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 invention provides nonlimiting examples of structures for and methods of dispensing droplets in a droplet actuator. The droplet actuator structures and methods of the invention exhibit numerous advantages over droplet actuators of the prior art. In various embodiments, the structures and methods of the invention provide, among other things, improved efficiency, throughput, scalability, and/or droplet uniformity, as compared with existing droplet actuators. Further, in some embodiments, the droplet actuators provide configurations for improved methods of loading and/or unloading fluid and/or droplets. In yet other embodiments, the droplet actuators provide fluid loading configurations for loading numerous fluid reservoirs in a substantially simultaneous and/or substantially sequential manner.

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: 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 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: Sample processing droplet actuators, systems and methods are provided. According to one embodiment, a stamping device including a droplet microactuator is provided and includes: (a) a first plate including a path or network of control electrodes for transporting droplets on a surface thereof; (b) a second plate mounted in a substantially parallel orientation with respect to the first plate providing an interior volume between the plates, the second plate including one or more stamping ports for transporting some portion or all of a droplet from the interior volume to an exterior location; (c) a port for introducing fluid into the interior volume between the plates; and (d) a path or network of reference electrodes corresponding to the path or network of control electrodes. Associated systems and methods including the stamping device are also provided.

Abstract: The invention provides droplet actuators with droplet operations surfaces for manipulating droplets, e.g., by conducting droplet operations. The droplet operations surfaces are typically exposed to a droplet operations gap. One or more regions of a droplet operation surface may include patterned topographic features. The invention also provides a droplet actuator in which one or both gap-facing droplet operations surfaces is formed using a removable film. The removable film may, in various embodiments, also include other components ordinarily associated with the droplet actuator substrate, such as the dielectric layer and the electrodes. Further, the invention provides droplet actuator devices and methods for coupling and/or sealing substrates of a droplet actuator, such as techniques for self-aligning assembly of droplet actuator substrates.

Abstract: The systems and methods of the invention provide a guided approach to pyrosequencing (i.e., hybrid pyrosequencing). A de novo nucleic acid sequence may compared to a library of possible results and the next nucleotide to be dispensed is selected based on the comparison of the de novo sequence and the library of possible results. In another example, at least the first nucleotide to be dispensed is selected based on a query of a database(s) of non-sequence parameters (e.g., incidence of infection, diagnostic symptoms, sample source) and subsequent dispensations determined based on a comparison of the de novo sequence and the library of possible results (e.g., candidate sequences). The systems and methods of the invention may be performed using a droplet actuator.

Abstract: The present invention relates to droplet-based particle sorting. According to one embodiment, a droplet microactuator is provided and includes: (a) a suspension of particles; and (b) electrodes arranged for conducting droplet operations using droplets comprising particles. A method of transporting a particle is also provided, wherein the method includes providing a droplet comprising the particle and transporting the droplet on a droplet microactuator.

Abstract: A method of preparing a sample for conducting an assay, the method including: providing an input sample including glycoproteins; capturing glycoproteins from the input sample on a solid support; and washing the sample support to remove unbound portions of the input sample.

Abstract: A method of amplifying a nucleic acid, the method comprising cycling an amplification-ready droplet through a temperature gradient to locations within the temperature gradient suitable for effecting steps in an amplification reaction.

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.

Abstract: The present invention relates to bead incubating and washing on a droplet actuator. Methods for incubating magnetically responsive beads that are labeled with primary antibody, a sample (i.e., analyte), and secondary reporter antibodies on a magnet, on and off a magnet, and completely off a magnet are provided. Also provided are methods for washing magnetically responsive beads using shape-assisted merging of droplets. Also provided are methods for shape-mediated splitting, transporting, and dispensing of a sample droplet that contains magnetically responsive beads. The apparatuses and methods of the invention provide for rapid time to result and optimum detection of an analyte in an immunoassay.

Abstract: A method of preparing a nucleic acid library in droplets in contact with oil, including: (a) blunt-ending nucleic acid fragments in a droplet in the oil to yield blunt-ended nucleic acid fragments; (b) phosphorylating the blunt-ended nucleic acid fragments in a droplet in the oil to yield phosphorylated nucleic acid fragments; coupling A-tails to the phosphorylated nucleic acid fragments in a droplet in the oil to yield A-tailed nucleic acid fragments; and (d) coupling nucleic acid adapters to the A-tailed nucleic acid fragments in a droplet in the oil to yield the nucleic acid library comprising adapter-ligated nucleic acid fragments.

Abstract: The invention provides droplet actuators and droplet actuator cassettes including reagent storage capabilities, as well as methods of making and using the droplet actuators and cassettes. The invention also provides continuous flow channel elements and techniques for using electrodes to manipulate droplets in flowing streams. The invention also discloses methods of separating compounds on a droplet actuator. Various other aspects of the invention are also disclosed.

Abstract: A droplet actuator configured to improve the throughput of droplet operations in a detection spot of the droplet actuator and/or to reduce carryover problems is provided. The droplet actuator may include electrodes configured for effecting droplet operations transporting droplets on a surface; a sensor arranged in proximity to one or more of the electrodes establishing a detection window on the surface for detection of one or more properties of one or more droplets on the surface; wherein the electrodes may establish at least two pathways for transport of droplets into the detection window.

Abstract: The invention provides a method for reducing or preventing droplet pinning as the droplet is transported across a boundary between a ground electrode region and a non-ground electrode region on a droplet actuator. The invention also provides a method for reducing or preventing droplet super-movement as the droplet is transported across a boundary between a ground electrode region and a non-ground electrode region on a droplet actuator.

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

Abstract: A microfluidic device having a substrate with an electrically conductive element made using a conductive ink layer underlying a hydrophobic layer.