Abstract: The invention relates to a droplet actuator device and methods for integrated sample preparation and analysis of a biological sample. A droplet actuator device is provided for conducting droplet operations. The droplet actuator device may include a bottom substrate and a top substrate separated from each other to form a gap therebetween; an arrangement of droplet operations electrodes arranged on one or both of the bottom and/or top substrates for conducting droplet operations thereon; a reagent storage layer comprising one or more compartments bound to the top substrate; and one or more openings arranged to provide a fluidic path from the one or more compartments into the gap, upon breach of a breachable seal separating the one or more compartments and openings.

Abstract: The present invention is directed to methods of improving accuracy of droplet metering using at least one on-actuator reservoir as the fluid input. In some embodiments, the on-actuator reservoir that is used for metering droplets includes a loading port, a liquid storage zone, a droplet metering zone, and a droplet dispensing zone. The on-actuator reservoirs are designed to prevent liquid flow-back into the loading port and to prevent liquid from flooding into the droplet operations gap in the dispensing zone.

Abstract: The present invention provides methods for on-actuator temperature measurement and temperature control, including where one or more of the temperature sensors are combined with one or more heaters that are formed of wiring traces and/or providing heaters designed for one-to-one correspondence to the temperature sensors to form temperature sensor-heater pairs. The present invention also provides methods for on-actuator temperature measurement and temperature control in which the temperature sensors comprise a connection comprising a plurality of terminals by which an amount of current can be applied and then a voltage measured, wherein the voltage that is measured across the temperature sensors can be accurately correlated to a temperature.

Abstract: The invention provides a droplet actuator designed to provide reliable electrical connections to droplets to reduce or eliminate gas bubble formation during droplet operations. The invention also provides methods and systems for reducing or eliminating the formation of bubbles in a droplet during droplet operations.

Abstract: The invention provides a droplet actuator designed for performing electroporation on cells in droplets. The invention also provides method and systems for performing electroporation on cells in droplets on a droplet actuator.

Abstract: The invention provides a method of circulating magnetically responsive beads within a droplet in a droplet actuator. The invention also provides methods for splitting droplets. The invention, in one embodiment, makes use of a droplet actuator with top and bottom substrates, a plurality of magnetic fields respectively present proximate the top and bottom substrates, wherein at least one of the magnet fields is selectively alterable, and a plurality of droplet operations electrodes positioned along at least one of the top and bottom surfaces. A droplet is positioned between the top and bottom surfaces and at least one of the magnetic fields is selectively altered.

Abstract: A method, circuit and apparatus for detecting capacitance on a droplet actuator, inter alia, for determining the presence, partial presence or absence of a droplet at an electrode on a droplet actuator by: (a) providing a droplet actuator comprising: (i) a substrate comprising electrodes arranged on the substrate for conducting droplet operations on a surface of the substrate; (ii) a capacitance detection circuit for detecting capacitance at the droplet operations surface at one or more of the electrodes; (b) detecting capacitance at the droplet operations surface at one or more of the electrodes; and (c) determining from the capacitance the presence, partial presence or absence of a droplet at the droplet operations surface at the electrode.

Abstract: A method of growing cells on a droplet actuator is provided. The method may include providing a droplet actuator including a cell culture droplet including a cell culture medium and one or more cells; and maintaining the droplet at a temperature suitable for causing the cells to grow in the cell culture medium on the droplet actuator. Related methods, droplet actuators, and systems are also provided.

Abstract: Droplet actuators that include molecular barrier coatings are provided. The molecular barrier coating may be provided atop the conductive layer of the top substrate, atop the droplet operations electrodes of the bottom substrate, or both. Where the conductive layer of the top substrate and/or the droplet operations electrodes of the bottom substrate are formed of an electrically conductive organic polymer, such as poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS), the molecular barrier coating helps to immobilize the contents of the PEDOT:PSS layer. Further, the molecular barrier coating reduces, preferably entirely eliminates, moisture from seeping into the electrically conducting organic polymer. Methods of conducting droplet operations using the disclosed droplet actuators are also provided.

Abstract: The invention provides pyrosequencing-based methods of analyzing and synthesizing DNA, including methods of DNA error correction, determining DNA size distribution, screening for nucleotide repeat disorders such as fragile X syndrome, determining size distribution and bias in a DNA library, and determining pyrosequencing read length. The methods include on-bench protocols as well as droplet-based protocols that may be conducted on a droplet actuator.

Abstract: During droplet operations in a droplet actuator, bubbles often form in the filler fluid in the droplet operations gap and interrupt droplet operations. The present invention provides methods and systems for performing droplet operations on a droplet in a droplet actuator comprising maintaining substantially consistent contact between the droplet and an electrical ground while conducting multiple droplet operations on the droplet in the droplet operations gap and/or reducing the accumulation of electrical charges in the droplet operations gap during multiple droplet operations. The methods and systems reduce or eliminate bubble formation in the filler fluid of the droplet operations gap, thereby permitting completion of multiple droplet operations without interruption by bubble formation in the filler fluid in the droplet operations gap.

Abstract: The invention provides a method of redistributing magnetically responsive beads in a droplet. The method may include conducting on a droplet operations surface one or more droplet operations using the droplet without removing the magnetically responsive beads from the region of the magnetic field. The droplet operations may in some cases be electrode-mediated. The droplet operations may redistribute and/or circulate the magnetically responsive beads within the droplet. In some cases, the droplet may include a sample droplet may include a target analyte. The redistributing of the magnetically responsive beads may cause target analyte to bind to the magnetically responsive beads. In some cases, the droplet may include unbound substances in a wash buffer. The redistributing of the magnetically responsive beads causes unbound substances to be freed from interstices of an aggregated set or subset of the magnetically responsive beads.

Abstract: Methods are provided for manipulating droplets. The methods include providing the droplet on a surface comprising an array of electrodes and a substantially co-planer array of reference elements, wherein the droplet is disposed on a first one of the electrodes, and the droplet at least partially overlaps a second one of the electrodes and an intervening one of the reference elements disposed between the first and second electrodes. The methods further include activating the first and second electrodes to spread at least a portion of the droplet across the second electrode and deactivating the first electrode to move the droplet from the first electrode to the second electrode.

Abstract: The invention provides a method of circulating magnetically responsive beads within a droplet in a droplet actuator. The invention also provides methods for splitting droplets. The invention, in one embodiment, makes use of a droplet actuator with top and bottom substrates, a plurality of magnetic fields respectively present proximate the top and bottom substrates, wherein at least one of the magnet fields is selectively alterable, and a plurality of droplet operations electrodes positioned along at least one of the top and bottom surfaces. A droplet is positioned between the top and bottom surfaces and at least one of the magnetic fields is selectively altered.

Abstract: Methods of conducting an assay for measuring enzymatic modification of a substrate by a glycoprotein having enzymatic activity are provided. The methods include concentrating the glycoprotein having enzymatic activity by capturing the glycoproteins from the input sample on a solid support, washing the glycoproteins captured on a solid support to remove unbound portions of the input sample, adding an enzyme substrate to the output sample comprising glycoproteins having enzymatic activity, and measuring enzymatic modification of the substrate. In some examples, the capturing of the glycoprotein and subsequent washing steps are performed in one or more droplets in oil.

Abstract: The present invention relates to filler fluids for droplet operations. According to one embodiment of this aspect, a droplet microactuator is provided and includes: (a) a first substrate comprising electrodes configured for conducting droplet operations on a surface of the substrate; (b) a second substrate spaced from the surface of the substrate by a distance sufficient to define an interior volume between the first substrate and second substrate, wherein the distance is sufficient to contain a droplet disposed in the space on the first substrate; and (c) a droplet arranged in the interior volume and arranged with respect to the electrodes in a manner which permits droplet operations to be effected on the droplet using the electrodes.

Abstract: The invention provides droplet actuator assemblies and systems and methods of manufacturing the droplet actuator assemblies. In certain embodiments, two-piece enclosures are used to form a droplet actuator assembly that houses a droplet operations substrate. In certain other embodiments, one-piece enclosures are used to form a droplet actuator assembly that houses a droplet operations substrate. In the plastic injection molding process for forming substrates of the droplet actuator assemblies of the present invention may utilize insert molding (or overmolding) processes for forming a gasket in at least one substrate, thereby avoiding the need for providing and installing a separate gasket component. Further, the droplet actuator assemblies may include features that allow ultrasonic welding processes to be used for bonding substrates together. The manufacturing systems of the present invention for fabricating the droplet actuator assemblies may utilize continuous flow reel-to-reel manufacturing processes.

Abstract: The invention is directed to an assay for acid ?-galactosidase activity. The invention may include combining in oil a sample droplet with a 4-methylumbelliferyl-B-galactose to yield a reaction droplet; splitting the reaction droplet to yield a first daughter droplet and a second daughter droplet; combining the first daughter droplet with a stop buffer droplet to yield a first stopped reaction droplet; incubating the second daughter droplet; combining the second daughter droplet with a stop buffer droplet to yield a second stopped reaction droplet; and measuring 4-methylumbelliferone released in the first and second stopped reaction droplets.

Abstract: The present invention relates to filler fluids for droplet operations. According to one embodiment of this aspect, a droplet microactuator is provided and includes: (a) a first substrate comprising electrodes configured for conducting droplet operations on a surface of the substrate; (b) a second substrate spaced from the surface of the substrate by a distance sufficient to define an interior volume between the first substrate and second substrate, wherein the distance is sufficient to contain a droplet disposed in the space on the first substrate; and (c) a droplet arranged in the interior volume and arranged with respect to the electrodes in a manner which permits droplet operations to be effected on the droplet using the electrodes.

Abstract: The present invention relates to droplet-based surface modification and washing. According to one embodiment, a method of splitting a droplet is provided, the method including providing a droplet microactuator including a droplet including one or more beads and immobilizing at least one of the one or more beads. The method further includes conducting one or more droplet operations to divide the droplet to yield a set of droplets including a droplet including the one or more immobilized beads and a droplet substantially lacking the one or more immobilized beads.