polystyrene microparticles and nanoparticles, functionalized polystyrene microparticles and nanoparticles, coated polystyrene microparticles and nanoparticles, silica microbeads, fluorescent microspheres and nanospheres, functionalized fluorescent microspheres and nanospheres, coated fluorescent microspheres and nanospheres, color dyed microparticles and nanoparticles, magnetic microparticles and nanoparticles, superparamagnetic microparticles and nanoparticles (e.g., DYNABEADSO particles, available from Invitrogen Group, Carlsbad, CA), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles, and those described in U.S. Patent Publication Nos.
20050260686, entitled "Multiplex flow assays preferably with magnetic particles as solid phase,"
published on November 24, 2005; 20030132538, entitled "Encapsulation of discrete quanta of fluorescent particles," published on July 17, 2003; 20050118574, entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," published on June 2, 2005; 20050277197.
Entitled "Microparticles with Multiple Fluorescent Signals and Methods of Using Same,"
published on December 15, 2005; 20060159962, entitled "Magnetic Microspheres for use in Fluorescence-based Applications," published on July 20, 2006; the entire disclosures of which are incorporated herein by reference for their teaching concerning beads and magnetically responsive materials and beads. Beads may be pre-coupled with a biomolecule or other substance that is able to bind Docket No. 212PCT
to and form a complex with a biomolecule. Beads may be pre-coupled with an antibody, protein or antigen, DNA/RNA probe or any other molecule with an affinity for a desired target.
Examples of droplet actuator techniques for immobilizing magnetically responsive beads and/or non-magnetically responsive 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/U52008/053545, entitled "Droplet Actuator Devices and Methods Employing Magnetic Beads," filed on February 11, 2008; International Patent Application No.
PCT/U52008/058018, entitled "Bead-based Multiplexed Analytical Methods and Instrumentation," filed on March 24, 2008; International Patent Application No.
PCT/U52008/058047, "Bead Sorting on a Droplet Actuator," filed on March 23, 2008; and International Patent Application No. PCT/U52006/047486, entitled "Droplet-based Biochemistry," filed on December 11, 2006; the entire disclosures of which are incorporated herein by reference. Bead characteristics may be employed in the multiplexing aspects of the invention. Examples of beads having characteristics suitable for multiplexing, as well as methods of detecting and analyzing signals emitted from such beads, may be found in U.S. Patent Publication No. 20080305481, entitled "Systems and Methods for Multiplex Analysis of PCR in Real Time," published on December 11, 2008; U.S. Patent Publication No.
20080151240, "Methods and Systems for Dynamic Range Expansion," published on June 26, 2008;
U.S. Patent Publication No. 20070207513, entitled "Methods, Products, and Kits for Identifying an Analyte in a Sample," published on September 6, 2007; U.S. Patent Publication No.
20070064990, entitled "Methods and Systems for Image Data Processing," published on March 22, 2007; U.S.
Patent Publication No. 20060159962, entitled "Magnetic Microspheres for use in Fluorescence-based Applications," published on July 20, 2006; U.S. Patent Publication No.
20050277197, entitled "Microparticles with Multiple Fluorescent Signals and Methods of Using Same,"
published on December 15, 2005; and U.S. Patent Publication No. 20050118574, entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," published on June 2, 2005.
"Droplet" means a volume of liquid on a droplet actuator. Typically, a droplet is at least partially bounded by a filler fluid. For example, a droplet may be completely surrounded by a filler fluid or may be bounded by filler fluid and one or more surfaces of the droplet actuator. As another example, a droplet may be bounded by filler fluid, one or more surfaces of the droplet actuator, Docket No. 212PCT
and/or the atmosphere. As yet another example, a droplet may be bounded by filler fluid and the atmosphere. 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, combinations of such shapes, 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.
For examples of droplet fluids that may be subjected to droplet operations using the approach of the invention, see International Patent Application No. PCT/US 06/47486, entitled, "Droplet-Based Biochemistry," filed on December 11, 2006. In various embodiments, a droplet may include a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs and biological washes. Moreover, a droplet may include a reagent, such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions and/or buffers. Other examples of droplet contents include reagents, such as a reagent for a biochemical protocol, such as a nucleic acid amplification protocol, an affinity-based assay protocol, an enzymatic assay protocol, a sequencing protocol, and/or a protocol for analyses of biological fluids.
"Droplet Actuator" means a device for manipulating droplets. For examples of droplet actuators, see Pamula et al., U.S. Patent 6,911,132, entitled "Apparatus for Manipulating Droplets by Electrowetting-Based Techniques," issued on June 28, 2005; 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; Pollack et al., International Patent Application No. PCT/US2006/047486, entitled "Droplet-Based Biochemistry,"
filed on December 11, 2006; Shenderov, 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; Kim and/or Shah et al., U.S. Patent Application Nos. 10/343,261, entitled "Electrowetting-driven Micropumping," filed on January 27, 2003, 11/275,668, entitled "Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle," filed on January 23, 2006, 11/460,188, entitled "Small Object Moving on Printed Circuit Board," filed on January 23, 2006, 12/465,935, entitled "Method for Using Magnetic Particles in Droplet Microfluidics," filed on May 14, 2009, and 12/513,157, entitled "Method and Apparatus for Real-time Feedback Control Docket No. 212PCT
of Electrical Manipulation of Droplets on Chip," filed on April 30, 2009;
Velev, U.S. Patent 7,547,380, entitled "Droplet Transportation Devices and Methods Having a Fluid Surface,"
issued on June 16, 2009; Sterling et al., U.S. Patent 7,163,612, entitled "Method, Apparatus and Article for Microfluidic Control via Electrowetting, for Chemical, Biochemical and Biological Assays and the Like," issued on January 16, 2007; Becker and Gascoyne et al., U.S. Patent Nos.
7,641,779, entitled "Method and Apparatus for Programmable fluidic Processing," issued on January 5, 2010, and 6,977,033, entitled "Method and Apparatus for Programmable fluidic Processing," issued on December 20, 2005; Decre et al., U.S. Patent 7,328,979, entitled "System for Manipulation of a Body of Fluid," issued on February 12, 2008; Yamakawa et al., U.S. Patent Pub. No. 20060039823, entitled "Chemical Analysis Apparatus," published on February 23, 2006; Wu, International Patent Pub. No. WO/2009/003184, entitled "Digital Microfluidics Based Apparatus for Heat-exchanging Chemical Processes," published on December 31, 2008; Fouillet et al., U.S. Patent Pub. No. 20090192044, entitled "Electrode Addressing Method," published on July 30, 2009; Fouillet et al., U.S. Patent 7,052,244, entitled "Device for Displacement of Small Liquid Volumes Along a Micro-catenary Line by Electrostatic Forces," issued on May 30, 2006;
Marchand et al., U.S. Patent Pub. No. 20080124252, entitled "Droplet Microreactor," published on May 29, 2008; Adachi et al., U.S. Patent Pub. No. 20090321262, entitled "Liquid Transfer Device," published on December 31, 2009; Roux et al., U.S. Patent Pub. No.
20050179746, entitled "Device for Controlling the Displacement of a Drop Between two or Several Solid Substrates," published on August 18, 2005; Dhindsa et al., "Virtual Electrowetting Channels:
Electronic Liquid Transport with Continuous Channel Functionality," Lab chip, 10:832-836 (2010); the entire disclosures of which are incorporated herein by reference, along with their priority documents. Certain droplet actuators will include one or more substrates arranged with a gap therebetween and electrodes associated with (e.g., layered on, attached to, and/or embedded in) the one or more substrates and arranged to conduct one or more droplet operations. For example, certain droplet actuators will include a base (or bottom) substrate, droplet operations electrodes associated with the substrate, one or more dielectric layers atop the substrate and/or electrodes, and optionally one or more hydrophobic layers atop the substrate, dielectric layers and/or the electrodes forming a droplet operations surface. A top substrate may also be provided, which is separated from the droplet operations surface by a gap, commonly referred to as a droplet operations gap. Various electrode arrangements on the top and/or bottom substrates are discussed in the above-referenced patents and applications and certain novel electrode arrangements are discussed in the description of the invention. During droplet operations it is preferred that droplets remain in continuous contact or frequent contact with a ground or reference electrode. A ground or reference electrode may be associated with the top substrate facing the gap, the bottom substrate facing the gap, in the gap. Where electrodes are provided on both substrates, electrical contacts for coupling the electrodes to a droplet actuator instrument for Docket No. 212PCT
controlling or monitoring the electrodes may be associated with one or both plates. In some cases, electrodes on one substrate are electrically coupled to the other substrate so that only one substrate is in contact with the droplet actuator. In one embodiment, a conductive material (e.g., an epoxy, such as MASTER BONDTM Polymer System EP79, available from Master Bond, Inc., Hackensack, NJ) provides the electrical connection between electrodes on one substrate and electrical paths on the other substrates, e.g., a ground electrode on a top substrate may be coupled to an electrical path on a bottom substrate by such a conductive material.
Where multiple substrates are used, a spacer may be provided between the substrates to determine the height of the gap therebetween and define dispensing reservoirs. The spacer height may, for example, be from about 5 um to about 600 um, or about 100 um to about 400 um, or about 200 um to about 350 um, or about 250 um to about 300 um, or about 275 um. The spacer may, for example, be formed of a layer of projections form the top or bottom substrates, and/or a material inserted between the top and bottom substrates. One or more openings may be provided in the one or more substrates for forming a fluid path through which liquid may be delivered into the droplet operations gap. The one or more openings may in some cases be aligned for interaction with one or more electrodes, e.g., aligned such that liquid flowed through the opening will come into sufficient proximity with one or more droplet operations electrodes to permit a droplet operation to be effected by the droplet operations electrodes using the liquid. The base (or bottom) and top substrates may in some cases be formed as one integral component. One or more reference electrodes may be provided on the base (or bottom) and/or top substrates and/or in the gap.
Examples of reference electrode arrangements are provided in the above referenced patents and patent applications. In various embodiments, the manipulation of droplets by a droplet actuator may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated or Coulombic force mediated. Examples of other techniques for controlling droplet operations that may be used in the droplet actuators of the invention include using devices that induce hydrodynamic fluidic pressure, such as those that operate on the basis of mechanical principles (e.g. external syringe pumps, pneumatic membrane pumps, vibrating membrane pumps, vacuum devices, centrifugal forces, piezoelectric/ultrasonic pumps and acoustic forces); electrical or magnetic principles (e.g. electroosmotic flow, electrokinetic pumps, ferrofluidic plugs, electrohydrodynamic pumps, attraction or repulsion using magnetic forces and magnetohydrodynamic pumps); thermodynamic principles (e.g. gas bubble generation/phase-change-induced volume expansion); other kinds of surface-wetting principles (e.g. electrowetting, and optoelectrowetting, as well as chemically, thermally, structurally and radioactively induced surface-tension gradients); gravity; surface tension (e.g., capillary action);
electrostatic forces (e.g., electroosmotic flow); centrifugal flow (substrate disposed on a compact disc and rotated);
magnetic forces (e.g., oscillating ions causes flow); magnetohydrodynamic forces; and vacuum or pressure differential. In certain embodiments, combinations of two or more of the foregoing Docket No. 212PCT
techniques may be employed to conduct a droplet operation in a droplet actuator of the invention.
Similarly, one or more of the foregoing may be used to deliver liquid into a droplet operations gap, e.g., from a reservoir in another device or from an external reservoir of the droplet actuator (e.g., a reservoir associated with a droplet actuator substrate and a fluid path from the reservoir into the droplet operations gap). Droplet operations surfaces of certain droplet actuators of the invention may be made from hydrophobic materials or may be coated or treated to make them hydrophobic. For example, in some cases some portion or all of the droplet operations surfaces may be derivatized with low surface-energy materials or chemistries, e.g., by deposition or using in situ synthesis using compounds such as poly- or per-fluorinated compounds in solution or polymerizable monomers. Examples include TEFLON AF (available from DuPont, Wilmington, DE), members of the cytop family of materials, coatings in the FLUOROPELO
family of hydrophobic and superhydrophobic coatings (available from Cytonix Corporation, Beltsville, MD), silane coatings, fluorosilane coatings, hydrophobic phosphonate derivatives (e.g.., those sold by Aculon, Inc), and NOVECTM electronic coatings (available from 3M
Company, St. Paul, MN), and other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD). In some cases, the droplet operations surface may include a hydrophobic coating having a thickness ranging from about 10 nm to about 1,000 nm.
Moreover, in some embodiments, the top substrate of the droplet actuator includes an electrically conducting organic polymer, which is then coated with a hydrophobic coating or otherwise treated to make the droplet operations surface hydrophobic. For example, the electrically conducting organic polymer that is deposited onto a plastic substrate may be poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS). Other examples of electrically conducting organic polymers and alternative conductive layers are described in Pollack et al., International Patent Application No. PCT/U52010/040705, entitled "Droplet Actuator Devices and Methods," the entire disclosure of which is incorporated herein by reference. One or both substrates may be fabricated using a printed circuit board (PCB), glass, indium tin oxide (ITO)-coated glass, and/or semiconductor materials as the substrate. When the substrate is ITO-coated glass, the ITO
coating is preferably a thickness in the range of about 20 to about 200 nm, preferably about 50 to about 150 nm, or about 75 to about 125 nm, or about 100 nm. In some cases, the top and/or bottom substrate includes a PCB substrate that is coated with a dielectric, such as a polyimide dielectric, which may in some cases also be coated or otherwise treated to make the droplet operations surface hydrophobic. When the substrate includes a PCB, the following materials are examples of suitable materials: MITSUITm BN-300 (available from MITSUI
Chemicals America, Inc., San Jose CA); ARLONTM 11N (available from Arlon, Inc, Santa Ana, CA).;
NELCOO
N4000-6 and N5000-30/32 (available from Park Electrochemical Corp., Melville, NY); ISOLATM
FR406 (available from Isola Group, Chandler, AZ), especially IS620;
fluoropolymer family (suitable for fluorescence detection since it has low background fluorescence); polyimide family;

Docket No. 212PCT
polyester; polyethylene naphthalate; polycarbonate; polyetheretherketone;
liquid crystal polymer;
cyclo-olefin copolymer (COC); cyclo-olefin polymer (COP); aramid; THERMOUNTO
nonwoven aramid reinforcement (available from DuPont, Wilmington, DE); NOMEXO
brand fiber (available from DuPont, Wilmington, DE); and paper. Various materials are also suitable for use as the dielectric component of the substrate. Examples include: vapor deposited dielectric, such as PARYLENETM C (especially on glass) and PARYLENETM N
(available from Parylene Coating Services, Inc., Katy, TX); TEFLON AF coatings; cytop;
soldermasks, such as liquid photoimageable soldermasks (e.g., on PCB) like TAIYOTm PSR4000 series, TAIYOTm PSR and AUS series (available from Taiyo America, Inc. Carson City, NV) (good thermal characteristics for applications involving thermal control), and PROBIMERTm 8165 (good thermal characteristics for applications involving thermal control (available from Huntsman Advanced Materials Americas Inc., Los Angeles, CA); dry film soldermask, such as those in the VACRELO dry film soldermask line (available from DuPont, Wilmington, DE); film dielectrics, such as polyimide film (e.g., KAPTONO polyimide film, available from DuPont, Wilmington, DE), polyethylene, and fluoropolymers (e.g., FEP), polytetrafluoroethylene;
polyester;
polyethylene naphthalate; cyclo-olefin copolymer (COC); cyclo-olefin polymer (COP); any other PCB substrate material listed above; black matrix resin; and polypropylene.
Droplet transport voltage and frequency may be selected for performance with reagents used in specific assay protocols. Design parameters may be varied, e.g., number and placement of on-actuator reservoirs, number of independent electrode connections, size (volume) of different reservoirs, placement of magnets/bead washing zones, electrode size, inter-electrode pitch, and gap height (between top and bottom substrates) may be varied for use with specific reagents, protocols, droplet volumes, etc. In some cases, a substrate of the invention may derivatized with low surface-energy materials or chemistries, e.g., using deposition or in situ synthesis using poly- or per-fluorinated compounds in solution or polymerizable monomers. Examples include TEFLON AF coatings and FLUOROPELO coatings for dip or spray coating, and other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD).
Additionally, in some cases, some portion or all of the droplet operations surface may be coated with a substance for reducing background noise, such as background fluorescence from a PCB
substrate. For example, the noise-reducing coating may include a black matrix resin, such as the black matrix resins available from Toray industries, Inc., Japan. Electrodes of a droplet actuator are typically controlled by a controller or a processor, which is itself provided as part of a system, which may include processing functions as well as data and software storage and input and output capabilities. Reagents may be provided on the droplet actuator in the droplet operations gap or in a reservoir fluidly coupled to the droplet operations gap. The reagents may be in liquid form, e.g., droplets, or they may be provided in a reconstitutable form in the droplet operations gap or in a reservoir fluidly coupled to the droplet operations gap. Reconstitutable reagents may Docket No. 212PCT
typically be combined with liquids for reconstitution. An example of reconstitutable reagents suitable for use with the invention includes those described in Meathrel, et al., U.S. Patent 7,727,466, entitled "Disintegratable films for diagnostic devices," granted on June 1, 2010.
"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; 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 that are 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 volume of the resulting droplets (i.e., the volume 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. Droplet operations may be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and/or hydrophobic regions on surfaces and/or by physical obstacles. For examples of droplet operations, see the patents and patent applications cited above under the definition of "droplet actuator." Impedance or capacitance sensing or imaging techniques may sometimes be used to determine or confirm the outcome of a droplet operation. Examples of such techniques are described in Sturmer et al., International Patent Pub. No. WO/2008/101194, entitled "Capacitance Detection in a Droplet Actuator," published on August 21, 2008, the entire disclosure of which is incorporated herein by reference. Generally speaking, the sensing or imaging techniques may be used to confirm the presence or absence of a droplet at a specific electrode. For example, the presence of a dispensed droplet at the destination electrode following a droplet dispensing operation confirms that the droplet dispensing operation was effective. Similarly, the presence of a droplet at a detection spot Docket No. 212PCT
at an appropriate step in an assay protocol may confirm that a previous set of droplet operations has successfully produced a droplet for detection. Droplet transport time can be quite fast. For example, in various embodiments, transport of a droplet from one electrode to the next may exceed about 1 sec, or about 0.1 sec, or about 0.01 sec, or about 0.001 sec.
In one embodiment, the electrode is operated in AC mode but is switched to DC mode for imaging.
It is helpful for conducting droplet operations for the footprint area of droplet to be similar to electrowetting area;
in other words, lx-, 2x- 3x-droplets are usefully controlled operated using 1, 2, and 3 electrodes, respectively. If the droplet footprint is greater than the number of electrodes available for conducting a droplet operation at a given time, the difference between the droplet size and the number of electrodes should typically not be greater than 1; in other words, a 2x droplet is usefully controlled using 1 electrode and a 3x droplet is usefully controlled using 2 electrodes.
When droplets include beads, it is useful for droplet size to be equal to the number of electrodes controlling the droplet, e.g., transporting the droplet.
"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. For example, the gap of a droplet actuator is typically filled with a filler fluid. The filler fluid may, for example, be a low-viscosity oil, such as silicone oil or hexadecane filler fluid. The filler fluid may fill the entire gap of the droplet actuator or may coat one or more surfaces of the droplet actuator. Filler fluids may be conductive or non-conductive. Filler fluids may, for example, be doped with surfactants or other additives. For example, additives may be selected to improve droplet operations and/or reduce loss of reagent or target substances from droplets, formation of microdroplets, cross contamination between droplets, contamination of droplet actuator surfaces, degradation of droplet actuator materials, etc. Composition of the filler fluid, including surfactant doping, may be selected for performance with reagents used in the specific assay protocols and effective interaction or non-interaction with droplet actuator materials. Examples of filler fluids and filler fluid formulations suitable for use with the invention are provided in Srinivasan et al, International Patent Pub.
Nos.
WO/2010/027894, entitled "Droplet Actuators, Modified Fluids and Methods,"
published on March 11, 2010, and WO/2009/021173, entitled "Use of Additives for Enhancing Droplet Operations," published on February 12, 2009; Sista et al., International Patent Pub. No.
WO/2008/098236, entitled "Droplet Actuator Devices and Methods Employing Magnetic Beads,"
published on August 14, 2008; and Monroe et al., U.S. Patent Publication No.
20080283414, entitled "Electrowetting Devices," filed on May 17, 2007; the entire disclosures of which are incorporated herein by reference, as well as the other patents and patent applications cited herein.

Docket No. 212PCT
"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 in a droplet to permit execution of a droplet splitting operation, 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. "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 Fe304, BaFe12019, CoO, NiO, Mn203, Cr203, and CoMnP.
"Reservoir" means an enclosure or partial enclosure configured for holding, storing, or supplying liquid. A droplet actuator system of the invention may include on-cartridge reservoirs and/or off-cartridge reservoirs. On-cartridge reservoirs may be (1) on-actuator reservoirs, which are reservoirs in the droplet operations gap or on the droplet operations surface;
(2) off-actuator reservoirs, which are reservoirs on the droplet actuator cartridge, but outside the droplet operations gap, and not in contact with the droplet operations surface; or (3) hybrid reservoirs which have on-actuator regions and off-actuator regions. An example of an off-actuator reservoir is a reservoir in the top substrate. An off-actuator reservoir is typically in fluid communication with an opening or fluid path arranged for flowing liquid from the off-actuator reservoir into the droplet operations gap, such as into an on-actuator reservoir. An off-cartridge reservoir may be a reservoir that is not part of the droplet actuator cartridge at all, but which flows liquid to some portion of the droplet actuator cartridge. For example, an off-cartridge reservoir may be part of a system or docking station to which the droplet actuator cartridge is coupled during operation.
Similarly, an off-cartridge reservoir may be a reagent storage container or syringe which is used to force fluid into an on-cartridge reservoir or into a droplet operations gap. A system using an off-cartridge reservoir will typically include a fluid passage means whereby liquid may be transferred from the off-cartridge reservoir into an on-cartridge reservoir or into a droplet operations gap.
"Transporting into the magnetic field of a magnet," "transporting towards a magnet," and the like, as used herein to refer to droplets and/or magnetically responsive beads within droplets, is intended to refer to transporting into a region of a magnetic field capable of substantially attracting magnetically responsive beads in the droplet. Similarly, "transporting away from a magnet or magnetic field," "transporting out of the magnetic field of a magnet," and the like, as Docket No. 212PCT
used herein to refer to droplets and/or magnetically responsive beads within droplets, is intended to refer to transporting away from a region of a magnetic field capable of substantially attracting magnetically responsive beads in the droplet, whether or not the droplet or magnetically responsive beads is completely removed from the magnetic field. It will be appreciated that in any of such cases described herein, the droplet may be transported towards or away from the desired region of the magnetic field, and/or the desired region of the magnetic field may be moved towards or away from the droplet. Reference to an electrode, a droplet, or magnetically responsive beads being "within" or "in" a magnetic field, or the like, is intended to describe a situation in which the electrode is situated in a manner which permits the electrode to transport a droplet into and/or away from a desired region of a magnetic field, or the droplet or magnetically responsive beads is/are situated in a desired region of the magnetic field, in each case where the magnetic field in the desired region is capable of substantially attracting any magnetically responsive beads in the droplet. Similarly, reference to an electrode, a droplet, or magnetically responsive beads being "outside of' or "away from" a magnetic field, and the like, is intended to describe a situation in which the electrode is situated in a manner which permits the electrode to transport a droplet away from a certain region of a magnetic field, or the droplet or magnetically responsive beads is/are situated away from a certain region of the magnetic field, in each case where the magnetic field in such region is not capable of substantially attracting any magnetically responsive beads in the droplet or in which any remaining attraction does not eliminate the effectiveness of droplet operations conducted in the region. In various aspects of the invention, a system, a droplet actuator, or another component of a system may include a magnet, such as one or more permanent magnets (e.g., a single cylindrical or bar magnet or an array of such magnets, such as a Halbach array) or an electromagnet or array of electromagnets, to form a magnetic field for interacting with magnetically responsive beads or other components on chip. Such interactions may, for example, include substantially immobilizing or restraining movement or flow of magnetically responsive beads during storage or in a droplet during a droplet operation or pulling magnetically responsive beads out of a droplet.
"Washing" with respect to washing a bead means reducing the amount and/or concentration of one or more substances in contact with the bead or exposed to the bead from a droplet in contact with the 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 Docket No. 212PCT
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. Examples of suitable washing techniques are described in Pamula et al., U.S. Patent 7,439,014, entitled "Droplet-Based Surface Modification and Washing,"
granted on October 21, 2008, the entire disclosure of which is incorporated herein by reference.
The terms "top," "bottom," "over," "under," and "on" are used throughout the description with reference to the relative positions of components of the droplet actuator, such as relative positions of top and bottom substrates of the droplet actuator. It will be appreciated that the droplet actuator is functional regardless of its orientation in space.
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/an-ay/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.
4 Brief Description of the Drawings Figure 1 illustrates a top view of an example of a portion of a droplet actuator and also shows an example of an array of reagent droplets dried on certain droplet operations electrodes;
Figure 2 illustrates a top view of an example of a portion of a droplet actuator and also shows an example of dried reagent droplets on an on-actuator reagent dispensing electrode;
Figures 3A, 3B, and 3C illustrate top views of an example of a portion of an electrode arrangement of a droplet actuator and show a process of performing a reagent reconstitution protocol on a droplet actuator; and Docket No. 212PCT
Figures 4A through 4E illustrate top views of the electrode arrangement of Figure 3A and show another process of performing a reagent reconstitution protocol on a droplet actuator.
Description The invention provides methods for storage and reconstitution (i.e., reagent recovery) of assay reagents on a droplet actuator. In one embodiment, the invention provides methods for drying one or more assay reagents on a solid surface of a droplet actuator. In another embodiment, the invention provides methods for storing one or more liquid reagents on a droplet actuator. In yet another embodiment, the invention provides methods for recovery of one or more dried assay reagents from a solid surface of a droplet actuator using digital microfluidic liquid handling protocols. The dried reagent and the surface may be covered with a filler fluid, such as a silicone oil.
Assay reagents may be preloaded and stored on a droplet actuator as dried reagents, liquid reagents, and/or any combination thereof Storage format (i.e., dried reagent or liquid reagent) may be selected to provide maximum stability of stored reagents (e.g., shelf-life of 12 months or more). Storage format may be selected such that no special handling, precautions or storage conditions are required. User intervention is minimized because assay reagents are preloaded on a droplet actuator and digital microfluidic liquid handling protocols are used for reconstitution of dried reagent.
In one embodiment, the methods of the invention are used to provide a preloaded, disposable droplet actuator that is suitable for point-of-care (POC) and sample-to-answer diagnostic testing.
In one example, the methods of the invention may be used for reagent storage and reconstitution on a droplet actuator configured for POC and sample-to-answer testing for HIV.
In this example, dried reagents for sample preparation, immunoassays for antibodies to HIV, and reverse transcription quantitative PCR (RT-qPCR) for HIV viral load may be preloaded and stored on a droplet actuator. Liquid reagents, such as wash buffers and oil filler fluid, may also be stored on the droplet actuator.

5.1 Reagent Storage on a Droplet Actuator The invention provides methods for reagent storage and reconstitution (i.e., reagent recovery) on a droplet actuator. Available reagent drying technologies for preserving and storing chemical reagents (e.g., sample preparation reagents, immunoassay reagents, and RT-qPCR
reagents) on a solid surface may be selected and adapted for use on a droplet actuator.
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liquid handling protocols may be used for recovery of dried reagents. Liquid reagents (e.g., oil filler fluid, rehydration buffers, and certain assay reagents) may also be stored on a droplet actuator, and may coat the reagents prior to reconstitution. Reagents may be dried in a reservoir or fluid passage leading to a droplet operations gap or surface. Reagents may be dried on a droplet operations surface, such as a hydrophobic surface of a droplet actuator.
Reagents stored on a droplet actuator may be dried reagents, liquid reagents, and/or any combination thereof and are suitable for conducting one or more sample preparation protocols and/or one or more assay protocols. In one example, all reagents for conducting one or more sample preparation protocols and/or one or more assay protocols may be provided as dried reagents on a droplet actuator. In another example, a single reagent may be provided as a dried reagent and all other reagents may be provided as liquid reagents.
Sample preparation on a droplet actuator typically involves purifying a sample and/or lysing a sample to release molecular targets for one or more molecular assays. Sample preparation protocols that may be performed on a droplet actuator using one or more dried and reconstituted reagents and/or any combination of dried and liquid reagents may include, but are not limited to, blood preparation (e.g., agglutinating blood cells, agglutinating red blood cells, and lysing blood cells), and various lysing protocols for cells, spores, bacteria, fungi, virus, armored RNA, and armored DNA.
Molecular assays that may be performed on a droplet actuator using one or more dried and reconstituted reagents and/or any combination of dried and liquid reagents may include, but are not limited to, immunoassays, electrochemical assays, enzymatic assays, polymerase chain reaction (PCR) assays, and/or reverse transcriptase (RT)-PCR assays.
The invention provides methods for drying one or more assay reagents on a solid surface of a droplet actuator and for reconstituting the dried reagent(s). In one example, the methods of the invention may include, but are not limited to, the following steps:
1. Depositing an aqueous droplet(s) that contains one or more reagents on a surface of a droplet actuator;
2. Drying the droplet(s) to yield a dried composition on the surface of the droplet actuator;
3. Covering the dried composition with an oil filler fluid; and Docket No. 212PCT
4. Transporting a second aqueous droplet (i.e., rehydration droplet) in the oil filler fluid to contact the dried composition and thereby resuspend one or more dried reagents.
A droplet actuator may, for example, include a bottom substrate and a top substrate that are separated by a gap. The bottom substrate may include an electrode arrangement, such as a path and/or array of droplet operations electrodes (e.g., electrowetting electrodes) and one or more fluid reservoir electrodes that may be coated with a hydrophobic material (e.g., Cytop). The hydrophobic coating is provided for efficient electrowetting of droplets. The top substrate may include a single large ground reference electrode that may also be coated with a hydrophobic material. One or more droplets (e.g., sample droplets, reagent droplets) may be positioned in the gap between the two substrates. The gap between top and bottom substrates may be filled with a filler fluid, such as an oil filler fluid, to prevent evaporation of the droplets and to facilitate droplet operations. Examples of suitable oil filler fluids include silicone oil, perfluorinated oil, and hexadecane. The viscosity of the oil filler fluid may range from about 0.5 cSt to about 15 cSt. The oil filler fluid may, for example, be a silicone oil that has a viscosity ranging from about 1 cSt to about 10 cSt. In one example, the oil filler fluid may be 7cSt silicone oil with 0.005%
Span 85.
In one embodiment, reagent droplets may be loaded onto and dried onto electrodes on a droplet actuator. For example, reagent droplets may be deposited atop and dried onto certain droplet operations electrodes (e.g., electrowetting electrodes) of the droplet actuator. Droplet operations are conducted atop droplet operations electrodes on a droplet operations surface. In another example, reagent droplets may be deposited atop and dried onto one or more reservoir electrodes on the droplet actuator. In another embodiment, reagent solutions may be deposited onto a plastic surface of the droplet actuator. In another embodiment, reagent solutions may be deposited into a fluid passage of the droplet actuator leading to a droplet operations surface or gap. In another embodiment, reagent solutions may be deposited in a liquid reservoir of the droplet actuator, where the reservoir has a shape or feature designed to permit the liquid to settle and dry without flowing into and potentially clogging a fluid passage leading from the reservoir onto a droplet operations surface or into a droplet operations gap.
A reagent droplet may include one or more reagents. A reagent droplet may have a volume ranging from about 1 nanoliter (nL) to about 3 milliliters (mL) or from about 5 nL to about 1 mL.
Reagent droplets may be dried directly on a surface of the droplet actuator or combined with reagent stabilization and/or protection compounds prior to drying on the surface. Examples of reagents may include, but are not limited to, beads, proteins, nucleic acids, salts, sugars, and/or surfactants. Specific examples of reagents may include, but are not limited to, an antibody, an Docket No. 212PCT
antibody attached to a bead, a protease (e.g., protease K), a lectin (e.g., Phaseoulus vulgaris agglutinin), a virus, a spore, a bacteria, a fungus, an armored RNA, an armored DNA, a bacteriophage (e.g., MS2), a polymer (e.g., a temperature sensitive polymer), a fluorophore, a lysis reagent, a buffer (e.g., a wash buffer, an elution buffer), a surfactant, and/or a magnetically responsive bead.
A rehydration droplet may include a rehydration buffer and a surfactant for efficient reconstitution of dried reagent droplets. Examples of rehydration buffers include PBS with 0.1%
Tween0 20, PBS with 0.02% Tween0 20, and water with 0.02% Tween0 20. Higher concentrations of surfactant (e.g., 0.1% Tween0 20) provide for more rapid reconstitution of dried reagent spots. In another example, a rehydration droplet may be a droplet of sample fluid to be analyzed. In various embodiments, the volume of a rehydration droplet may range from about picoliters (pL) to about 10 mL or from about 100 pL to about 5 mL or from about 50 nL to about 2 mL or from about 100 nL to about 0.5 mL. In various embodiments, the ratio of a rehydration droplet volume to one electrode may range from about 1 pL:1 electrode to about 5 mL: 1 electrode or from about 10pL: 1 electrode to about 3 mL:1 electrode or from about 1 nL:1 electrode to about 1 mL: 1 electrode or from about 10 nL:1 electrode to about 0.5 mL:1 electrode or from about 50 nL:1 electrode to about 0.3 mL:1 electrode.
Liquid storage modules may be assembled onto a droplet actuator during manufacturing and used to store liquid reagents, such as oil filler fluid (e.g., 7cSt oil with 0.005%
Span 85) and rehydration buffers.
5.1.1 Dry Reagent Storage Existing technologies for drying reagent fluids on a solid surface may be selected and adapted for use on a droplet actuator. In one example, a reagent drying technology may be selected for efficient recovery of dry reagents in an oil-filled droplet actuator. In another example, reagent stabilization and/or protection compounds may be selected such that they do not substantially interfere with the assay and/or droplet operations. In yet another example, a reagent drying technology may be selected for long term stability (shelf-life) at different environmental conditions (e.g., shipping temperatures, humidity, etc). In some cases, reagent droplets are so small that no special drying techniques are required. In yet another example, dispensing operations for reagent reconstitution may be automated such that user intervention is not required.
In one embodiment, the methods of the invention provide for drying reagent solution droplets directly on the surface of a droplet actuator without the addition of reagent stabilization and/or Docket No. 212PCT
protection compounds. Examples of reagent solutions that may be dried directly on a surface and reconstituted via droplet operations using a rehydration droplet may include, but are not limited to, magnetically responsive bead solutions, wash buffers, lysis buffers, elution buffers, IgG (0.6-1.2 mg/mL), BSA (20 mg/mL), MS2 phage stock solutions (1:10 and 1:100 dilutions), and/or lectin solutions (200 pg/mL in PBS).
In another embodiment, the methods of the invention use one or more stabilizing agents in the aqueous reagent droplet for preservation and controlled release of dried assay reagents in a droplet actuator. In one example, the stabilizing agent may be a polymer. In another example, the stabilizing agent may be a sugar matrix. Examples of suitable sugars may include dextrans, sucroses, and/or trehaloses. Trehalose and dextran are two sugars commonly used to stabilize proteins (i.e., preserve enzymatic activity) in dried reagent preparations.
Trehalose has also been shown to enhance reverse transcription (RT)-PCR reactions by reducing DNA
secondary structures and DNA melting temperature by about 2-3 C. Trehalose also provides thermostability to enzymes at higher temperatures. Examples of reagent solutions that may be combined with a stabilizing agent (e.g., trehalose/dextran matrices) prior to drying on a droplet actuator and reconstituted via droplet operations using a rehydration droplet may include, but are not limited to, M52 phage stock solution (1:10 and 1:100 dilutions), lectins (200 pg/mL in PBS) for sample preparation, and/or PCR master mix solutions that may include enzymes, salts, probes, primers, and/or deoxynueleotides.
An example of a generic protocol used to evaluate the use of sugar matrices in storing dried assay reagents on a droplet actuator included the following steps: An aliquot (e.g., about 0.6 ',IL to about 1.5 1.11 or greater) of each test solution was spotted onto a droplet operations electrode (storage electrode) on a Cytop coated bottom substrate of a droplet actuator.
Test solutions included a trehalose/dextran matrix and rhodamine dye for visualization of the test spots.
Examples of trehalose/dextran matrix compositions are shown in Table 1. The test solutions may also include one or more assay reagents (e.g., RT-PCR master mix, magnetically responsive beads, M52). The bottom substrate was incubated in an oven at about 35-37 C
overnight to dry the test spots onto the bottom substrate surface. The following day, the droplet actuator was assembled and stored in a desiccator at ambient conditions until use. After a certain period of time, oil filler fluid and rehydration buffer (e.g., water or PBS) were loaded onto the droplet actuator. Dried reagent spots were rehydrated by electrowetting a droplet of rehydration buffer to the location of the dried reagent. An example of a reconstitution protocol is described in reference to Figure 3.

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Table 1. Sugar matrices Sugar solution Trehalose % (w/v)* Dextran % (w/v)*

* Final concentration after reconstitution Parameters assessed in evaluating sugar matrices for dry reagent storage on a droplet actuator included reagent droplet volume, microfluidic transport (e.g., viscosity, inadvertent droplet splitting), and reconstitution time. The volume of reagent droplet that can be loaded on a droplet operations electrode is dependent on the composition of the trehalose/dextran sugar matrix. For example, greater than about 1.5 uL of reagent solution containing 40%
trehalose/20% dextran may be larger than the typical gap height of a droplet actuator (i.e., 275 pm). To maintain droplet integrity after reconstitution of a dried reagent, the reconstituted droplet may be transported away from the storage electrode as a 2X droplet. Reconstituted droplets may be evaluated using an appropriate digital microfluidic assay protocol and/or removed from the droplet actuator and evaluated using an appropriate on-bench protocol.
In one example, RT-PCR master mix was combined with sugar matrices and evaluated for reconstitution and electrowetting on a droplet actuator as described in reference to Table 1. Two RT-PCR stock solutions containing different concentrations of enzyme and master mix were used. One RT-PCR stock solution included 10x enzyme and 5x master mix with very little glycerol and the second RT-PCR stock solution included 2.3x enzyme and 2.3x master mix with glycerol. Dilutions of the 10x enzyme/5x master mix stock solution are shown in Table 2.
Aliquots of stock solution and dilutions were combined with aliquots of the sugar matrices shown in Table 1. A 0.6 L aliquot of each combined solution was spotted onto individual droplet operations electrodes of a droplet actuator as described in reference to Table 1 and rehydrated using an electrode pulsing protocol described in reference to Figure 3. Over 180 droplets of dried RT-PCR master mix/sugar matrix solutions were assessed. All dried droplets were reconstituted in about 3 minutes or less. Each type of dried master mix solution was reconstituted in approximately similar time frames based on sugar content as shown in Table 3.

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Table 2. RT-PCR master mix dilutions Master mix solution 1 6x enzyme 3x master mix (no glycerol) 2 2.73x enzyme 1.365x master mix (no glycerol) 3 1.365x enzyme 0.6825x master mix (no glycerol) 4 1.365x enzyme 1.365x master mix (glycerol) * Final concentration after reconstitution Table 3. Sugar matrices Sugar solution Trehalose % (w/v)* Dextran % (w/v)* Pulses (seconds) A 40 0 ¨50 20 0 ¨50 0 ¨40 40 20 ¨150 10 ¨125 0 0 ¨50 Figure 1 illustrates a top view of a portion of a droplet actuator 100 and also shows an example of an array of reagent droplets dried on certain droplet operations electrodes. In this example, the reagent droplets were loaded onto a droplet actuator and dried as described in reference to Table 1. Droplet actuator 100 may include a bottom substrate 110 and a top substrate (not shown) that are separated by a gap. The gap may be filled with a filler fluid, such as silicone oil (not shown).
Bottom substrate 110 may, for example, be a printed circuit board (PCB). An arrangement, such as a path and/or array of droplet operations electrodes 112 (e.g., electrowetting electrodes) may be formed as a part of the bottom substrate 110, and arranged to conduct droplet operations in the droplet operations gap. Droplet operations are generally conducted atop droplet operations electrodes 112 in the droplet operations gap. One or more reagent solution droplets 114 may be loaded onto certain droplet operations electrodes 112 (storage electrodes) and dried for storage.
Reagent solution droplets 114 may, for example, be from about 0.6 1.IL to about 1 L in volume.
As reagent solution droplets 114 were dried, they became localized between adjacent electrodes.
This effect is likely due to the topography of droplet operations electrodes 112. Divots, patterned surfaces, hydrophilic regions, or other features may be used to localize dried reagents directly atop electrodes; however, localization of the dried reagent droplets between electrodes does not appear to affect reconstitution of the dried reagent droplet.

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Loading and drying a reagent solution droplet onto an on-actuator reservoir was also evaluated.
Fi2ure 2 illustrates a top view of a portion of a droplet actuator 200 and also shows an example of dried reagent droplets on an on-actuator reagent dispensing electrode.
Droplet actuator 200 may include a bottom substrate 210 and a top substrate 212 that are separated by a gap. The gap may be filled with a filler fluid, such as silicone oil (not shown). Bottom substrate 210 may, for example, be a PCB. Bottom substrate 210 may include a reagent dispensing electrode 214.
Reagent dispensing electrode 214 may be segmented into multiple individually controlled electrodes. The combined features of bottom substrate 210 and top substrate 212 form an on-actuator reagent reservoir 216. On-actuator reagent reservoir 216 has, for example two input ports 218 (e.g., input ports 218a and 218b). Therefore, input ports 218a and 218b are integrated into top substrate 112 in proximity to at least a portion of reagent dispensing electrode 214. A
port (e.g., input ports 218a and 218b) provides an entrance/exit (opening) to the droplet operations gap. Liquid may flow through the port into any portion of the gap, e.g., into a reservoir region of the gap or onto a droplet operations pathway. A port may be used to fill the gap with filler fluid. However, in most cases, a reagent fluid or sample fluid flowing through a port should come into sufficient proximity with an electrode, such that the electrode can be used to conduct one or more droplet operations using the liquid, such as droplet transport, splitting, and/or dispensing.
On-actuator reagent reservoir 216 and input ports 218a and 218b are designed such that the dried reagent droplet is accessible to electrowetting for rehydration. One or more reagent solution droplets 220 may be loaded onto reagent dispensing electrode 214 and dried for storage. For example, a reagent solution droplet 220a may be loaded via input port 218a onto reagent dispensing electrode 214 and dried for storage. Further, a reagent solution droplet 220b may be loaded via input port 218b onto reagent dispensing electrode 214 and dried for storage. Reagent solution droplets 220 may, for example, be about 20 L in volume.
5.1.2 Reagent Reconstitution Protocols The reagent reconstitution methods of the invention use digital microfluidic liquid handling protocols for recovery of dried reagents. In one embodiment, the methods of the invention use electrowetting mediated droplet operations for manipulating an aqueous droplet for recovery of a dried reagent droplet. Importantly, the inventors have discovered that the dried reagents can be coated with an oil filler fluid and substantially fully reconstituted by transporting a droplet through the oil and into contact with the dried reagent.

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Droplet voltages in electrowetting mediated droplet operations may, for example, range from about 0.5 volts to about 1000 volts; or from about 2 volts to about 700 volts;
or from about 4 volts to about 500 volts. Electrowetting mediated droplet operations use AC voltage with frequencies ranging, for example, from about 0.1 Hz to about 10000 Hz; or from about 1 Hz to about 1000 Hz; or from about 2 Hz to about 500 Hz. In another embodiment, the methods of the invention use dielectrophoresis mediated droplet operations.
The reagent reconstitution methods of the invention provide for recovery of greater than about 50% of the dried reagent droplet; or greater than about 80% of the dried reagent droplet; or greater than about 90% of the dried reagent droplet; or greater than about 95%
of the dried reagent droplet; or greater than about 99% of the dried reagent droplet.
The reagent reconstitution methods of the invention provide for reconstitution (recovery) of substantially all of a dried reagent droplet in less than about 15 minutes; or less than about 7 minutes; or less than about 5 minutes; or less than about 3 minutes.
In a preferred embodiment, an electrode pulsing protocol (i.e., electrowetting mediated droplet pulsing) may be used to manipulate an aqueous droplet for reconstitution of a dried reagent droplet. In various embodiments, the pulsing may have an ON/OFF pulsing ratio from about 1:1 to about 20:1; or from about 5:1 to about 15:1; or from about 8:1 to 12:1. In various embodiments, the electrowetting mediated pulsing may have an ON/OFF pulsing ratio from about 1:1 to about 20:1 wherein each pulsing cycle is from about 1 nanosecond to 1 minute; or from about 1 millisecond to about 30 seconds; or from about 100 milliseconds to about 5 seconds.
Figures 3A, 3B, and 3C illustrate top views of an example of a portion of an electrode arrangement 300 of a droplet actuator and show a process of performing a reagent reconstitution protocol on a droplet actuator. The method of the invention of Figures 3A
through 3C is an example of a reagent reconstitution protocol in which electrode pulsing is used to reconstitute a dried reagent stored on a certain droplet operations electrode on a droplet actuator.
Electrode arrangement 300 may include a path and/or array of droplet operations electrodes 310 (e.g., electrowetting electrodes) that is configured to conduct droplet operations. Droplet operations are conducted atop droplet operations electrodes 310 on a droplet operations surface.
A dried concentrated reagent droplet 312 may be present at a certain droplet operations electrode 310. In one example, dried reagent 312 may be a reverse transcription-polymerase chain reaction (RT-PCR) master mix droplet that includes enzyme, salts, primer pairs, deoxynucleotides, and probes that are sufficient for RT-PCR amplification. Dried reagent 312 may, for example, be Docket No. 212PCT
dried in place by manual spotting or by an automated printing device. An example of a process of reconstituting dried assay reagents using electrode pulsing may include, but is not limited to, the following steps. Reagent droplet 312 may be coated with a filler fluid, such as an oil filler fluid, such as a silicone oil filler fluid.
In one step and referring to Figure 3A, a dried reagent 312 is present at a certain droplet operations electrode 310 and is ready for reconstitution. An oil filler fluid (not shown) coats the surface including the dried reagent 312. Dried reagent 312 provides a high concentration of dried regent. A rehydration droplet 314 is present at another droplet operations electrode 310 and is ready to be transported via droplet operations toward dried reagent 312.
Rehydration droplet 314 may be a lx droplet, meaning that its footprint is approximately equal to the area of one droplet operations electrode 310. In one example, rehydration droplet 314 may be a rehydration buffer (e.g., water with 0.02% Tween0 20) that is used to reconstitute a dried reagent droplet prior to mixing and incubation with a sample droplet (not shown). In another example, rehydration droplet 314 may be a sample droplet that is used to reconstitute dried reagent 312.
In another step and referring to Figure 3B, an incubation process is provided in which rehydration droplet 314 is transported using droplet operations along droplet operations electrodes 310 and into contact with dried reagent 312. In doing so, dried reagent 312 is rehydrated to form a reaction droplet 316. To facilitate rehydration of reagent droplet 312, the droplet operations electrode 310 holding reagent droplet 312 may be repeatedly pulsed ON and OFF.
For example, the droplet operations electrode 310 may be pulsed ON for about 0.9 seconds (e.g., 300 V, 30 Hz) and OFF for about 0.1 second. The pulsing may be repeated from about 40 to about 150 times.
Increased time with the droplet operations electrode 310 ON provides for more efficient wetting of the electrode and increases the time the rehydration droplet has to resuspend the dried reagent.
Pulsing also introduces convection within the droplet which further facilitates rehydration of a dried reagent droplet. In an alternative embodiment, the dried reagent may be provided in a conventional hydrophobic microfluidic channel, and an electrode positioned adjacent the channel may be used to pulse a droplet in the channel (surrounded by an oil filler fluid) in the presence of a dried reagent on a surface of the channel to cause the reagent to be reconstituted in the channel.
In another step and referring to Figure 3C, after an incubation period sufficient to reconstitute the dried reagent (e.g., 3 minutes or less), reaction droplet 316 is transported using droplet operations along droplet operations electrodes 310 for further processing. Additionally, reaction droplet 316 is transported as a 2X droplet, meaning that its footprint is approximately 2 times the area of one droplet operations electrode 310. Transport of reaction droplet 316 as a 2X
droplet prevents inadvertent droplet splitting.

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In another embodiment, a droplet shuttling protocol (i.e., electrowetting mediated droplet shuttling) may be used to manipulate an aqueous droplet for reconstitution of a dried reagent droplet. In one example, an aqueous droplet may be shuttled across a dried reagent droplet by turning ON two adjacent droplet operations electrodes. In another example, an aqueous droplet may be shuttled across a dried reagent droplet by turning ON one adjacent droplet operations electrode. The frequency of droplet shuttling across a dried reagent droplet may, for example, range from about once every 10 milliseconds to about once every 20 seconds; or from about once every 100 milliseconds to about once every 15 seconds; or from about once every 200 milliseconds to about once every 10 seconds.
Fi2ures 4A throu2h 4E illustrate top views of electrode arrangement 300 of Figure 3A and show another process of performing a reagent reconstitution protocol on a droplet actuator. The method of the invention of Figures 4A through 4E is an example of a reagent reconstitution protocol in which shuttling an aqueous droplet back and forth is used to reconstitute a dried reagent droplet stored on a certain droplet operations electrode on a droplet actuator. An example of a process of reconstituting dried assay reagents using droplet shuttling may include, but is not limited to, the following steps.
In one step and referring to Figure 4A, a dried reagent 312 is present at a certain droplet operations electrode 310 and is ready for reconstitution. Dried reagent 312 provides a high concentration of dried regent. A rehydration droplet 314 is present at another droplet operations electrode 310 and is ready to be transported via droplet operations toward dried reagent 312.
Rehydration droplet 314 may be a lx droplet.
In another step and referring to Figure 4B, rehydration droplet 314 is transported using droplet operations along droplet operations electrodes 310 and into contact with dried reagent 312. In doing so, dried reagent 312 is rehydrated to form a reaction droplet 316.
In other steps and referring to Figures 4C, 4D, and 4E, a process is provided of shuttling reaction droplet 316 back and forth over the droplet operations electrode 310 holding the dried reagent 312 in order to fully rehydrate dried reagent 312. Reaction droplet 316 is shuttled as a 2X droplet by turning ON the two droplet operations electrodes 310 adjacent to dried reagent 312. Transport of reaction droplet 316 as a 2X droplet prevents inadvertent droplet splitting. As reaction droplet 316 is shuttled back and forth over the droplet operations electrode 310 holding the dried reagent 312, dried reagent 312 is rehydrated. The shuttling process may be repeated any number of times sufficient for rehydration of dried reagent 312. Reaction droplet 316 is now ready for further processing.

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5.1.3 Liquid Reagent Storage Reconstitution buffers as well as certain other reagents may be stored in a liquid form on a droplet actuator. One or more physical structures and/or features may be incorporated into the droplet actuator and used for containment of the liquid reagents. In one example, the physical structures and/or features may be selected to provide containment of liquid reagents during shipment. Large volumes of reagent fluids, such as wash buffers, may be stored in separate reservoirs (e.g., reservoirs atop the top substrate). Reagents suitable for liquid storage may be selected based on shelf-life studies.
Liquid reagents preloaded and stored on a droplet actuator may be surrounded by a protective layer so that they are contained until the droplet actuator is used. For example, the protective layer may be a frangible layer (pressure sensitive) such that insertion of the droplet actuator into the instrument actuates breakage of the protective layer. As the protective layer is broken, liquid reagents may, for example, be released into proximity of certain droplet operations electrodes where they may be dispensed by droplet operations. In another example, liquid reagents may be released into one or more adjacent compartments in which dried reagents are stored. As the liquid enters the compartment the dried reagent is reconstituted (i.e., rehydrated).
5.2 Example Application The methods of the invention are used to provide a point-of-care (POC) diagnostic device for integrated (i.e., sample-to-answer) sample preparation and multiplexed detection of HIV. In this example, dried reagents for sample preparation, immunoassays for antibodies to HIV, and RT-PCR for determination of HIV viral load may be stored on a droplet actuator.
Liquid reagents, e.g., wash buffers and oil filler fluid may also be stored on the droplet actuator.
5.3 Systems It will be appreciated that various aspects of the invention may be embodied as a method, system, computer readable medium, and/or computer program product. Aspects of the invention may take the form of hardware embodiments, software embodiments (including firmware, resident software, micro-code, etc.), or embodiments combining software and hardware aspects that may all generally be referred to herein as a "circuit," "module" or "system."
Furthermore, the methods of the invention may take the form of a computer program product on a computer-usable storage medium having computer-usable program code embodied in the medium.

Docket No. 212PCT
Any suitable computer useable medium may be utilized for software aspects of the invention.
The computer-usable or computer-readable medium may be, for example but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. The computer readable medium may include transitory and/or non-transitory embodiments. More specific examples (a non-exhaustive list) of the computer-readable medium would include some or all of the following: an electrical connection having one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a transmission medium such as those supporting the Internet or an intranet, or a magnetic storage device. Note that the computer-usable or computer-readable medium could even be paper or another suitable medium upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner, if necessary, and then stored in a computer memory. In the context of this document, a computer-usable or computer-readable medium may be any medium that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.
Program code for carrying out operations of the invention may be written in an object oriented programming language such as Java, Smalltalk, C++ or the like. However, the program code for carrying out operations of the invention may also be written in conventional procedural programming languages, such as the "C" programming language or similar programming languages. The program code may be executed by a processor, application specific integrated circuit (ASIC), or other component that executes the program code. The program code may be simply referred to as a software application that is stored in memory (such as the computer readable medium discussed above). The program code may cause the processor (or any processor-controlled device) to produce a graphical user interface ("GUI").
The graphical user interface may be visually produced on a display device, yet the graphical user interface may also have audible features. The program code, however, may operate in any processor-controlled device, such as a computer, server, personal digital assistant, phone, television, or any processor-controlled device utilizing the processor and/or a digital signal processor.
The program code may locally and/or remotely execute. The program code, for example, may be entirely or partially stored in local memory of the processor-controlled device. The program code, however, may also be at least partially remotely stored, accessed, and downloaded to the processor-controlled device. A user's computer, for example, may entirely execute the program code or only partly execute the program code. The program code may be a stand-alone software Docket No. 212PCT
package that is at least partly on the user's computer and/or partly executed on a remote computer or entirely on a remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through a communications network.
The invention may be applied regardless of networking environment. The communications network may be a cable network operating in the radio-frequency domain and/or the Internet Protocol (IP) domain. The communications network, however, may also include a distributed computing network, such as the Internet (sometimes alternatively known as the "World Wide Web"), an intranet, a local-area network (LAN), and/or a wide-area network (WAN). The communications network may include coaxial cables, copper wires, fiber optic lines, and/or hybrid-coaxial lines. The communications network may even include wireless portions utilizing any portion of the electromagnetic spectrum and any signaling standard (such as the IEEE 802 family of standards, GSM/CDMA/TDMA or any cellular standard, and/or the ISM
band). The communications network may even include powerline portions, in which signals are communicated via electrical wiring. The invention may be applied to any wireless/wireline communications network, regardless of physical componentry, physical configuration, or communications standard(s).
Certain aspects of invention are described with reference to various methods and method steps. It will be understood that each method step can be implemented by the program code and/or by machine instructions. The program code and/or the machine instructions may create means for implementing the functions/acts specified in the methods.
The program code may also be stored in a computer-readable memory that can direct the processor, computer, or other programmable data processing apparatus to function in a particular manner, such that the program code stored in the computer-readable memory produce or transform an article of manufacture including instruction means which implement various aspects of the method steps.
The program code may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed to produce a processor/computer implemented process such that the program code provides steps for implementing various functions/acts specified in the methods of the invention.

Docket No. 212PCT

6 Concluding Remarks The foregoing detailed description of embodiments refers to the accompanying drawings, which illustrate specific embodiments of the invention. Other embodiments having different structures and operations do not depart from the scope of the present invention. The term "the invention" or the like is used with reference to certain specific examples of the many alternative aspects or embodiments of the applicants' invention set forth in this specification, and neither its use nor its absence is intended to limit the scope of the applicants' invention or the scope of the claims. This specification is divided into sections for the convenience of the reader only.
Headings should not be construed as limiting of the scope of the invention. The definitions are intended as a part of the description of the invention. It will be understood that various details of the present invention may be changed without departing from the scope of the present invention.
Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

1. A method of providing a droplet comprising one or more reagents, the method comprising:
(a) depositing a first aqueous droplet comprising the one or more reagents on a surface;
(b) drying the droplet to yield a dried composition on the surface comprising the one or more reagents;
(c) covering the dried composition with oil; and (d) causing a second aqueous droplet in the oil to contact the dried composition and thereby resuspend one or more reagents.

2. The method of any of claims 1 and following, wherein the aqueous droplet further comprises a stabilizing agent.

3. The method of any of claims 2 and following, wherein the stabilizing agent comprises a sugar.

4. The method of any of claims 3 and following, wherein the sugar is selected from the group consisting of: dextrans, sucroses, and trehaloses.

5. The method of any of claims 2 and following, wherein the stabilizing agent comprises a polymer.

6. The method of any of claims 2 and following, wherein the one or more reagents comprise a reagent selected from the group consisting of: beads, proteins, nucleic acids, salts, sugars, and surfactants.

7. The method of any of claims 1 and following, wherein the one or more reagents comprise comprises reagents selected for amplifying a target nucleic acid when combined with a sample comprising the target.

8. The method of any of claims 1 and following, wherein the one or more reagents comprises an antibody.

9. The method of any of claims 1 and following, wherein the one or more reagents comprises an antibody attached to a bead.

10. The method of any of claims 1 and following, wherein the one or more reagents comprises a protease.

11. The method of any of claims 1 and following, wherein the one or more reagents comprises protease K.

12. The method of any of claims 1 and following, wherein the one or more reagents comprises a lectin.

13. The method of any of claims 1 and following, wherein the one or more reagents comprises a phaseoulus vulgaris agglutinin.

14. The method of any of claims 1 and following, wherein the one or more reagents comprises a bead.

15. The method of any of claims 1 and following, wherein the one or more reagents comprises a virus.

16. The method of any of claims 1 and following, wherein the one or more reagents comprises a spore.

17. The method of any of claims 1 and following, wherein the one or more reagents comprises a bacteria.

18. The method of any of claims 1 and following, wherein the one or more reagents comprises a fungus.

19. The method of any of claims 1 and following, wherein the one or more reagents comprises an armored RNA.

20. The method of any of claims 1 and following, wherein the one or more reagents comprises an armored DNA.

21. The method of any of claims 1 and following, wherein the one or more reagents comprises a bacteriophage.

22. The method of any of claims 1 and following, wherein the one or more reagents comprises MS2.

23. The method of any of claims 1 and following, wherein the one or more reagents comprises a polymer.

24. The method of any of claims 1 and following, wherein the one or more reagents comprises a temperature sensitive polymer.

25. The method of any of claims 1 and following, wherein the one or more reagents comprises a fluorophore.

26. The method of any of claims 1 and following, wherein the one or more reagents comprises a nucleic acid.

27. The method of any of claims 1 and following, wherein the one or more reagents comprises a lysis reagent.

28. The method of any of claims 1 and following, wherein the one or more reagents comprises a buffer.

29. The method of any of claims 1 and following, wherein the one or more reagents comprises a magnetically responsive bead.

30. The method of any of claims 1 and following, wherein the second aqueous droplet has a volume ranging from about 10 picoliters to about 10 milliliters.

31. The method of any of claims 1 and following, wherein the first aqueous droplet has a volume ranging from about one nanoliter to about 3 milliliters.

32. The method of any of claims 1 and following, wherein the first aqueous droplet has a volume ranging from about five nanoliters to about one milliliter.

33. The method of any of claims 1 and following, wherein the second aqueous droplet has a volume ranging from about 10 picoliters to about 10 milliliters.

34. The method of any of claims 1 and following, wherein the second aqueous droplet has a volume ranging from about 100 picoliters to about 5 milliliters.

35. The method of any of claims 1 and following, wherein the second aqueous droplet has a volume ranging from about 50 nanoliters to about 2 milliliters.

36. The method of any of claims 1 and following, wherein the second aqueous droplet has a volume ranging from about 100 nanoliters to about 0.5 milliliters.

37. The method of any of claims 1 and following, wherein the first aqueous droplet comprises a surfactant.

38. The method of any of claims 1 and following, wherein the second aqueous droplet comprises a surfactant.

39. The method of any of claims 1 and following, wherein the surface comprises an electrode.

40. The method of any of claims 1 and following, wherein the surface comprises a plastic surface.

41. The method of any of claims 1 and following, wherein the surface is hydrophobic.

42. The method of any of claims 1 and following, wherein the surface comprises an electrode and the dried composition is positioned on the electrode.

43. The method of any of claims 42 and following, wherein the electrode is a component of an array of electrodes.

44. The method of any of claims 43 and following, wherein the array of electrodes is configured to conduct droplet operations on the surface.

45. The method of any of claims 42 in which the second aqueous droplet and the electrode have a drolet volume:electrode ratio ranging from about 1 picoliter: 1 electrode to about 5 milliliters: 1 electrode.

46. The method of any of claims 42 in which the second aqueous droplet and the electrode have a drolet volume:electrode ratio ranging from about 10 picoliters: 1 electrode to about 3 milliliters: 1 electrode.

47. The method of any of claims 42 in which the second aqueous droplet and the electrode have a drolet volume:electrode ratio ranging from about 1 nanoliter: 1 electrode to about 1 milliliter: 1 electrode.

48. The method of any of claims 42 in which the second aqueous droplet and the electrode have a drolet volume:electrode ratio ranging from about 10 nanoliters: 1 electrode to about 0.5 milliliters: 1 electrode.

49. The method of any of claims 42 in which the second aqueous droplet and the electrode have a drolet volume:electrode ratio ranging from about 50 nanoliters: 1 electrode to about 0.3 milliliters: 1 electrode.

50. The method of any of claims 1 and following, wherein step (d) is mediated by electrowetting droplet operations.

51. The method of any of claims 50 and following, wherein the electrowetting droplet operations are mediated using voltages ranging from about 0.5 volt to about 1000 volts.

52. The method of any of claims 50 and following, wherein the electrowetting droplet operations are mediated using voltages ranging from about 2 volts to about 700 volts.

53. The method of any of claims 50 and following, wherein the electrowetting droplet operations are mediated using voltages ranging from about 4 volts to about 500 volts.

54. The method of any of claims 50 and following, wherein the electrowetting droplet operations are mediated using AC voltage at a frequency ranging from about 0.1 Hz to about 10000 Hz.

55. The method of any of claims 50 and following, wherein the electrowetting droplet operations are mediated using AC voltage at a frequency ranging from about 1 Hz to about 1000 Hz.

56. The method of any of claims 50 and following, wherein the electrowetting droplet operations are mediated using AC voltage at a frequency ranging from about 2 Hz to about 500 Hz.

57. The method of any of claims 1 and following, wherein step (d) is mediated by dielectrophoresis mediated droplet operations.

58. The method of any of claims 1 and following, further comprising pulsing the second droplet.

59. The method of any of claims 58 and following, wherein the pulsing is mediated by an electrode.

60. The method of any of claims 59 and following, wherein the pulsing is electrowetting mediated.

61. The method of any of claims 60 and following, wherein the pulsing has an on/off pulsing ratio from 1:1 to 20:1.

62. The method of any of claims 60 and following, wherein the pulsing has an on/off pulsing ratio from 5:1 to 15:1.

63. The method of any of claims 60 and following, wherein the pulsing has an on/off pulsing ratio from 8:1 to 12:1.

64. The method of any of claims 60 and following, wherein the pulsing has an on/off pulsing ratio from 1:1 to 20:1 and following, wherein each pulsing cycle is 1 nanosecond to 1 minute.

65. The method of any of claims 60 and following, wherein the electrowetting mediated pulsing has an on/off pulsing ratio and following, wherein each pulsing cycle is 1 millisecond to 30 seconds.

66. The method of any of claims 60 and following, wherein the electrowetting mediated pulsing has an on/off pulsing ratio and following, wherein each pulsing cycle is 100 milliseconds to 5 seconds.

67. The method of any of claims 1 and following, wherein the dried composition is substantially dissolved in less than about 15 minutes.

68. The method of any of claims 1 and following, wherein the dried composition is substantially dissolved in less than about 7 minutes.

69. The method of any of claims 1 and following, wherein the dried composition is substantially dissolved in less than about 5 minutes.

70. The method of any of claims 1 and following, wherein the dried composition is substantially dissolved in less than about 3 minutes.

71. The method of any of claims 1 and following, wherein the method further comprises shuttling the second droplet back and forth across the dried composition.

72. The method of any of claims 71 and following, wherein the shuttling of the second droplet across the dried composition is mediated by electrodes.

73. The method of any of claims 71 and following, wherein the shuttling of the second droplet is electrowetting mediated.

74. The method of any of claims 71 and following, wherein the shuttling of the second droplet across the dried composition is mediated by turning on two adjacent electrodes.

75. The method of any of claims 71 and following, wherein the shuttling of the second droplet across the dried composition is mediated by turning on just one electrode.

76. The method of any of claims 71 and following, wherein the frequency of second droplet passing the dried composition ranges from about once every 10 milliseconds to once every 20 seconds.

77. The method of any of claims 71 and following, wherein the frequency of second droplet passing the dried composition ranges from about once every 100 milliseconds to once every 15 seconds.

78. The method of any of claims 71 and following, wherein the frequency of second droplet passing the dried composition ranges from about once every 200 milliseconds to once every 10 seconds.

79. (78) The method in claim 1 and following, wherein the oil is an oil with a viscosity of ranging from about 0.5 cSt to about 15 cSt.

80. The method of any of claims 1 and following, wherein the oil is a silicone oil with a viscosity of ranging from about 1 cSt to about 10 cSt.

81. The method of any of claims 1 and following, wherein the oil is selected from the group consisting of silicone oil, perfluorinated oil, and hexadecane.

82. The method of any of claims 1 and following, wherein greater than 50%
of the dried composition is resuspended by the second droplet.

83. The method of any of claims 1 and following, wherein greater than 80%
of the dried composition is resuspended by the second droplet.

84. The method of any of claims 1 and following, wherein greater than 90%
of the dried composition is resuspended by the second droplet.

85. The method of any of claims 1 and following, wherein greater than 95%
of the dried composition is resuspended by the second droplet.

86. The method of any of claims 1 and following, wherein greater than 99%
of the dried composition is resuspended by the second droplet.

87. The method of any of claims 1 and following, further comprising conducting an assay with the resuspended dried composition.

88. The method of any of claims 1 and following, further comprising conducting an immunoassay with the resuspended dried composition.

89. The method of any of claims 1 and following, further comprising conducting cell handling methods with the resuspended dried composition.

90. The method of any of claims 1 and following, further comprising an electrochemical assay with the resuspended dried composition.

91. The method of any of claims 1 and following, further comprising conducting an enzymatic assay with the resuspended dried composition.

92. The method of any of claims 1 and following, further comprising conducting a polymerase chain reaction assay (PCR) with the resuspended dried composition.

93. The method of any of claims land following, further comprising conducting a reverse transcriptase polymerase chain reaction (RT-PCR) assay with the resuspended dried composition.

94. The method of any of claims 1 and following, further comprising conducting sample preparation with the resuspended dried composition.

95. The method of any of claims 1 and following, further comprising conducting sample preparation on blood with the resuspended dried composition.

96. The method of any of claims 1 and following, further comprising agglutinating blood cells with the resuspended dried composition.

97. The method of any of claims 1 and following, further comprising agglutinating red blood cells with the resuspended dried composition.

98. The method of any of claims 1 and following, further comprising lysing cells with the resuspended dried composition.

99. The method of any of claims 1 and following, further comprising lysing spores with the resuspended dried composition.

100. The method of any of claims 1 and following, further comprising lysing bacteria with the resuspended dried composition.

101. The method of any of claims 1 and following, further comprising lysing fungi with the resuspended dried composition.

102. The method of any of claims 1 and following, further comprising lysing blood cells with the resuspended dried composition.

103. The method of any of claims 1 and following, further comprising lysing virus with the resuspended dried composition.

104. The method of any of claims 1 and following, further comprising lysing armored RNA
with the resuspended dried composition.

105. The method of any of claims 1 and following, further comprising lysing armored DNA
with the resuspended dried composition.

106. The method of any of claims 1 and following, further comprising binding nucleic acids to beads with the resuspended dried composition.

107. The method of any of claims 1 and following, further comprising binding antigens to beads with the resuspended dried composition.

108. The method of any of claims 1 and following, further comprising washing the sample with the resuspended dried composition.

109. The method of any of claims 1 and following, further comprising washing beads with the resuspended dried composition.

110. The method of any of claims 1 and following, further comprising eluting nucleic acids with the resuspended dried composition.

111. The method of claim 1 wherein the first aqueous droplet comprises reagents for nucleic acid amplification having a volume in a range of about 1 nanoliter to about 20 microliters .

112. The method of claim 111 wherein the first aqueous droplet comprises trehalose with a concentration in a range of about 0%w/v to about 70% w/v.

113. The method of claim 111 wherein the first aqueous droplet comprises trehalose with a concentration in a range of about 0%w/v to about 40% w/v.

114. The method of claim 111 wherein the first aqueous droplet comprises dextran with a concentration in a range of about 0%w/v to about 50% w/v.

115. The method of claim 111 wherein the first aqueous droplet comprises dextran with a concentration in a range of about 0%w/v to about 30% w/v.

116. The method of claim 111 wherein the first aqueous droplet comprises primers with a concentration in a range of about 0.05 µM to about 5 µM.

117. The method of claim 111 wherein the first aqueous droplet comprises probes with a concentration in a range of about 0.05 µM to about 2 µM.

118. The method of claim 111 wherein the first aqueous droplet comprises manganese acetate with a concentration in a range of about 0.1 mM to about 10 mM.

119. The method of claim 111 wherein the first aqueous droplet comprises enzymes capable of PCR with a concentration in a range of about 0.2x to about 15x.

120. The method of claim 111 wherein the first aqueous droplet comprises enzymes capable of RT-PCR with a concentration in a range of about 0.2x to about 15x.

121. The method of claim 111 wherein the first aqueous droplet comprises RNase inhibitors with a concentration in a range of about 0.2x to about 20x.

122. The method of claim 111 wherein the first aqueous droplet comprises DNase inhibitors with a concentration in a range of about 0.2x to about 20x.

123. The method of claim 1 wherein the first aqueous droplet comprises armored RNA with a volume that ranges between about 20 nanoliters and about 100 microliters.

124. The method of claim 123 wherein the concentration of armored RNA is about 10^9 to about 10^2/mL.

125. The method of claim 1 wherein the first aqueous droplet comprises armored RNA and one or more sugars having a volume between about 20 nanoliters and about 100 microliters .

126. The method of claim 125 wherein the concentration of armored RNA is in the range of about 10^9 to about 10^2/mL.

127. The method of claim 125 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

128. The method of claim 125 wherein the one or more sugars comprise dextran and/or trehalose .

129. The method of claim 125 wherein the one or more sugars comprise trehalose with a concentration ranging from about 0% w/v to about 70% w/v.

130. The method of claim 125 wherein the one or more sugars comprises trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

131. The method of claim 125 wherein the one or more sugars comprises dextran having a concentration ranging from about 0% w/v to about 60% w/v.

132. The method of claim 125 wherein the one or more sugars comprises dextran having a concentration ranging from about 0% w/v to about 20% w/v.

133. The method of claim 1 wherein the first aqueous droplet comprises a virus with a volume that ranges between about 20 nanoliters and about 100 microliters.

134. The method of claim 133 wherein the concentration of virus is in the range of about 10^9 to about 10^2 cfu /mL.

135. The method of claim 1 wherein the first aqueous droplet comprises a virus and one or more sugars with a volume that ranges between about 20 nanoliters and about microliters .

136. The method of claim 135 wherein the concentration of virus is in the range of about 10^9 to about 10^2 cfu /mL.

137. The method of claim 135 wherein the concentration of the one or more sugars ranges from about 0% w/v to about 70% w/v.

138. The method of claim 135 wherein the one or more sugars comprise dextran and/or trehalose .

139. The method of claim 135 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

140. The method of claim 135 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

141. The method of claim 135 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

142. The method of claim 135 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

143. The method of claim 1 wherein the first aqueous droplet comprises MS2 with a volume that ranges between about 20 nanoliters and about 100 microliters.

144. The method of claim 143 wherein the concentration of MS2 is in the range of about 10^9 to about 10^2 cfu /mL.

145. The method of claim 1 wherein the first aqueous droplet comprises MS2 and one or more sugars with a volume that ranges between about 20 nanoliters and about 100 microliters.

146. The method of claim 145 wherein the concentration of MS2 is in the range of about 10^9 to about 10^2 cfu /mL.

147. The method of claim 145 wherein the concentration of the one or more sugars ranges from about 0% w/v to about 70% w/v.

148. The method of claim 145 wherein the one or more sugars comprise dextran and/or trehalose .

149. The method of claim 145 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

150. The method of claim 145 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

151. The method of claim 145 wherein the one or more sugars comprise dextran with a concentration ranging from about 0% w/v to about 60% w/v.

152. The method of claim 145 wherein the one or more sugars comprise dextran with a concentration ranging from about 0% w/v to about 20% w/v.

153. The method of claim 1 wherein the first aqueous droplet comprises armored DNA with a volume that ranges between about 20 nanoliters and about 100 microliters.

154. The method of claim 153 wherein the concentration of armored DNA is in the range of about 10^9 to about 10^2 copies/mL.

155. The method of claim 1 wherein the first aqueous droplet comprises armored DNA with a volume that ranges between 20 nanoliters and 100 microliters and one or more sugars having a volume between about 20 nanoliters and about 100 microliters.

156. The method of claim 155 wherein the concentration of armored DNA is in the range of about 10^9 to about 10^2 copies/mL.

157. The method of claim 155 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

158. The method of claim 155 wherein the one or more sugars comprise dextran and/or trehalose .

159. The method of claim 155 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

160. The method of claim 155 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

161. The method of claim 155 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

162. The method of claim 155 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

163. The method of claim 1 wherein the first aqueous droplet comprises a spore having a volume that ranges between about 20 nanoliters and about 100 microliters.

164. The method of claim 163 wherein the concentration of the spore is in the range of about 10^9 to about 10^1/mL.

165. The method of claim 1 wherein the first aqueous droplet comprises a spore and one or more sugars having a volume that ranges between about 20 nanoliters and about microliters .

166. The method of claim 165 wherein the concentration of the spore is in the range of about 10^9 to about 10^1/mL.

167. The method of claim 165 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

168. The method of claim 165 wherein the one or more sugars comprise dextran and/or trehalose .

169. The method of claim 165 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

170. The method of claim 165 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

171. The method of claim 165 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

172. The method of claim 165 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

173. The method of claim 1 wherein the first aqueous droplet comprises bacteria with a volume that ranges between about 20 nanoliters and about 100 microliters.

174. The method of claim 173 wherein the concentration of bacteria is in the range of about 10^9 to about 10^ 1 /mL.

175. The method of claim 1 wherein the first aqueous droplet comprises bacteria and one or more sugars with a volume that ranges between about 20 nanoliters and about microliters .

176. The method of claim 175 wherein the concentration of a bacteria is in the range of about 10^9 to about 10^ 1 /mL.

177. The method of claim 175 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

178. The method of claim 175 wherein the one or more sugars comprise dextran and/or trehalose .

179. The method of claim 175 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

180. The method of claim 175 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

181. The method of claim 175 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

182. The method of claim 175 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

183. The method of claim 1 wherein the first aqueous droplet comprises a lectin having a volume that ranges between about 20 nanoliters and about 100 microliters.

184. The method of claim 183 wherein the concentration of lectin is in the range of about 10 ng/mL to about 1 mg/ mL.

185. The method of claim 1 wherein the first aqueous droplet comprises a lectin and one or more sugars having a volume that ranges between about 20 nanoliters and about microliters .

186. The method of claim 185 wherein the concentration of lectin is in the range of about 10 ng/mL to about 1 mg/ mL.

187. The method of claim 185 wherein the concentration of the one or more sugars ranges from about 0% w/v to about 70% w/v.

188. The method of claim 185 wherein the one or more sugars comprise dextran and/or trehalose .

189. The method of claim 185 wherein the one or more sugars comprise trehalose with a concentration ranging from about 0% w/v to about 70% w/v.

190. The method of claim 185 wherein the one or more sugars comprise trehalose with a concentration ranging from about 0% w/v to about 40% w/v.

191. The method of claim 185 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

192. The method of claim 185 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

193. The method of claim 1 wherein the first aqueous droplet comprises phaseoulus vulgaris agglutinin having a volume that ranges between about 20 nanoliters and about microliters .

194. The method of claim 193 wherein the concentration of phaseoulus vulgaris agglutinin is in a range of about 10 ng/mL to about 1 mg/ mL.

195. The method of claim 1 wherein the first aqueous droplet comprises phaseoulus vulgaris agglutinin and one or more sugars having a volume that ranges between about 20 nanoliters and about 100 microliters.

196. The method of claim 195 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

197. The method of claim 195 wherein the one or more sugars comprise dextran and/or trehalose .

198. The method of claim 195 wherein the one or more sugars comprise trehalose with a concentration ranging from about 0% w/v to about 70% w/v.

199. The method of claim 195 wherein the one or more sugars comprise trehalose with a concentration ranging from about 0% w/v to about 40% w/v.

200. The method of claim 195 wherein the one or more sugars comprise dextran with a concentration ranging from about 0% w/v to about 60% w/v.

201. The method of claim 195 wherein the one or more sugars comprise dextran with a concentration ranging from about 0% w/v to about 20% w/v.

202. The method of claim 1 wherein the first aqueous droplet comprises proteinase K with a volume that ranges between about 20 nanoliters and about 30 microliters.

203. The method of claim 202 wherein the concentration of proteinase K is in the range of about 1 ng/mL to about 40 mg/ mL.

204. The method of claim 1 wherein the first aqueous droplet comprises proteinase K and one or more sugars having a volume that ranges between about 20 nanoliters and about 30 microliters .

205. The method of claim 204 wherein the concentration of proteinase K is in the range of about 1 ng/mL to about 40 mg/ mL.

206. The method of claim 204 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

207. The method of claim 204 wherein the one or more sugars comprise dextran and/or trehalose .

208. The method of claim 204 wherein the one or more sugars comprise trehalose with a concentration ranging from about 0% w/v to about 70% w/v.

209. The method of claim 204 wherein the one or more sugars comprise trehalose with a concentration ranging from about 0% w/v to about 40% w/v.

210. The method of claim 204 wherein the one or more sugars comprise dextran with a concentration ranging from about 0% w/v to about 60% w/v.

211. The method of claim 204 wherein the one or more sugars comprise dextran with a concentration ranging from about 0% w/v to about 20% w/v.

212. The method of claim 1 wherein the first aqueous droplet comprises lysis buffer with a volume that ranges between about 20 nanoliters and about 1 mL.

213. The method of claim 1 wherein the fn-st aqueous droplet comprises lysis buffer and one or more sugars with a volume that ranges between about 20 nanoliters and about 1 mL.

214. The method of claim 213 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

215. The method of claim 213 wherein the one or more sugars comprise dextran and/or trehalose .

216. The method of claim 213 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

217. The method of claim 213 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

218. The method of claim 213 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

219. The method of claim 213 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

220. The method of claim 1 wherein the first aqueous droplet comprises magnetically sensitive beads having a volume that ranges between about 20 nanoliters and about 10 microliters.

221. The method of claim 1 wherein the first aqueous droplet comprises magnetically sensitive beads and one or more sugars having a volume that ranges between about 20 nanoliters and about 10 microliters.

222. The method of claim 221 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

223. The method of claim 221 wherein the one or more sugars comprise dextran and/or trehalose.

224. The method of claim 221 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

225. The method of claim 221 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

226. The method of claim 221 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

227. The method of claim 221 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

228. The method of claim 1 wherein the first aqueous droplet comprises beads having a volume that ranges between about 20 nanoliters and about 10 microliters.

229. The method of claim 1 wherein the first aqueous droplet comprises beads and one or more sugars having a volume that ranges between about 20 nanoliters and about microliters .

230. The method of claim 229 wherein the concentration of the one or more sugars ranges from about 0% w/v to about 70% w/v.

231. The method of claim 229 wherein the one or more sugars comprise dextran and/or trehalose.

232. The method of claim 229 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

233. The method of claim 229 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

234. The method of claim 229 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

235. The method of claim 229 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

236. The method of claim 1 wherein the first aqueous droplet comprises a buffer that enables beads to bind to nucleic acids having a volume that ranges between about 20 nanoliters and about 1 mL.

237. The method of claim 1 wherein the first aqueous droplet comprises a buffer that enables beads to bind to nucleic acids and one or more sugars having a volume that ranges between about 20 nanoliters and about 1 mL.

238. The method of claim 237 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

239. The method of claim 237 wherein the one or more sugars comprise dextran and/or trehalose .

240. The method of claim 237 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

241. The method of claim 237 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

242. The method of claim 237 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

243. The method of claim 237 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

244. The method of claim 1 wherein the first aqueous droplet comprises a buffer that enables proteins to bind to beads with a volume that ranges between about 20 nanoliters and about 1 mL.

245. The method of claim 1 wherein the first aqueous droplet comprises a buffer that enables proteins to bind to beads and one or more sugars with a volume that ranges between about 20 nanoliters and about 1 mL.

246. The method of claim 245 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

247. The method of claim 245 wherein the one or more sugars comprise dextran and/or trehalose .

248. The method of claim 245 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

249. The method of claim 245 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

250. The method of claim 245 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

251. The method of claim 245 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

252. The method of claim 1 wherein the first aqueous droplet comprises a buffer that washes beads bound to nucleic acids having a volume that ranges between about 20 nanoliters and about 50 microliters.

253. The method of claim 1 wherein the first aqueous droplet comprises a buffer that washes beads bound to nucleic acids and one or more sugars with a volume that ranges between about 20 nanoliters and about 50 microliters.

254. The method of claim 253 wherein the concentration of the sugars ranges from 0% w/v to 70% w/v.

255. The method of claim 253 wherein the one or more sugars comprise dextran and/or trehalose .

256. The method of claim 253 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

257. The method of claim 253 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

258. The method of claim 253 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

259. The method of claim 253 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

260. The method of claim 1 wherein the first aqueous droplet comprises a buffer that washes beads bound to protein with a volume that ranges between about 20 nanoliters and about 50 microliters.

261. The method of claim 1 wherein the first aqueous droplet comprises a buffer that washes beads bound to protein and one or more sugars having a volume that ranges between about 20 nanoliters and about 50 microliters.

262. The method of claim 261 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

263. The method of claim 261 wherein the one or more sugars comprise dextran and/or trehalose .

264. The method of claim 261 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

265. The method of claim 261 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

266. The method of claim 261 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

267. The method of claim 261 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

268. The method of claim 1 wherein the first aqueous droplet comprises a buffer that elutes nucleic acids from beads with a volume that ranges between about 20 nanoliters and about 20 microliters.

269. The method of claim 1 wherein the first aqueous droplet comprises a buffer that elutes nucleic acids from beads and one or more sugars having a volume that ranges between about 20 nanoliters and about 20 microliters.

270. The method of claim 269 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

271. The method of claim 269 wherein the one or more sugars comprise dextran and/or trehalose .

272. The method of claim 269 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

273. The method of claim 269 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

274. The method of claim 269 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

275. The method of claim 269 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

276. The method of claim 1 wherein the first aqueous droplet comprises a temperature sensitive polymer with a volume that ranges between about 20 nanoliters and about 20 microliters .

277. The method of claim 276 wherein the temperature sensitive polymer has a concentration ranging from about 0% w/v to about 50% w/v.

278. The method of claim 276 wherein the temperature sensitive polymer comprises poly(N-isopropylacrylamide).

279. The method of claim 1 wherein the first aqueous droplet comprises antibody with a volume that ranges between about 20 nanoliters and about 10 microliters.

280. The method of claim 279 wherein the concentration of antibody is in the range of about 1 ng/mL to about 1 mg/ mL.

281. The method of claim 1 wherein the first aqueous droplet comprises antibody and one or more sugars having a volume that ranges between about 20 nanoliters and about microliters .

282. The method of claim 281 wherein the concentration of antibody is in the range of about 1 ng/mL to about 1 mg/ mL.

283. The method of claim 281 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

284. The method of claim 281 wherein the one or more sugars comprise dextran and/or trehalose .

285. The method of claim 281 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

286. The method of claim 281 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

287. The method of claim 281 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

288. The method of claim 281 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

289. The method of claim 1 wherein the first aqueous droplet comprises .beta.-galactosidase with a volume that ranges between about 20 nanoliters and about 10 microliters.

290. The method of claim 289 wherein the concentration of .beta.-galactosidase is in a range of about 0.01 nM to about 10 µM.

291. The method of claim 1 wherein the first aqueous droplet comprises .beta.-galactosidase and one or more sugars with a volume that ranges between about 20 nanoliters and 10 about microliters .

292. The method of claim 291 wherein the concentration of .beta.-galactosidase is in a range of about 0.01 nM to about 10 µM.

293. The method of claim 291 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

294. The method of claim 291 wherein the one or more sugars comprise dextran and/or trehalose .

295. The method of claim 291 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

296. The method of claim 291 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

297. The method of claim 291 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

298. The method of claim 291 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

299. The method of claim 1 wherein the first aqueous droplet comprises bovine serum albumin with a volume that ranges between about 20 nanoliters and about 30 microliters.

300. The method of claim 299 wherein the concentration of bovine serum albumin is in a range of about 100 ng/mL to about 50 mg/ mL.

301. The method of claim 1 wherein the first aqueous droplet comprises bovine serum albumin and one or more sugars with a volume that ranges between about 20 nanoliters and about 30 microliters.

302. The method of claim 301 wherein the concentration of bovine serum albumin is in a range of about 100 ng/mL to about 50 mg/ mL.

303. The method of claim 301 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

304. The method of claim 301 wherein the one or more sugars comprise dextran and/or trehalose .

305. The method of claim 301 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

306. The method of claim 301 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

307. The method of claim 301 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

308. The method of claim 301 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

309. The method of claim 1 wherein the first aqueous droplet comprises serum having a volume that ranges between about 20 nanoliters and about 30 microliters.

310. The method of claim 309 wherein the concentration of serum is in a range of about 1% to about 30%.

311. The method of claim 1 wherein the fn-st aqueous droplet comprises serum and one or more sugars with a volume that ranges between about 20 nanoliters and about 30 microliters .

312 . The method of claim 311 wherein the concentration of serum is in a range of about 1% to about 30%.

313. The method of claim 311 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

314. The method of claim 311 wherein the one or more sugars comprise dextran and/or trehalose .

315. The method of claim 311 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

316. The method of claim 311 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

317. The method of claim 311 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

318. The method of claim 311 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

319. The method of claim 1 wherein the first aqueous droplet comprises 4-Methylumbelliferone having a volume that ranges between about 20 nanoliters and about 30 microliters.

320. The method of claim 319 wherein the concentration of 4-Methylumbelliferone is in a range of about 11.1M to about 1 mM.

321. The method of claim 319 wherein the 4-Methylumbelliferone is dissolved in 2-Amino-2-methyl-1-propanol (AMP) buffer.

322. The method of claim 1 wherein the first aqueous droplet comprises 4-Methylumbelliferone and one or more sugars with a volume that ranges between about 20 nanoliters and about 30 microliters.

323. The method of claim 322 wherein the concentration of 4¨Methylumbelliferone is in a range of about 11.1M to about 1 mM.

324. The method of claim 322 wherein the 4-Methylumbelliferone is dissolved in 2-Amino-2-methyl-l-propanol (AMP) buffer.

325. The method of claim 322 wherein the concentration of the one or more sugars range from about 0% w/v to about 70% w/v.

326. The method of claim 322 wherein the one or more sugars comprise dextran and/or trehalose .

327. The method of claim 322 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 70% w/v.

328. The method of claim 322 wherein the one or more sugars comprise trehalose having a concentration ranging from about 0% w/v to about 40% w/v.

329. The method of claim 322 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 60% w/v.

330. The method of claim 322 wherein the one or more sugars comprise dextran having a concentration ranging from about 0% w/v to about 20% w/v.

331. The method of claim 1 wherein the one or more reagents comprise an RNase inhibitor.

332. The method of claim 1 wherein the one or more reagents comprise a protease inhibitor.

333. The method of claim 1 wherein the one or more reagents comprise a DNase inhibitor.

334. The method of claim 1 wherein the one or more reagents comprise a trehalase inhibitor.

335. The method of claim 1 wherein the one or more reagents comprise a molecule or compound that inhibits the digestion of sugars.

336. The method of claim 1 wherein the one or more reagents comprise a molecule or compound that inhibits the digestion of carbohydrates.

337. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet can amplify nucleic acids.

338. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet can perform RT-PCR.

339. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet can perform PCR.

340. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet comprises armored RNA.

341. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet comprises armored DNA.

342. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet comprises a virus.

343. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet comprises MS2.

344. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet comprises lectins.

345. A hydrophobic surface upon which three unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, and the third resuspended droplet comprises MS2.

346. A hydrophobic surface upon which four unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises MS2, and the fourth resuspended droplet comprises lectins.

347. A hydrophobic surface upon which four unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises MS2, and the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin.

348. A hydrophobic surface upon which five unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises MS2, the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin, and the fifth resuspended droplet comprises proteinase K.

349. A hydrophobic surface upon which eight unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises MS2, the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin, the fifth resuspended droplet comprises proteinase K, the sixth resuspended droplet comprises a buffer that washes beads, the seventh resuspended droplet comprises a buffer that elutes nucleic acid from beads, and the eighth droplet contains a buffer that lysis blood.

350. A hydrophobic surface upon which three unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, and the third resuspended droplet comprises armored RNA.

351. A hydrophobic surface upon which four unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises armored RNA, and the fourth resuspended droplet comprises lectins.

352. A hydrophobic surface upon which four unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises armored RNA, and the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin.

353. A hydrophobic surface upon which five unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises armored RNA, the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin, and the fifth resuspended droplet comprises proteinase K.

354. A hydrophobic surface upon which eight unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises armored RNA, the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin, the fifth resuspended droplet comprises proteinase K, the sixth resuspended droplet comprises a buffer that washes beads, the seventh resuspended droplet comprises a buffer that elutes nucleic acid from beads, and the eighth droplet contains a buffer that lysis blood.

355. A hydrophobic surface upon which three unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, and the third resuspended droplet comprises bacteriophage.

356. A hydrophobic surface upon which four unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises bacteriophage, and the fourth resuspended droplet comprises lectins.

357. A hydrophobic surface upon which four unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises bacteriophage, and the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin.

358. A hydrophobic surface upon which five unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises bacteriophage, the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin, and the fifth resuspended droplet comprises proteinase K.

359. A hydrophobic surface upon which eight unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform RT-PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises bacteriophage, the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin, the fifth resuspended droplet comprises proteinase K, the sixth resuspended droplet comprises a buffer that washes beads, the seventh resuspended droplet comprises a buffer that elutes nucleic acid from beads, and the eighth droplet contains a buffer that lysis blood.

360. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet contains magnetically sensitive beads.

361. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet contains beads.

362. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet contains a temperature sensitive polymer.

363. A hydrophobic surface upon which one unique droplet is provided as described in claim 1 in which the resuspended droplet contains a temperature sensitive polymer.

364. A hydrophobic surface upon which eight unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises armored DNA, the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin, the fifth resuspended droplet comprises proteinase K, the sixth resuspended droplet comprises a buffer that washes beads, the seventh resuspended droplet comprises a buffer that elutes nucleic acid from beads, and the eighth droplet contains a buffer that lysis blood.

365. A hydrophobic surface upon which eight unique droplets are provided as described in claim 1 in which the first resuspended droplet can perform PCR, the second resuspended droplet comprises magnetic beads, the third resuspended droplet comprises DNA, the fourth resuspended droplet comprises phaseoulus vulgaris agglutinin, the fifth resuspended droplet comprises proteinase K, the sixth resuspended droplet comprises a buffer that washes beads, the seventh resuspended droplet comprises a buffer that elutes nucleic acid from beads, and the eighth droplet contains a buffer that lysis blood.

366. A hydrophobic surface upon which five unique droplets are provided as described in claim 1 in which the first resuspended droplet comprises antibodies, the second resuspended droplet comprises beads, the third resuspended droplet comprises a lectin, the fourth resuspended droplet comprises a buffer that washes beads, and the fifth resuspended droplet comprises 4-Methylumbelliferone-Pi.