WO2015023745A1

WO2015023745A1 - Droplet actuator test cartridge for a microfluidics system

Info

Publication number: WO2015023745A1
Application number: PCT/US2014/050883
Authority: WO
Inventors: Keith BRADFORD
Original assignee: Advanced Liquid Logic, Inc.
Priority date: 2013-08-13
Filing date: 2014-08-13
Publication date: 2015-02-19

Abstract

The present invention is directed to a droplet actuator test cartridge for verifying the operability of a microfluidics system, wherein the test cartridge provides a mechanism for performing diagnostics on a microfluidics system in an automated fashion. In particular, the test cartridge substantially mimics the functions of a droplet actuator and, in so doing, the driving and sensing/receiving functions of a microfluidics system with respect to a droplet actuator can be verified under program control.

Description

Droplet Actuator Test Cartridge for a Microfluidics System

Field of the Invention

The invention relates to droplet actuator test cartridges for verifying the operability of a microfluidics system, wherein the test cartridge provides a mechanism for performing diagnostics on a microfluidics system in an automated fashion.

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.

Microfluidics systems are used for controlling droplet actuators. There is an initial debug of any microfluidics system as well as ongoing maintenance. Therefore, what is needed is a way to verify that a microfluidics system is in good working order. Namely, automated methods of performing diagnostics on a microfluidics system are needed.

Brief Description of the Invention

A droplet actuator test cartridge is provided, including a mechanism for performing diagnostics on a microfluidics system in an automated fashion, in which the mechanism for performing diagnostics on a microfluidics system in an automated fashion provides means for verifying the operability of the microfluidics system. In one embodiment, the droplet actuator test cartridge includes: a) an instrument deck, including an instrument deck interface; b) channel voltage measurement circuitry; c) temperature measurement circuitry; d) magnetic field strength measurement circuitry; e) data storage; and f) one or more diagnostic device. In another embodiment, the droplet actuator test cartridge is configured to substantially mimic one or more functions of a droplet actuator. In one embodiment, the one or more functions of the droplet actuator include driving and sensing or receiving functions. In another embodiment, the one or more functions of the droplet actuator are verified under program control. In one embodiment, the droplet actuator test cartridge includes a printed circuit board (PCB). In another embodiment, the PCB includes one or more mechanical supports along one or more edges of the PCB. In one embodiment, the PCB further includes a plastic cover mounted thereon.

In another embodiment, the instrument deck interface includes one or more sets of input/output (I/O) pads or pins. In one embodiment, the microfluidics system includes an instrument. In another embodiment, the one or more sets of I/O pads or pins are connected to the instrument. In one embodiment, the instrument deck interface includes both signal and power I/O pads or pins.

In another embodiment, the instrument deck interface is configured to facilitate one or more sets of channel electrowetting voltages, one or more sense lines, or one or more serial communications lines. In one embodiment, the microfluidics system supplies one or more sets of electrowetting voltages. In another embodiment, the microfluidics system supplies one or more sets of electrowetting voltages designed to mimic each of one or more channels of a droplet actuator.

In one embodiment, the channel voltage measurement circuitry is configured to verify that each of the one or more channels of the droplet actuator can be turned on and off. In another embodiment, the channel voltage measurement circuitry is configured to verify one or more characteristics of the one or more sets of electrowetting voltages. In one embodiment, the characteristic of the one or more sets of electrowetting voltages is an electrowetting voltage waveform for each of the one or more channels of the droplet actuator. In another embodiment, the characteristic of the one or more sets of electrowetting voltages is voltage amplitude, shape, and/or timing for each of the one or more channels of the droplet actuator. In one embodiment, the characteristic of the one or more sets of electrowetting voltages is voltage shape for each of the one or more channels of the droplet actuator, and in which the voltage shape includes duty cycle and/or rise/fall time. In another embodiment, the channel voltage measurement circuitry is configured to verify capacitance and/or impedance measurements for each of the one or more channels of the droplet actuator. In one embodiment, the channel voltage measurement circuitry is configured for either AC mode or DC mode.

In another embodiment, the instrument includes one or more heat sources. In one embodiment, the one or more heat sources mimic heating zones in a droplet actuator. In another embodiment, the droplet actuator test cartridge includes one or more set of contact pads configured to press against the heat sources when the droplet actuator test cartridge is installed in the instrument deck. In one embodiment, the droplet actuator test cartridge includes a printed circuit board (PCB) configured to press against the heat sources when the droplet actuator test cartridge is installed in the instrument deck. In another embodiment, the one or more heat sources include one or more heater bars. In one embodiment, the temperature measurement circuitry of the test cartridge is configured to measure the heat that is generated by the one or more heat sources, thereby verifying whether the heat sources are working properly. In another embodiment, the temperature measurement circuitry of the test cartridge is configured to measure the heat generated by the one or more heat sources, thereby allowing for verification that the one or more heat sources are working properly. In one embodiment, the temperature measurement circuitry is further configured to drive one or more heater sense lines back to the microfluidics system, thereby allowing for verification that the one or more heater sense lines are working properly. In another embodiment, the temperature measurement circuitry is further configured to determine a temperature profile of the instrument deck.

In one embodiment, the instrument includes one or more magnets, in which the one or more magnets generate one or more magnetic fields. In another embodiment, the one or more magnets are configured to manipulate magnetically responsive beads in the droplet actuator. In one embodiment, the magnetic field strength measurement circuitry is configured to be located within the one or more magnetic fields of the magnets when the droplet actuator test cartridge is installed in the instrument deck. In another embodiment, each position of the one or more magnets is adjustable. In one embodiment, strength of the one or more magnetic fields is adjustable. In another embodiment, the instrument includes one or more motors for adjusting each position of the one or more magnets. In one embodiment, the magnetic field strength measurement circuitry is configured to determine a magnetic field strength profile of the instrument deck, thereby verifying whether the one or more motors for adjusting each position of the one or more magnets are operating properly. In another embodiment, the magnetic field strength measurement circuitry is further configured to drive one or more magnetic field strength sense lines back to the microfluidics system, thereby verifying whether the magnetic field strength sense lines are operating properly.

In one embodiment, the data storage includes any volatile or non-volatile memory device capable of storing electronic information. In another embodiment, calibration data are stored in the data storage. In one embodiment, calibration data include any information collected or otherwise generating during any process of the microfluidics system. In another embodiment, calibration data include channel data, temperature data, or magnetic field strength data. In one embodiment, a unique cartridge ID is stored in the data storage. In another embodiment, a unique cartridge ID is hardcoded into the droplet actuator test cartridge.

In one embodiment, the one or more diagnostics device includes any passive or active device useful for performing diagnostics on the microfluidics system. In another embodiment, the one or more diagnostics device and the microfluidics system interact via the instrument deck interface. In one embodiment, one or more additional diagnostics devices and the microfluidics system interact via optical means, magnetic means, sonic means, or thermal means.

In another embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices include a fluorimeter test site. In one embodiment, the fluorimeter test site is configured to calibrate fluorescence measurements. In another embodiment, the fluorimeter test site includes a piece of calibration glass over gold. In one embodiment, the piece of calibration glass has a known fluorescence, the fluorimeter is configured to emit light onto the calibration glass, and the one or more diagnostics devices or the one or more additional diagnostics devices include means for measuring the fluorescence of the calibration glass.

In another embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices include a light detector. In one embodiment, the light detector is configured to test external light sources that are directed at the droplet actuator test cartridge. In another embodiment, the light detector includes a light- emitting diode (LED). In one embodiment, the light detector is a calibrated light detector configured to calibrate one or more light sources on the instrument.

In another embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices include a light emitter. In one embodiment, the light emitter includes a light- emitting diode (LED). In another embodiment, the LED is configured to be turned off and on to calibrate one or more external light detectors.

In one embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices include a chip including a microphone configured to detect sounds that indicate one or more components of the microfluidics system are not working properly. In another embodiment, the microphone is configured to detect rattling sounds from motors or from imbalanced fans, thereby verifying that one or more components of the microfluidics system are not working properly. In one embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices include an accelerometer. In another embodiment, the instrument includes one or more instrument decks configured to mimic agitation of the droplet actuator. In one embodiment, the accelerometer is configured to measure movement of the one or more instrument decks, thereby verifying agitation of the droplet actuator.

In another embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices include an inclinometer. In one embodiment, the inclinometer is configured to measure angles of slope or inclination, thereby verifying that the instrument deck is level.

In another embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices include a liquid sensor. In one embodiment, the liquid sensor is configured to detect spills within the instrument deck.

In another embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices include an environmental sensor. In one embodiment, the environmental sensor is selected from the group consisting of: a light sensor configured to measure ambient light; a temperature sensor configured to measure ambient temperature; and a humidity sensor configured to measure ambient humidity. In another embodiment, the environmental sensor includes a light sensor configured to measure ambient light; whereby an enclosed darkened environment is verified. In one embodiment, the microfluidics system can be calibrated using environmental information measured by the environmental sensor.

In another embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices is selected from the group consisting of: an inertial measurement unit (IMU); a proximity sensor; an infrared (IR) sensor; an image capture device; an audio recorder; an electronic compass; and a location tracking system. In one embodiment, the image capture device includes a digital camera. In another embodiment, the audio recorder includes a digital recorder. In one embodiment, the location tracking system includes a global positioning system (GPS).

In another embodiment, the one or more diagnostics devices or the one or more additional diagnostics devices are configured to collect digital information. In one embodiment, the digital information includes calibration data. In another embodiment, the digital information is stored in the data storage. In one embodiment, the droplet actuator test cartridge and the data storage are configured for processing of the digital information separately from the microfluidics system after the droplet actuator test cartridge is removed from the microfluidics system. In another embodiment, the digital information is transmitted to an external computing device.

In one embodiment, the droplet actuator test cartridge further includes a wireless communications link. In one embodiment, the wireless communications link includes a wireless communication interface configured to connect to a network. In another embodiment, the wireless communication interface is configured to exchange information with one or more devices connected to the network. In one embodiment, the wireless communication interface is configured for a sealed environment. In another embodiment, the wireless communication interface is configured for collecting real-time data. In one embodiment, the wireless communication interface is selected from the group consisting of: an Intranet connection; Internet; Personal Area Networks (PANs); Wi-Fi; Wi-Max; IEEE 802.1 1 technology; radio frequency ( F); Infrared Data Association (IrDA) compatible protocols; Local Area Networks (LANs); Wide Area Networks (WANs); and Shared Wireless Access Protocol (SWAP); or any combinations thereof.

In another embodiment, the droplet actuator test cartridge further includes one or more interfaces for one or more mobile devices.

A method of using a droplet actuator test cartridge in a microfluidics system is also provided, the method including the steps of: a) inserting any of the droplet actuator test cartridges disclosed herein into an instrument deck of the microfluidics system, whereby the microfluidics system automatically detects the presence of the droplet actuator test cartridge in the instrument deck and initiates a test sequence; and b) removing the droplet actuator test cartridge from the instrument deck. In one embodiment, the instrument deck includes an instrument deck interface and when the droplet actuator test cartridge is inserted into the instrument deck, the instrument deck interface is electrically connected to the droplet actuator test cartridge. In another embodiment, automatic detection of the presence of the droplet actuator test cartridge in the instrument deck by the microfluidics system includes reading a unique cartridge ID from the data storage of the droplet actuator test cartridge.

In one embodiment, the test sequence includes performance of diagnostics on one or more sets of electrowetting voltages supplied by the microfluidics system, in which the one or more sets of electrowetting voltages are designed to mimic each of one or more channels of a droplet actuator. In another embodiment, the performance of diagnostics on the one or more sets of electrowetting voltages includes use of the channel voltage measurement circuitry to verify that each of the one or more channels of the droplet actuator can be turned on and off. In one embodiment, the performance of diagnostics on the one or more sets of electrowetting voltages includes use of the channel voltage measurement circuitry to verify one or more characteristics of the one or more sets of electrowetting voltages. In one embodiment, the characteristic of the one or more sets of electrowetting voltages is an electrowetting voltage waveform for each of the one or more channels of the droplet actuator. In another embodiment, the characteristic of the one or more sets of electrowetting voltages is voltage amplitude, shape, and/or timing for each of the one or more channels of the droplet actuator. In one embodiment, the characteristic of the one or more sets of electrowetting voltages is voltage shape for each of the one or more channels of the droplet actuator, and in which the voltage shape includes duty cycle and/or rise/fall time. In another embodiment, the performance of diagnostics on the one or more sets of electrowetting voltages includes use of the channel voltage measurement circuitry to verify capacitance and/or impedance measurements for each of the one or more channels of the droplet actuator. In one embodiment, the performance of diagnostics on the one or more sets of electrowetting voltages is performed in either AC mode or DC mode.

In another embodiment, the test sequence includes performance of diagnostics on one more heat sources of the instrument, in which the one or more heat sources mimic heating zones in a droplet actuator. In one embodiment, the performance of diagnostics on the one or more heat sources includes use of the temperature measurement circuitry to measure the temperature of the droplet actuator test cartridge, thereby verifying whether the heat sources are working properly. In another embodiment, the one more heat sources are activated. In one embodiment, the heat sources are ramped up and down in a controlled fashion, further in which the temperature measurement circuitry is used to determine a temperature profile of the instrument deck. In another embodiment, the temperature measurement circuitry drives one or more heater sense lines back to the microfluidics system, thereby allowing for verification that the one or more heater sense lines are working properly.

In one embodiment, the test sequence includes performance of diagnostics on one or more magnets of the instrument, in which the one or more magnets generate one or more magnetic fields. In another embodiment, the magnetic field strength measurement circuitry is configured to be located within the one or more magnetic fields of the magnets when the droplet actuator test cartridge is installed in the instrument deck. In one embodiment, the method includes adjusting each position of the one or more magnets. In another embodiment, the method includes adjusting the strength of the one or more magnetic fields. In one embodiment, the method includes the use of one or more motors to adjust each position of the one or more magnets. In another embodiment, each position of the one or more magnets is adjusted in a controlled fashion. In one embodiment, adjusting each position of the one or more magnets in a controlled fashion includes stepped adjustment of each position of the one or more magnets. In another embodiment, magnetic field strength measurement circuitry is used to determine a magnetic field strength profile of the instrument deck, thereby verifying whether the one or more motors for adjusting each position of the one or more magnets are operating properly. In one embodiment, the magnetic field strength measurement circuitry is further used to drive one or more magnetic field strength sense lines back to the microfluidics system, thereby verifying whether the magnetic field strength sense lines are operating properly.

In another embodiment, the test sequence includes performance of diagnostics with one or more diagnostic devices of Figure 1 through Figure 6.

In one embodiment, digital information collected from the performance of diagnostics is stored in the data storage of the droplet actuator test cartridge and/or transmitted to an external computing device. In another embodiment, the digital information includes calibration data. In one embodiment, the droplet actuator test cartridge is removed from the instrument deck following the storage of digital information in the data storage and/or transmittal to an external computing device.

In another embodiment, channel voltage measurement circuitry of the droplet actuator test cartridge is used to map instrument channels to droplet actuator channels. In one embodiment, the instrument deck interface includes one or more sets of input/output (I/O) pads or pins, and further in which the one or more sets of I/O pads or pins are connected to the instrument. In another embodiment, channel voltage measurement circuitry of the droplet actuator test cartridge is used to determine which bits in software correspond to which of the one or more sets of I/O pads or pins of the instrument deck. In one embodiment, each of the one or more sets of I/O pads or pins of the instrument deck correspond to an electrowetting channel of the instrument. In another embodiment, information relating to mapping of the instrument channels to the droplet actuator channels is used to generate an interface file for the instrument deck. In one embodiment, the test sequence includes methods for testing hardware, firmware, and/or software upgrades to the microfluidics system.

In another embodiment, the droplet actuator test cartridge is used to write or assign a unique serial number to a line of instruments during a manufacturing process.

In one embodiment, chain of custody of information is stored in digital information stored in the data storage. In another embodiment, an encryption key is provided on the droplet actuator test cartridge, in which only an authorized entity with the encryption key can access and/or collect any or all digital information stored in the data storage.

In one embodiment, the method includes accessing and/or collecting digital information from the droplet actuator test cartridge via a mobile device.

A microfluidics system is also provided programmed to execute any of the methods disclosed herein on a droplet actuator test cartridge. In another embodiment, the droplet actuator test cartridge includes any of the droplet actuator test cartridges disclosed herein.

A storage medium is also provided including program code embodied in the medium for executing any of the methods disclosed herein on a droplet actuator test cartridge. In one embodiment, the droplet actuator test cartridge includes any of the droplet actuator test cartridges disclosed herein.

A microfluidics system is also provided including any of the droplet actuator test cartridges disclosed herein, in which the droplet actuator test cartridge is coupled to a processor. In another embodiment, the processor executes program code embodied in a storage medium for executing any of the methods disclosed herein on the droplet actuator test cartridge.

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 1000 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 10 MHz, or from about 10 Hz to about 60 Hz, or from about 20 Hz to about 40 Hz, or about 30 Hz.

"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 flow 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., DYNABEADS® 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; 200501 18574, 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 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. 1 1/639,566, entitled "Droplet-Based Particle Sorting," filed on December 15, 2006; U.S. Patent Application No. 61/039,183, entitled "Multiplexing Bead Detection in a Single Droplet," filed on March 25, 2008; U.S. Patent Application No. 61/047,789, entitled "Droplet Actuator Devices and Droplet Operations Using Beads," filed on April 25, 2008; U.S. Patent Application No. 61/086, 183, entitled "Droplet Actuator Devices and Methods for Manipulating Beads," filed on August 5, 2008; International Patent Application No. PCT/US2008/053545, entitled "Droplet Actuator Devices and Methods Employing Magnetic Beads," filed on February 11, 2008; International Patent Application No. PCT/US2008/058018, entitled "Bead-based Multiplexed Analytical Methods and Instrumentation," filed on March 24, 2008; International Patent Application No. PCT/US2008/058047, "Bead Sorting on a Droplet Actuator," filed on March 23, 2008; and International Patent Application No. PCT/US2006/047486, entitled "Droplet-based Biochemistry," filed on December 1 1, 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 1 1, 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. 200501 18574, 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, 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 1 1, 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. A droplet may include one or more beads.

"Droplet Actuator" means a device for manipulating droplets. For examples of droplet actuators, see Pamula et al., U.S. Patent 6,91 1, 132, entitled "Apparatus for Manipulating Droplets by Electrowetting-Based Techniques," issued on June 28, 2005; Pamula et al., U.S. Patent Application No. 1 1/343,284, 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 1 1, 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, 1 1/275,668, entitled "Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle," filed on January 23, 2006, 1 1/460, 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 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; Deere 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 droplet operations 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 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 BOND™ 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 μηι to about 600 μηι, or about 100 μηι to about 400 μηι, or about 200 μηι to about 350 μηι, or about 250 μηι to about 300 μηι, or about 275 μηι. 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 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 flow 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 FLUOROPEL® 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 NOVEC™ electronic coatings (available from 3M Company, St. Paul, MN), other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxane (e.g., SiOC) for 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/US2010/040705, 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: MITSUI™ BN-300 (available from MITSUI Chemicals America, Inc., San Jose CA); ARLON™ 1 IN (available from Arlon, Inc, Santa Ana, CA).; NELCO® N4000-6 and N5000-30/32 (available from Park Electrochemical Corp., Melville, NY); ISOLA™ FR406 (available from Isola Group, Chandler, AZ), especially IS620; fluoropolymer family (suitable for fluorescence detection since it has low background fluorescence); polyimide family; polyester; polyethylene naphthalate; polycarbonate; polyetheretherketone; liquid crystal polymer; cyclo-olefm copolymer (COC); cyclo-olefm polymer (COP); aramid; THERMOUNT® nonwoven aramid reinforcement (available from DuPont, Wilmington, DE); NOMEX® 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 PARYLENE™ C (especially on glass), PARYLENE™ N, and PARYLENE™ HT (for high temperature, ~300°C) (available from Parylene Coating Services, Inc., Katy, TX); TEFLON® AF coatings; cytop; soldermasks, such as liquid photoimageable soldermasks (e.g., on PCB) like TAIYO™ PSR4000 series, TAIYO™ PSR and AUS series (available from Taiyo America, Inc. Carson City, NV) (good thermal characteristics for applications involving thermal control), and PROBIMER™ 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 VACREL® dry film soldermask line (available from DuPont, Wilmington, DE); film dielectrics, such as polyimide film (e.g., KAPTON® polyimide film, available from DuPont, Wilmington, DE), polyethylene, and fluoropolymers (e.g., FEP), polytetrafluoroethylene; polyester; polyethylene naphthalate; cyclo-olefm copolymer (COC); cyclo-olefm 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 FLUOROPEL® coatings for dip or spray coating, other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxane (e.g., SiOC) for 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 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., U.S. Patent Application Publication No. US20100194408, entitled "Capacitance Detection in a Droplet Actuator," published on Aug. 5, 2010, 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 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 droplet operations gap of a droplet actuator is typically filled with a filler fluid. The filler fluid may, for example, be or include a low- viscosity oil, such as silicone oil or hexadecane filler fluid. The filler fluid may be or include a halogenated oil, such as a fluorinated or perfluorinated oil. 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 be selected to improve droplet operations and/or reduce loss of reagent or target substances from droplets, improve formation of microdroplets, reduce cross contamination between droplets, reduce contamination of droplet actuator surfaces, reduce degradation of droplet actuator materials, etc. For example, filler fluids may be selected for compatibility with droplet actuator materials. As an example, fluorinated filler fluids may be usefully employed with fluorinated surface coatings. Fluorinated filler fluids are useful to reduce loss of lipophilic compounds, such as umbelliferone substrates like 6- hexadecanoylamido-4-methylumbelliferone substrates (e.g., for use in Krabbe, Niemann-Pick, or other assays); other umbelliferone substrates are described in U.S. Patent Pub. No. 201 101 18132, published on May 19, 201 1, the entire disclosure of which is incorporated herein by reference. Examples of suitable fluorinated oils include those in the Galden line, such as Galden HT170 (bp = 170 °C, viscosity = 1.8 cSt, density = 1.77), Galden HT200 (bp = 200C, viscosity = 2.4 cSt, d = 1.79), Galden HT230 (bp = 230C, viscosity = 4.4 cSt, d = 1.82) (all from Solvay Solexis); those in the Novec line, such as Novec 7500 (bp = 128C, viscosity = 0.8 cSt, d = 1.61), Fluorinert FC- 40 (bp = 155 °C, viscosity = 1.8 cSt, d = 1.85), Fluorinert FC-43 (bp = 174 °C, viscosity = 2.5 cSt, d = 1.86) (both from 3M). In general, selection of perfluorinated filler fluids is based on kinematic viscosity (< 7 cSt is preferred, but not required), and on boiling point (> 150 °C is preferred, but not required, for use in DNA/RNA-based applications (PCR, etc.)). 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 1 1, 2010, and WO/2009/021 173, 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. Fluorinated oils may in some cases be doped with fluorinated surfactants, e.g., Zonyl FSO-100 (Sigma-Aldrich) and/or others.

"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, femmagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as Fe304, BaFel2019, 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 flow 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 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 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/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface. In one example, filler fluid can be considered as a film between such 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.

Brief Description of the Drawings

Figure 1 illustrates a functional block diagram of an example of a droplet actuator test cartridge for verifying the operability of a microfluidics system;

Figures 2 and 3 illustrate a plan view and a perspective view, respectively, of an example of the test cartridge of Figure 1 that is implemented on a printed circuit board (PCB);

Figure 4 illustrates a perspective view of the PCB of Figures 2 and 3 with a cover mounted thereon;

Figures 5 and 6 illustrate a top perspective view and a bottom perspective view, respectively, of the cover shown in Figure 4;

Figure 7 illustrates a flow diagram of an example of a method of using the test cartridge in a microfluidics system; and

Figure 8 illustrates a functional block diagram of an example of a microfluidics system whose operation can be verified using the presently disclosed test cartridge.

Description

The present invention is directed to a droplet actuator test cartridge for verifying the operability of a microfluidics system, wherein the test cartridge provides a mechanism for performing diagnostics on a microfluidics system in an automated fashion. Namely, the test cartridge substantially mimics the functions of a droplet actuator and, in so doing, the driving and sensing/receiving functions of a microfluidics system with respect to a droplet actuator can be verified under program control. Figure 1 illustrates a functional block diagram of an example of a droplet actuator test cartridge 100 for verifying the operability of a microfluidics system (not shown). In this example, test cartridge 100 includes an instrument deck interface 1 10, channel voltage measurement circuitry 1 15, temperature measurement circuitry 120, magnetic field strength measurement circuitry 125, data storage 130, and diagnostics devices 135. Optionally, test cartridge 100 can include a wireless communications link 140. In one example, the droplet actuator test cartridge 100 is implemented on a printed circuit board (PCB) 160.

Figures 2 and 3 illustrate a plan view and a perspective view, respectively, of an example of the test cartridge 100 of Figure 1 that is implemented on PCB 160. Figures 2 and 3 also show an example of mechanical supports 165 that are installed along the edges of PCB 160. Additionally, Figure 4 shows a perspective view of PCB 160 with a plastic cover 170 mounted thereon, while Figures 5 and 6 show a top perspective view and a bottom perspective view, respectively, of the plastic cover 170 alone.

Instrument deck interface 1 10 is, for example, a set of input/output (I/O) pads or pins that can be mechanically and electrically connected to the instrument (not shown) of a microfluidics system. Instrument deck interface 1 10 includes both signal and power I/O pads or pins. For example, instrument deck interface 1 10 facilitates a set of channel electrowetting voltages, various sense lines, a serial communications line, and I/O for any other functions.

The microfluidics system (not shown) supplies a set of electrowetting voltages, i.e., one for each channel of the droplet actuator. Channel voltage measurement circuitry 1 15 of test cartridge 100 is used to verify that each channel can be turned on and off. Additionally, channel voltage measurement circuitry 1 15 is used to verify any characteristics of the electrowetting voltage for each channel in both AC mode and DC mode. For example, channel voltage measurement circuitry 1 15 is used to verify the electrowetting voltage waveform for each channel; namely, to measure the voltage amplitude, shape (e.g., duty cycle and rise/fall time), and timing for each channel.

Further, test cartridge 100 can include controlled capacitance and impedance, whereby channel voltage measurement circuitry 1 15 can be used for verifying the capacitance and/or impedance measurements of the electrowetting channels. Associated with the instrument may be one or more heat sources (not shown) for providing heating zones in the droplet actuator. When test cartridge 100 is installed in the instrument deck, PCB or contact pads of test cartridge 100 press against the heat sources (e.g., heater bars) of the microfluidics system. Temperature measurement circuitry 120 of test cartridge 100 is then used to measure the heat that is generated by the heat sources and thereby verify whether the heat sources of the microfluidics system are working properly. Temperature measurement circuitry 120 also drives the heater sense lines back to the microfluidics system. In so doing, the heat sense lines of the microfluidics system are verified. Further, temperature measurement circuitry 120 may be used to determine a temperature profile of the instrument deck.

Associated with the instrument may be one or more magnets (not shown) for manipulating, for example, magnetically responsive beads in the droplet actuator. When test cartridge 100 is installed in the instrument deck, magnetic field strength measurement circuitry 125 may be within the magnetic field of the magnets. Further, the position of the magnets may be adjustable. Therefore, the magnetic field strength can be adjusted. Accordingly, magnetic field strength measurement circuitry 125 may be used to determine a magnetic field strength profile of the instrument deck. In so doing, magnetic field strength measurement circuitry 125 is also used to verify that the motors for moving the magnets are operating properly. Magnetic field strength measurement circuitry 125 also drives the magnetic field strength sense lines back to the microfluidics system. In so doing, the magnetic field strength sense lines of the microfluidics system are verified.

Data storage 130 can be any volatile or non-volatile memory device capable of storing electronic information. In one example, a unique cartridge ID 145 and calibration data 150 are stored in data storage 130. Alternatively, the unique cartridge ID 145 may be hardcoded into test cartridge 100 rather than written into data storage 130. Calibration data 150 can be any information collected or otherwise generating during any process of the microfluidics system. Calibration data 150 includes, for example, any channel data, temperature data, magnetic field strength data, and the like.

Diagnostics devices 135 can be any other passive or active devices that may be useful for performing diagnostics with respect to the microfluidics system. Interaction with certain types of diagnostics devices 135 may be via instrument deck interface 110, whereas interaction with other types of diagnostics devices 135 may be by other means, such as by optical means, magnetic means, sonic means, thermal means, and the like. One example of a diagnostics device 135 is a fluorimeter test site that can be used to calibrate fluorescence measurements. For example, a piece of calibration glass over gold is provided on test cartridge 100, wherein the glass has known fluorescence. A fluorimeter emits light onto the glass and then the fluorescence is measured.

Another example of a diagnostics device 135 is a light detector that can be used to test external light sources (e.g., light- emitting diodes (LEDs)) that are directed at test cartridge 100. In this example, the light detector is a calibrated light detector that can be used to calibrate light sources of the instrument.

Yet another example of a diagnostics device 135 is a light emitter, such as an LED, that can be turned off and on to calibrate external light detectors.

Yet another example of a diagnostics device 135 is a chip with a microphone that can be used to detect certain sounds that may indicate the health of certain components. For example, a microphone can be used to detect rattling sounds from motors or from imbalanced fans.

Yet another example of a diagnostics device 135 is an accelerometer. For example, certain instrument decks are designed to agitate the droplet actuator. In this case, an accelerometer can be used to measure and verify the deck movement.

Yet another example of a diagnostics device 135 is an inclinometer. For example, an inclinometer can be used to measure angles of slope (or tilt) or inclination in order to ensure that the instrument deck is level.

Yet another example of a diagnostics device 135 is a liquid sensor that can be used to, for example, detect spills within the instrument deck.

Further, the diagnostics devices 135 may include any types of environmental sensors, such as a light sensor for measuring the ambient light, a temperature sensor for measuring the ambient temperature, and a humidity sensor for measuring the ambient humidity. For example, some protocols require an enclosed darkened environment. Therefore, the ambient light sensor can be used to determine whether any light is leaking into the instrument deck. Further, environmental information is useful for calibrating the microfluidics system. Still other types of diagnostics devices 135 can include, for example, an inertial measurement unit (IMU), a proximity sensor, an infrared (IR) sensor, an image capture device (e.g., digital camera), an audio recorder (e.g., digital recorder), an electronic compass, a location tracking system (e.g., a global positioning system (GPS)), and the like.

The digital information collected from any of the diagnostics devices 135 of test cartridge 100 can be stored in calibration data 150 of data storage 130 or transmitted to any external computing device. Further, when testing is complete and test cartridge 100 is removed from the micro fluidics system, any information present in data storage 130 can be post-processed separately from the microfluidics system.

The optional wireless communications link 140 is any wireless communication interface for connecting to a network (not shown) and by which information may be exchanged with other devices connected to the network. The wireless communications link 140 can be useful in sealed environments and useful for collecting real-time data. Examples of wireless communication interfaces may include, but are not limited to, an Intranet connection, Internet, Personal Area Networks (PANs) for the exchange of data over short distances, e.g., using short-wavelength radio transmissions in the industrial, scientific, and medical (ISM) band ISM band from 2400- 2480 MHz) from fixed and mobile devices (e.g., Bluetooth® technology), Wi-Fi, Wi-Max, IEEE 802.1 1 technology, radio frequency (RF), Infrared Data Association (IrDA) compatible protocols, Local Area Networks (LANs), Wide Area Networks (WANs), Shared Wireless Access Protocol (SWAP), any combinations thereof, and other types of wireless networking protocols.

Figure 7 illustrates a flow diagram of an example of a method 700 of using test cartridge 100 in a microfluidics system. Method 700 may include, but is not limited to, the following steps.

At a step 710, test cartridge 100 is inserted into the instrument deck of the microfluidics system. In so doing, instrument deck interface 1 10 is electrically connected within the instrument deck of the microfluidics system.

At a step 715, the microfluidics system automatically detects the presence of test cartridge 100 in the instrument deck and initiates a test sequence. For example, the microfluidics system is able to read the unique cartridge ID 145 from data storage 130, which is an indicator that test cartridge 100 is plugged into the instrument deck. At a step 720, diagnostics are performed with respect to the channel electrowetting voltages. For example, channel voltage measurement circuitry 1 15 of test cartridge 100 is used to verify that each channel can be turned on and off and to verify any characteristics of the electrowetting voltage for each channel in both AC mode and DC mode. Additionally, channel voltage measurement circuitry 1 15 can be used for measuring the capacitance and/or impedance of the electrowetting channels.

At a step 725, diagnostics are performed with respect to temperature. For example, heat sources of the micro fluidics system may be activated. Then, temperature measurement circuitry 120 is used to measure the temperature at test cartridge 100, thereby verifying whether the heat sources are working properly. The heat sources can be ramped up and down in a controlled fashion, whereas temperature measurement circuitry 120 can be used to determine a temperature profile at test cartridge 100. Further, temperature measurement circuitry 120 also drives the heater sense lines back to the microfluidics system. In so doing, the heat sense lines of the microfluidics system are verified.

At a step 730, diagnostics are performed with respect to the magnetic field strength. For example, the magnets may be positioned in close proximity to test cartridge 100 and the magnetic field strength measured using magnetic field strength measurement circuitry 125. Further, the position of the magnets may be stepped in a controlled fashion, whereas magnetic field strength measurement circuitry 125 can be used to determine the magnetic field strength profile of the instrument deck at test cartridge 100. In so doing, magnetic field strength measurement circuitry 125 can verify that the motors for moving the magnets are operating properly.

At a step 735, diagnostics are performed with respect to any other diagnostics devices 135 that are present on test cartridge 100, such as any of the diagnostics devices 135 that are described with reference to Figures 1 through 6.

At a step 740, electronic information is stored and/or transmitted and then test cartridge 100 is removed from the instrument deck. For example, any electronic information collected or otherwise generated in steps 715, 720, 725, 730, and 735 is stored in, for example, calibration data 150 of data storage 130 or transmitted to any external computing device. Then, test cartridge 100 is removed from the instrument deck of the microfluidics system. Additionally, the channel assignments from one instrument deck to another may differ. Therefore, another use of channel voltage measurement circuitry 1 15 of test cartridge 100 is to map the instrument channels to the droplet actuator channels. That is, test cartridge 100 can be used to determine which bits in software correspond to which electrowetting channel I/Os of the instrument. This information can then be used to generate the interface file for the instrument deck.

Another use of test cartridge 100 is to test hardware, firmware, and/or software upgrades to the microfluidics system. Yet another use of test cartridge 100 is to write or assign a unique serial number to a line of instruments during the manufacturing process. Still another use of test cartridge 100 is to support the chain of custody of information and to comply with the Health Insurance Portability and Accountability Act (HIPAA) privacy rules. For example, an encryption key can be provided on test cartridge 100 by which only authorized entities can collect certain information stored in data storage 130 or stored at the microfluidics system that is using the test cartridge 100. Further, test cartridge 100 can include interfaces for common mobile devices. For example, test cartridge 100 can include an iPhone® dock.

6.1 Systems

Figure 8 illustrates a functional block diagram of an example of a microfluidics system 800 whose operation can be verified using the presently disclosed test cartridge 100. Microfluidics system 800 is designed for controlling a droplet actuator. However, in Figure 8, test cartridge 100 is installed in microfluidics system 800 in place of the droplet actuator. Test cartridge 100 may be designed to mimic the operations of a droplet actuator. Test cartridge 100 fits onto an instrument deck (not shown) of microfluidics system 800.

Generally, the instrument deck may hold the droplet actuator (or test cartridge 100) as well as house other droplet actuator features, such as, but not limited to, one or more magnets and one or more heating devices. For example, the instrument deck may house one or more magnets 810, which may be permanent magnets. Optionally, the instrument deck may house one or more electromagnets 815. Magnets 810 and/or electromagnets 815 are positioned in relation to the droplet actuator for immobilization of magnetically responsive beads. Optionally, the positions of magnets 810 and/or electromagnets 815 may be controlled by a motor 820. Additionally, the instrument deck may house one or more heating devices 825 for controlling the temperature within, for example, reaction and/or washing zones of the droplet actuator. In one example, heating devices 825 may be heater bars that are positioned in relation to the droplet actuator for providing thermal control thereof.

A controller 830 of microfluidics system 800 is electrically coupled to various hardware components of the invention, such as the droplet actuator, electromagnets 815, motor 820, and heating devices 825, as well as to a detector 835, an impedance sensing system 840, and any other input and/or output devices (not shown). Controller 830 controls the overall operation of microfluidics system 800. Controller 830 may, for example, be a general purpose computer, special purpose computer, personal computer, or other programmable data processing apparatus. Controller 830 serves to provide processing capabilities, such as storing, interpreting, and/or executing software instructions, as well as controlling the overall operation of the system. Controller 830 may be configured and programmed to control data and/or power aspects of these devices. For example, in one aspect, with respect to a droplet actuator, controller 830 controls droplet manipulation by activating and/or deactivating electrodes.

In one example, detector 835 may be an imaging system that is positioned in relation to test cartridge 100. In one example, the imaging system may include one or more light-emitting diodes (LEDs) (i.e., an illumination source) and a digital image capture device, such as a charge- coupled device (CCD) camera.

Impedance sensing system 840 may be any circuitry for detecting impedance at a specific electrode of test cartridge 100. In one example, impedance sensing system 840 may be an impedance spectrometer. Impedance sensing system 840 may be used to monitor the capacitive loading of any electrode, such as any droplet operations electrode, with or without a droplet thereon. For examples of suitable capacitance detection techniques, see Sturmer et al., U.S. Patent Application Publication No. US20100194408, entitled "Capacitance Detection in a Droplet Actuator," published on Aug. 5, 2010; and Bourn et al., U.S. Patent Publication No. US20030080143, entitled "System and Method for Dispensing Liquids," published on May 1, 2003; the entire disclosures of which are incorporated herein by reference.

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.

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 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 readonly 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 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.

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

The Claims We claim:

1. A droplet actuator test cartridge, comprising a mechanism for performing diagnostics on a microfluidics system in an automated fashion, wherein the mechanism for performing diagnostics on a microfluidics system in an automated fashion provides means for verifying the operability of the microfluidics system.

2. The droplet actuator test cartridge of claim 1, wherein the droplet actuator test cartridge comprises an instrument deck comprising an instrument deck interface, and wherein the mechanism for performing diagnostics is selected from the group consisting of: a. channel voltage measurement circuitry; b. temperature measurement circuitry; c. magnetic field strength measurement circuitry; d. data storage; and e. one or more diagnostic device.

3. The droplet actuator test cartridge of claim 2, wherein the droplet actuator test cartridge is configured to substantially mimic one or more functions of a droplet actuator.

4. The droplet actuator test cartridge of claim 3, wherein the one or more functions of the droplet actuator comprise driving and sensing or receiving functions.

5. The droplet actuator test cartridge of any of claims 3 to 4, wherein the one or more functions of the droplet actuator are verified under program control.

6. The droplet actuator test cartridge of any of claims 2 to 5, wherein the droplet actuator test cartridge comprises a printed circuit board (PCB).

7. The droplet actuator test cartridge of claim 6, wherein the PCB comprises one or more mechanical supports along one or more edges of the PCB.

8. The droplet actuator test cartridge of any of claims 6 to 7, wherein the PCB further comprises a plastic cover mounted thereon.

9. The droplet actuator test cartridge of any of claims 2 to 8, wherein the instrument deck interface comprises one or more sets of input/output (I/O) pads or pins.

10. The droplet actuator test cartridge of claim 9, wherein the microfluidics system comprises an instrument.

1 1. The droplet actuator test cartridge of claim 10, wherein the one or more sets of I/O pads or pins are connected to the instrument.

12. The droplet actuator test cartridge of any of claims 9 to 1 1, wherein the instrument deck interface includes both signal and power I/O pads or pins.

13. The droplet actuator test cartridge of any of claims 11 to 12, wherein the instrument deck interface is configured to facilitate one or more sets of channel electrowettmg voltages, one or more sense lines, or one or more serial communications lines.

14. The droplet actuator test cartridge of any of claims 2 to 13, wherein the microfluidics system supplies one or more sets of electrowettmg voltages.

15. The droplet actuator test cartridge of claim 14, wherein the microfluidics system supplies one or more sets of electrowettmg voltages designed to mimic each of one or more channels of a droplet actuator.

16. The droplet actuator test cartridge of claim 15, wherein the channel voltage measurement circuitry is configured to verify that each of the one or more channels of the droplet actuator can be turned on and off.

17. The droplet actuator test cartridge of claim 15, wherein the channel voltage measurement circuitry is configured to verify one or more characteristics of the one or more sets of electrowettmg voltages.

18. The droplet actuator test cartridge of claim 17, wherein the characteristic of the one or more sets of electrowetting voltages is an electrowetting voltage waveform for each of the one or more channels of the droplet actuator.

19. The droplet actuator test cartridge of claim 17, wherein the characteristic of the one or more sets of electrowetting voltages is voltage amplitude, shape, and/or timing for each of the one or more channels of the droplet actuator.

20. The droplet actuator test cartridge of claim 19, wherein the characteristic of the one or more sets of electrowetting voltages is voltage shape for each of the one or more channels of the droplet actuator, and wherein the voltage shape comprises duty cycle and/or rise/fall time.

21. The droplet actuator test cartridge of claim 15, wherein the channel voltage measurement circuitry is configured to verify capacitance and/or impedance measurements for each of the one or more channels of the droplet actuator.

22. The droplet actuator test cartridge of any of claims 15 to 21, wherein the channel voltage measurement circuitry is configured for either AC mode or DC mode.

23. The droplet actuator test cartridge of any of claims 10 to 22, wherein the instrument comprises one or more heat sources.

24. The droplet actuator test cartridge of claim 23, wherein the one or more heat sources mimic heating zones in a droplet actuator.

25. The droplet actuator test cartridge of any of claims 23 to 24, wherein the droplet actuator test cartridge comprises one or more set of contact pads configured to press against the heat sources when the droplet actuator test cartridge is installed in the instrument deck.

26. The droplet actuator test cartridge of any of claims 23 to 24, wherein the droplet actuator test cartridge comprises a printed circuit board (PCB) configured to press against the heat sources when the droplet actuator test cartridge is installed in the instrument deck.

27. The droplet actuator test cartridge of any of claims 23 to 26, wherein the one or more heat sources comprise one or more heater bars.

28. The droplet actuator test cartridge of any of claims 23 to 27, wherein the temperature measurement circuitry of the test cartridge is configured to measure the heat that is generated by the one or more heat sources, thereby verifying whether the heat sources are working properly.

29. The droplet actuator test cartridge of any of claims 23 to 28, wherein the temperature measurement circuitry of the test cartridge is configured to measure the heat generated by the one or more heat sources, thereby allowing for verification that the one or more heat sources are working properly.

30. The droplet actuator test cartridge of any of claims 23 to 29, wherein the temperature measurement circuitry is further configured to drive one or more heater sense lines back to the microfluidics system, thereby allowing for verification that the one or more heater sense lines are working properly.

31. The droplet actuator test cartridge of any of claims 23 to 30, wherein the temperature measurement circuitry is further configured to determine a temperature profile of the instrument deck.

32. The droplet actuator test cartridge of any of claims 10 to 31, wherein the instrument comprises one or more magnets, wherein the one or more magnets generate one or more magnetic fields.

33. The droplet actuator test cartridge of claim 32, wherein the one or more magnets are configured to manipulate magnetically responsive beads in the droplet actuator.

34. The droplet actuator test cartridge of any of claims 32 to 33, wherein the magnetic field strength measurement circuitry is configured to be located within the one or more magnetic fields of the magnets when the droplet actuator test cartridge is installed in the instrument deck.

35. The droplet actuator test cartridge of any of claims 32 to 33, wherein each position of the one or more magnets is adjustable.

36. The droplet actuator test cartridge of any of claims 32 to 33, wherein strength of the one or more magnetic fields is adjustable.

37. The droplet actuator test cartridge of any of claims 35 to 36, comprising one or more motors for adjusting each position of the one or more magnets.

38. The droplet actuator test cartridge of claim 37, wherein the magnetic field strength measurement circuitry is configured to determine a magnetic field strength profile of the instrument deck, thereby verifying whether the one or more motors for adjusting each position of the one or more magnets are operating properly.

39. The droplet actuator test cartridge of any of claims 32 to 38, wherein the magnetic field strength measurement circuitry is further configured to drive one or more magnetic field strength sense lines back to the microfluidics system, thereby verifying whether the magnetic field strength sense lines are operating properly.

40. The droplet actuator test cartridge of any of claims 2 to 39, wherein the data storage comprises any volatile or non-volatile memory device capable of storing electronic information.

41. The droplet actuator test cartridge of claim 40, wherein calibration data are stored in the data storage.

42. The droplet actuator test cartridge of claim 40, wherein calibration data comprise any information collected or otherwise generating during any process of the microfluidics system.

43. The droplet actuator test cartridge of claim 40, wherein calibration data comprise channel data, temperature data, or magnetic field strength data.

44. The droplet actuator test cartridge of any of claims 40 to 43, wherein a unique cartridge ID is stored in the data storage.

45. The droplet actuator test cartridge of any of claims 1 to 43, wherein a unique cartridge ID is hardcoded into the droplet actuator test cartridge.

46. The droplet actuator test cartridge of any of claims 2 to 45, wherein the one or more diagnostics device comprises any passive or active device useful for performing diagnostics on the microfluidics system.

47. The droplet actuator test cartridge of claim 46, wherein the one or more diagnostics device and the microfluidics system interact via the instrument deck interface.

48. The droplet actuator test cartridge of claim 46, wherein one or more additional diagnostics devices and the microfluidics system interact via optical means, magnetic means, sonic means, or thermal means.

49. The droplet actuator test cartridge of any of claims 46 to 48, wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise a fluorimeter test site.

50. The droplet actuator test cartridge of claim 49, wherein the fluorimeter test site is configured to calibrate fluorescence measurements.

51. The droplet actuator test cartridge of any of claims 49 to 50, wherein the fluorimeter test site comprises a piece of calibration glass over gold.

52. The droplet actuator test cartridge of claim 51, wherein the piece of calibration glass has a known fluorescence, wherein the fluorimeter is configured to emit light onto the calibration glass; and further wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise means for measuring the fluorescence of the calibration glass.

53. The droplet actuator test cartridge of any of claims 46 to 52, wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise a light detector.

54. The droplet actuator test cartridge of claim 53, wherein the light detector is configured to test external light sources that are directed at the droplet actuator test cartridge.

55. The droplet actuator test cartridge of claim 54, wherein the light detector comprises a light- emitting diode (LED).

56. The droplet actuator test cartridge of any of claims 53 to 55, wherein the light detector is a calibrated light detector configured to calibrate one or more light sources on the instrument.

57. The droplet actuator test cartridge of any of claims 46 to 56, wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise a light emitter.

58. The droplet actuator test cartridge of claim 57, wherein the light emitter comprises a light- emitting diode (LED).

59. The droplet actuator test cartridge of claim 57, wherein the LED is configured to be turned off and on to calibrate one or more external light detectors.

60. The droplet actuator test cartridge of any of claims 46 to 60, wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise a chip comprising a microphone configured to detect sounds that indicate one or more components of the microfluidics system are not working properly.

61. The droplet actuator test cartridge of claim 60, wherein the microphone is configured to detect rattling sounds from motors or from imbalanced fans, thereby verifying that one or more components of the microfluidics system are not working properly.

62. The droplet actuator test cartridge of any of claims 46 to 61, wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise an accelerometer.

63. The droplet actuator test cartridge of claim 62, wherein the instrument comprises one or more instrument decks configured to mimic agitation of the droplet actuator.

64. The droplet actuator test cartridge of claim 63, wherein the accelerometer is configured to measure movement of the one or more instrument decks, thereby verifying agitation of the droplet actuator.

65. The droplet actuator test cartridge of any of claims 46 to 64, wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise an inclinometer.

66. The droplet actuator test cartridge of claim 65, wherein the inclinometer is configured to measure angles of slope or inclination, thereby verifying that the instrument deck is level.

67. The droplet actuator test cartridge of any of claims 46 to 64, wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise a liquid sensor.

68. The droplet actuator test cartridge of claim 67, wherein the liquid sensor is configured to detect spills within the instrument deck.

69. The droplet actuator test cartridge of any of claims 46 to 68, wherein the one or more diagnostics devices or the one or more additional diagnostics devices comprise an environmental sensor.

70. The droplet actuator test cartridge of claim 70, wherein the environmental sensor is selected from the group consisting of: a light sensor configured to measure ambient light; a temperature sensor configured to measure ambient temperature; and a humidity sensor configured to measure ambient humidity.

71. The droplet actuator test cartridge of claim 70, wherein the environmental sensor comprises a light sensor configured to measure ambient light; whereby an enclosed darkened environment is verified.

72. The droplet actuator test cartridge of claim 71, whereby the microfluidics system can be calibrated using environmental information measured by the environmental sensor.

73. The droplet actuator test cartridge of any of claims 46 to 72, wherein the one or more diagnostics devices or the one or more additional diagnostics devices is selected from the group consisting of: an inertial measurement unit (IMU); a proximity sensor; an infrared (IR) sensor; an image capture device; an audio recorder; an electronic compass; and a location tracking system.

74. The droplet actuator test cartridge of claim 73, wherein the image capture device comprises a digital camera.

75. The droplet actuator test cartridge of claim 73, wherein the audio recorder comprises a digital recorder.

76. The droplet actuator test cartridge of claim 73, wherein the location tracking system comprises a global positioning system (GPS).

77. The droplet actuator test cartridge of any of claims 46 to 76, wherein the one or more diagnostics devices or the one or more additional diagnostics devices are configured to collect digital information.

78. The droplet actuator test cartridge of claim 77, wherein the digital information comprises calibration data.

79. The droplet actuator test cartridge of any of claims 77 to 78, wherein the digital information is stored in the data storage.

80. The droplet actuator test cartridge of claim 79, wherein the droplet actuator test cartridge and the data storage are configured for processing of the digital information separately from the microfluidics system after the droplet actuator test cartridge is removed from the microfluidics system.

81. The droplet actuator test cartridge of any of claims 77 to 78, wherein the digital information is transmitted to an external computing device.

82. The droplet actuator test cartridge of any of claims 2 to 81, wherein the droplet actuator test cartridge further comprises a wireless communications link.

83. The droplet actuator test cartridge of claim 82, wherein the wireless communications link comprises a wireless communication interface configured to connect to a network.

84. The droplet actuator test cartridge of claim 83, wherein the wireless communication interface is configured to exchange information with one or more devices connected to the network.

85. The droplet actuator test cartridge of any of claims 82 to 84, wherein the wireless communication interface is configured for a sealed environment.

86. The droplet actuator test cartridge of any of claims 82 to 85, wherein the wireless communication interface is configured for collecting real-time data.

87. The droplet actuator test cartridge of any of claims 82 to 86, wherein the wireless communication interface is selected from the group consisting of: an Intranet connection; Internet;

Personal Area Networks (PANs); Wi-Fi; Wi-Max; IEEE 802.1 1 technology; radio frequency (RF);

Infrared Data Association (IrDA) compatible protocols; Local Area Networks (LANs); Wide Area Networks (WANs); and Shared Wireless Access Protocol (SWAP); or any combinations thereof.

88. The droplet actuator test cartridge of any of claims 2 to 87, wherein the droplet actuator test cartridge further comprises one or more interfaces for one or more mobile devices.

89. The droplet actuator test cartridge of any of claims 2 to 88, wherein the droplet actuator test cartridge does not comprise a droplet.

90. The droplet actuator test cartridge of any of claims 2 to 88, wherein the droplet actuator test cartridge does not comprise a droplet in a filler fluid.

91. The droplet actuator test cartridge of any of claims 2 to 88, wherein the droplet actuator test cartridge is not configured to conduct a droplet operation.

92. A method of using a droplet actuator test cartridge in a microfluidics system, the method comprising the steps of: a. inserting the droplet actuator test cartridge of any of claims 2 to 91 into an instrument deck of the microfluidics system, whereby the microfluidics system automatically detects the presence of the droplet actuator test cartridge in the instrument deck and initiates a test sequence; and b. removing the droplet actuator test cartridge from the instrument deck.

93. The method of claim 92, wherein the instrument deck comprises an instrument deck interface, and further wherein when the droplet actuator test cartridge is inserted into the instrument deck, the instrument deck interface is electrically connected to the droplet actuator test cartridge.

94. The method of any of claims 92 to 93, wherein automatic detection of the presence of the droplet actuator test cartridge in the instrument deck by the microfluidics system comprises reading a unique cartridge ID from the data storage of the droplet actuator test cartridge.

95. The method of any of claims 92 to 94, wherein the test sequence comprises performance of diagnostics on one or more sets of electrowettmg voltages supplied by the microfluidics system, wherein the one or more sets of electrowettmg voltages are designed to mimic each of one or more channels of a droplet actuator.

96. The method of claim 95, wherein the performance of diagnostics on the one or more sets of electrowettmg voltages comprises use of the channel voltage measurement circuitry to verify that each of the one or more channels of the droplet actuator can be turned on and off.

97. The method of claim 95, wherein the performance of diagnostics on the one or more sets of electrowetting voltages comprises use of the channel voltage measurement circuitry to verify one or more characteristics of the one or more sets of electrowetting voltages.

98. The method of claim 97, wherein the characteristic of the one or more sets of electrowetting voltages is an electrowetting voltage waveform for each of the one or more channels of the droplet actuator.

99. The method of claim 97, wherein the characteristic of the one or more sets of electrowetting voltages is voltage amplitude, shape, and/or timing for each of the one or more channels of the droplet actuator.

100. The method of claim 99, wherein the characteristic of the one or more sets of electrowetting voltages is voltage shape for each of the one or more channels of the droplet actuator, and wherein the voltage shape comprises duty cycle and/or rise/fall time.

101. The method of claim 95, wherein the performance of diagnostics on the one or more sets of electrowetting voltages comprises use of the channel voltage measurement circuitry to verify capacitance and/or impedance measurements for each of the one or more channels of the droplet actuator.

102. The method of any of claims 95 to 101, wherein the performance of diagnostics on the one or more sets of electrowetting voltages is performed in either AC mode or DC mode.

103. The method of any of claims 92 to 94, wherein the test sequence comprises performance of diagnostics on one more heat sources of the instrument, wherein the one or more heat sources mimic heating zones in a droplet actuator.

104. The method of claim 103, wherein the performance of diagnostics on the one or more heat sources comprises use of the temperature measurement circuitry to measure the temperature of the droplet actuator test cartridge, thereby verifying whether the heat sources are working properly.

105. The method of any of claims 103 to 104, wherein the one more heat sources are activated.

106. The method of any of claims 103 to 105, wherein the heat sources are ramped up and down in a controlled fashion, further wherein the temperature measurement circuitry is used to determine a temperature profile of the instrument deck.

107. The method of any of claims 103 to 106, wherein the temperature measurement circuitry drives one or more heater sense lines back to the microfluidics system, thereby allowing for verification that the one or more heater sense lines are working properly.

108. The method of any of claims 92 to 107, wherein the test sequence comprises performance of diagnostics on one or more magnets of the instrument, wherein the one or more magnets generate one or more magnetic fields.

109. The method of claim 108, wherein the magnetic field strength measurement circuitry is configured to be located within the one or more magnetic fields of the magnets when the droplet actuator test cartridge is installed in the instrument deck.

1 10. The method of claim 108, comprising adjusting each position of the one or more magnets.

1 1 1. The method of any of claims 108 to 109, comprising adjusting strength of the one or more magnetic fields.

1 12. The method of any of claims 109 to 1 10, comprising the use of one or more motors to adjust each position of the one or more magnets.

1 13. The method of claim 112, wherein each position of the one or more magnets is adjusted in a controlled fashion.

1 14. The method of claim 113, wherein adjusting each position of the one or more magnets in a controlled fashion comprises stepped adjustment of each position of the one or more magnets.

1 15. The method of any of claims 108 to 1 14, wherein the magnetic field strength measurement circuitry is used to determine a magnetic field strength profile of the instrument deck, thereby verifying whether the one or more motors for adjusting each position of the one or more magnets are operating properly.

1 16. The method of any of claims 108 to 1 15, wherein the magnetic field strength measurement circuitry is further used to drive one or more magnetic field strength sense lines back to the microfluidics system, thereby verifying whether the magnetic field strength sense lines are operating properly.

1 17. The method of any of claims 92 to 116, wherein the test sequence comprises performance of diagnostics with one or more diagnostic devices 135 of Figure 1 through Figure 6.

1 18. The method of any of claims 92 to 1 17, wherein digital information collected from the performance of diagnostics is stored in the data storage of the droplet actuator test cartridge and/or transmitted to an external computing device.

1 19. The method of claim 1 18, wherein the digital information comprises calibration data.

120. The method of any one of claims 1 18 to 1 19, wherein the droplet actuator test cartridge is removed from the instrument deck following the storage of digital information in the data storage and/or transmittal to an external computing device.

121. The method of any of claims 92 to 120, wherein channel voltage measurement circuitry of the droplet actuator test cartridge is used to map instrument channels to droplet actuator channels.

122. The method of claim 121, wherein the instrument deck interface comprises one or more sets of input/output (I/O) pads or pins, and further wherein the one or more sets of I/O pads or pins are connected to the instrument.

123. The method of claim 122, wherein channel voltage measurement circuitry of the droplet actuator test cartridge is used to determine which bits in software correspond to which of the one or more sets of I/O pads or pins of the instrument deck.

124. The method of claim 123, wherein each of the one or more sets of I/O pads or pins of the instrument deck correspond to an electrowetting channel of the instrument.

125. The method of any of claims 121 to 124, wherein information relating to mapping of the instrument channels to the droplet actuator channels is used to generate an interface file for the instrument deck.

126. The method of any of claims 92 to 125, wherein the test sequence comprises methods for testing hardware, firmware, and/or software upgrades to the microfluidics system.

127. The method of any of claims 92 to 125, further wherein the droplet actuator test cartridge is used to write or assign a unique serial number to a line of instruments during a manufacturing process.

128. The method of any of claims 92 to 127, further wherein chain of custody of information is stored in digital information stored in the data storage.

129. The method of any of claims 92 to 128, further wherein an encryption key is provided on the droplet actuator test cartridge, wherein only an authorized entity with the encryption key can access and/or collect any or all digital information stored in the data storage.

130. The method of any of claims 92 to 129, further wherein the method comprises accessing and/or collecting digital information from the droplet actuator test cartridge via a mobile device.

131. A microfluidics system programmed to execute the method of any of claims 92 to 130 on a droplet actuator test cartridge.

132. The microfluidics system of claim 131, wherein the droplet actuator test cartridge comprises the droplet actuator test cartridge of any of claims 1 to 91.

133. A storage medium comprising program code embodied in the medium for executing the method of any of claims 92 to 130 on a droplet actuator test cartridge.

134. The storage medium of claim 133, wherein the droplet actuator test cartridge comprises the droplet actuator test cartridge of any of claims 1 to 91.

135. A microfluidics system comprising the droplet actuator test cartridge of any of claims 1 to 91 , wherein the droplet actuator test cartridge is coupled to a processor.

136. The microfluidics system of claim 135, wherein the processor executes program code embodied in a storage medium for executing the method of any of claims 92 to 130 on the droplet actuator test cartridge.

137. The droplet actuator test cartridge of any of claims 1 to 91, wherein the test cartridge does not comprise a droplet.

138. The droplet actuator test cartridge of any of claims 1 to 91, wherein the test cartridge does not comprise a droplet in a filler fluid.

139. The droplet actuator test cartridge of any of claims 1 to 91, wherein the test cartridge is not configured to conduct a droplet operation.