Abstract: A method of inoculating a culture medium including providing a droplet including a single cell type on a droplet actuator and inoculating a culture medium with the droplet. A method of providing a metabolically useful substance to a cell culture, including providing a droplet actuator including a cell culture droplet loaded thereon, the sample droplet including cells and a cell culture medium, and a second droplet comprising a metabolically useful substance. The method also includes conducting one or more droplet operations to combine the cell culture droplet with the second droplet on the droplet actuator. Related methods, droplet actuators, and systems are also provided.

Abstract: Droplet actuator for conducting droplet operations, such as droplet transport and droplet dispensing, is provided. In one embodiment, the droplet actuator may include an electrode that is rotationally but not reflectively symmetrical.

Abstract: A method of loading a droplet actuator is provided. In one embodiment, the method may include, providing: a droplet actuator loaded with a filler fluid; a reservoir comprising a droplet fluid; and a fluid path extending from the reservoir into the droplet actuator; and forcing filler fluid from one locus in the droplet actuator to another locus in the droplet actuator; or out of the droplet actuator; thereby causing droplet fluid to flow through the fluid path and into the droplet actuator.

Abstract: The present invention provides a droplet actuator device and methods for multiplexed PCR amplification and detection of target amplicons within a single droplet. The methods of the invention combine quantitative real-time PCR (qPCR) amplification with fluorescence-based sequence specific detection technologies for amplified DNA. In one embodiment, fluorescently-labeled oligonucleotide probes may be used for hybridization-based multiplexed detection of target amplicons. The methods of the invention generally involve combining the necessary reactants to form a PCR-ready droplet and thermal cycling the droplet at temperatures sufficient to result in amplification of one or more target nucleic acids. Fluorescence-based detection techniques may be used for end-point or real-time analysis of DNA amplification. For end-point analysis, the accumulation of a signal, e.g., a fluorescence signal, is measured after the amplification of the target sequence is complete.

Abstract: The invention is directed to droplet actuator devices and assay methods. The invention includes assay methods of conducting an assay comprising combining a sample with an umbelliferyl derivative, wherein the sample potentially comprises an enzyme capable of cleaving the umbelliferyl derivative and where the umbelliferyl derivative comprises an umbelliferyl core modified with one or more modifying moieties.

Abstract: The invention is directed to certain droplet actuated molecular techniques. In one embodiment, the invention provides droplet actuator methods for detection of single nucleotide polymorphisms (SNPs) in a DNA sequence using digital microfluidics, including droplet actuator-based sample preparation and SNP analysis. In another embodiment, the invention provides droplet actuator devices and methods for providing integrated sample preparation and multiplexed detection of an infectious agent, such as HIV. In yet another embodiment, the invention provides droplet actuator devices and techniques for PCR amplification and detection of specific nucleic acid sequences using digital microfluidics, including droplet actuator-based sample preparation and target nucleic acid analysis. In yet another embodiment the invention provides methods for performing hot-start PCR on a droplet actuator.

Abstract: Droplet actuators for conducting droplet operations, such as droplet transport and droplet dispensing, are provided. In one embodiment, the droplet actuator may include a substrate including, droplet operations electrodes arranged for conducting droplet operations on a surface of the substrate; and reference electrodes associated with the droplet operations electrodes and extending above the surface of the substrate. Other embodiments of droplet actuators and methods of loading and using such droplet actuators are also provided.

Abstract: The present invention relates to droplet-based surface modification and washing. According to one embodiment, a method of modifying a surface on a droplet microactuator is provided, wherein the method includes executing one or more droplet operations to bring a droplet comprising a surface-modifying agent into contact with the surface. According to another embodiment, a droplet microactuator is provided and includes a sample or reagent immobilized on a surface thereof and arranged such that a droplet on the droplet microactuator may contact the surface.

Abstract: A method is provided for conducting a droplet-based enzymatic assay, e.g., for diagnostic purposes. On a droplet actuator, a droplet comprising an enzyme of interest is provided along with a droplet comprising a substrate which is potentially modified in the presence of the enzyme. Droplet operations are executed to combine the enzyme and substrate droplets on the droplet actuator, thereby yielding an assay droplet on the droplet actuator. Detecting modification of the substrate by the enzyme in the assay droplet occurs on the droplet actuator. Modified substrate preparations for conducting such enzymatic assays are also provided.

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: 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 relates to a droplet actuator for conducting droplet operations. The actuator includes a bottom substrate and a top substrate separated from the bottom substrate to form a gap. An arrangement of droplet operations electrodes may be located on a surface of the bottom substrate and/or top substrate. Optionally, a sample reservoir may hold a quantity of a sample fluid containing cells. A disruption device which can take various forms is used to lyse the cells in the sample or in a sample droplet to thereby conduct operations on samples having lysed cells therein.

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 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 providing improved hydrogen peroxide assays, as well as droplet actuators for conducting such assays. The droplet actuators of the invention may be used to conduct droplet-based hydrogen peroxide assays. They may also be associated with detectors for analyzing the results of the hydrogen peroxide assays of the invention. They may be provided as components of systems which control droplet operations and/or detection for conducting the hydrogen peroxide assays. Measurement by the detector may be used to quantify the presence of an analyte in a sample.

Abstract: An integrated droplet actuator device and methods are provided for performing PCR amplification and high-resolution melting (HRM) analysis on a single droplet actuator. HRM analysis can be used in combination with PCR amplification for detection of sequence variations (e.g., single-nucleotide polymorphisms, nucleotide-repeat polymorphisms, mutation scanning and assessment of DNA methylation) within one or more genes of interest. The PCR amplicons can be fluorescently labeled during amplification using a saturating DNA intercalating fluorescent dye, a 5?-labeled primer, or labeled probes. Also provided are a droplet actuator device and methods for sample preparation using the droplet actuator and detection of sequence variations on the same droplet actuator.

Abstract: The present invention relates to droplet-based surface modification and washing. According to one embodiment, a method of providing a bead-containing droplet with a reduced concentration of a substance is provided, the method including providing a droplet microactuator including a droplet having a starting volume and including one or more beads and a starting concentration and starting quantity of the substance. The method further includes conducting one or more droplet operations to merge a wash droplet with the droplet provided in the above step to yield a combined droplet and including one or more droplet operations to divide the combined droplet to yield a set of droplets including a droplet including substantially all of the one or more beads and having a decreased concentration of the substance relative to the starting concentration and a droplet which is substantially lacking in the beads.

Abstract: The present invention relates to droplet-based surface modification and washing. According to one embodiment, a method of modifying a surface on a droplet microactuator is provided, wherein the method includes executing one or more droplet operations to bring a droplet comprising a surface-modifying agent into contact with the surface. According to another embodiment, a droplet microactuator is provided and includes a sample or reagent immobilized on a surface thereof and arranged such that a droplet on the droplet microactuator may contact the surface.

Abstract: The invention relates to a microfluidic platform and methods of using the platform for conducting enzyme assays using a droplet actuator. The enzyme assays of the invention are useful for, among other things, identifying and/or characterizing disorders resulting from conditions in which enzymes are defective or are produced in inappropriate amounts. Enzyme assays of the invention may, for example, be used to detect altered activity of a particular enzyme in a sample, which may serve as an indicator of a particular disease. Altered activity may, for example, be caused by conditions which result in the increased or reduced production of a certain enzyme or its substrate and/or conditions which result in defective enzymes and/or substrates exhibiting increased or decreased effectiveness relative to corresponding normal enzymes and/or substrates.

Abstract: The invention relates to certain novel approaches to reducing or eliminating the movement of contaminants from one droplet to another on a droplet actuator via liquid filler fluid. In one application, droplet actuators are used to conduct genetic analysis using polymerase chain reaction (PCR) techniques. The invention addresses the need for improved methods of performing PCR on a droplet actuator that provide for optimum amplification and detection of a sample target.