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: The present invention relates to droplet-based affinity assays. According to one embodiment, a method of detecting a target analyte in a sample is provided, wherein the method includes: (a) executing droplet operations to combine affinity-based assay reagents on a droplet microactuator with a sample potentially comprising the target analyte to generate a signal indicative of the presence, absence and/or quantity of analyte; and (b) detecting the signal, wherein the signal corresponds to the presence, absence and/or quantity of the analyte in the sample.

Abstract: Molecular adsorption to the microfluidic device surfaces can be passively and actively mitigated by mixing certain hydrophilic polymers (organic polymers with repeating hydrophilic groups—the preferred polymers being amphipathic surfactants—with the sample liquid during or prior to relevant microfluidic operations. Nonionic surfactants such as polyoxyethylene sorbitan monooleate and polyoxyethylene octyl phenyl ether are especially effective. High molecular weight polyethylene polymers are also effective. The hydrophilic polymers appear to prevent binding of the fouling molecules to the microfluidic by occupying the surface sites in place of the fouling molecules or by interacting with the fouling molecules to prevent binding of the fouling molecules the surface. When surface adsorption is thus mitigated, microfluidic devices can readily handle samples containing biomolecules to enable active sample concentration, filtering, washing, transport, mixing and other sample handling operations.

Abstract: The present invention relates to droplet-based pyrosequencing including a method of identifying a base at a target position in a sample nucleic acid. The method includes: (a) providing a droplet microactuator including a first droplet including a sample nucleic acid immobilized on a bead; and (b) on the droplet microactuator: (i) contacting the first droplet with one or more reagent droplets to yield a second droplet, wherein the one or more reagent droplets include reagents for extending a double stranded portion of the sample nucleic acid by incorporating a nucleotide at the target position; (ii) splitting the second droplet to yield a third droplet including the bead and a fourth droplet lacking the bead; and (iii) assaying the third droplet to determine whether the nucleotide was incorporated at the target position.

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: On-board reagent storage, handling, and dispensing apparatuses and methods for droplet actuator systems are provided. In one embodiment, a fluidic analyzer is provided and includes a droplet actuator including a substrate including a plurality of electrodes configured to actuate a droplet and a reagent storage component in selective fluid communication with the droplet actuator and including a reagent configured to combine with a solvent at a time of use. In another embodiment, a method of conducting fluidic analysis is provided and includes storing a reagent within a reagent storage component in fluid communication with a droplet actuator including a plurality of electrodes configured to actuate a droplet and combining the reagent with a solvent at a time for use within the droplet actuator.

Abstract: A droplet actuator comprising a substrate comprising an electrode coupled to a voltage source, wherein the droplet actuator is configured such that when voltage is applied to the electrode, an electrostatic energy gradient is established at a surface of the substrate which causes a droplet to be transported in a direction established by the energy gradient. Related methods and other embodiments are also provided.

Abstract: A method of conducting a droplet-based enzymatic assay is provided. The method generally makes use of a droplet actuator. A droplet comprising an enzyme of interest is provided on the droplet actuator along with a droplet comprising a substrate which is potentially modified in the presence of the enzyme. The method involves executing droplet operations on the droplet actuator to combine the droplets, thereby yielding an assay droplet, and detecting modification of the substrate by the enzyme in the assay droplet on the droplet actuator. The enzyme of interest may, for example, be a potentially mutated or improperly folded enzyme exhibiting altered enzyme activity as compared to a corresponding normal enzyme.

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: The invention is directed to droplet actuator devices and assay methods. The method may include immobilization of the enzymatic substrate including forming an inclusion complex with the substrate within an aqueous environment in contact with an oil.

Abstract: The present invention relates to a droplet-based affinity assay device and system. According to one embodiment, a droplet microactuator is provided and includes an antibody immobilized on a surface. According to another embodiment, a droplet microactuator is provided and includes a droplet on the droplet microactuator, the droplet comprising an antibody.

Abstract: The present invention provides modified droplet actuator systems, software, and software-executed methods for use in droplet actuator operation and droplet actuator systems that are configured and programmed to execute such software. An aspect of the software components of the invention is an interface description file for each hardware component of a microfluidics system that allows hardware components to be changed without modifying the program for performing droplet operations protocols. Another aspect of the software components of the invention is the establishment of electrode-to-electrode relationships and other aspects of droplet actuator configurations, which may be used when programming droplet operations protocols. Another aspect of the software components of the invention is a physical design library of predefined electrode elements that may be used by a droplet actuator designer when constructing a layout of electrodes.

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: A droplet actuator and method of providing a droplet comprising an target substance on the droplet actuator, and including discrete flow and continuous flow functionality. Discrete flow function is controlled by electrodes arranged for conducting droplet operations on a substrate surface. The continuous flow function includes a fluid path arranged for flowing a fluid therethrough. The discrete flow and continuous flow functions may be by a barrier, including a second fluid path through the barrier. The continuous flow function may include a capture surface comprising a component having an affinity for the target substance. Methods of the invention may include flowing a fluid comprising the target substance through the fluid path; capturing the target substance on the capture surface; and forming a droplet in the discrete flow region via the second fluid path and the electrodes comprising the captured target substance.

Abstract: The present invention relates to a droplet-based nucleic acid amplification apparatus and system. According to one embodiment, a droplet microactuator is provided made using a first substrate including a fluorescing material and including a detection region for detecting a fluorescence signal from a droplet, which detection region is coated with a light absorbing, low fluorescence or non-fluorescing material.

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: A refill droplet facilitates the extraction of a droplet laterally from a channel in a microfluidic apparatus. Such extraction allows a discrete band of separated particles or solute molecules to be excised from a fluid stream and processed and analyzed separately. An extraction point is located along the length of the channel and includes an EWOD surface or similar microfluidic technology to extract a droplet. An opening in the channel opposite the extraction means is equipped with microfluidic technology to transport a refill droplet to the opening. The refill droplet is moved into the channel or column to occupy the area previously occupied by the extracted droplet. This prevents distortion or mixing of the bands of particles or molecules within the channel and prevents the draining of any portion of the fluidic system.

Abstract: Alternative approaches to fabricating printed circuit boards for use in droplet actuator operations are provided. In one embodiment, a method of manufacturing a droplet actuator for conducting droplet operations includes positioning a dielectric material between a first metal layer configured to include an electrode and a second metal layer configured to include an interconnect pad. The method additionally includes forming a connection between the first and second metal layers. Droplet actuators and methods of fabricating and supporting printed circuit boards of droplet actuators are also provided.

Abstract: Devices, configurations and methods for improving absorbance detection are provided. For example, methods and devices are provided for determining the absorbance of a droplet, e.g., a droplet on a droplet actuator, by providing an elongated light path through the droplet.