Droplet-based surface modification and washing
Inventors
Pamula, Vamsee K. • Srinivasan, Vijay • Pollack, Michael G. • Fair, Richard B.
Assignees
Advanced Liquid Logic Inc • Duke University
Publication Number
US-12332205-B2
Publication Date
2025-06-17
Expiration Date
2026-12-15
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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.
Core Innovation
The invention concerns droplet-based surface modification and washing, specifically employing droplet microactuators to execute protocols using discrete droplets. One of the main embodiments described is a method for splitting a droplet that contains one or more beads, immobilizing at least one bead, and conducting droplet operations to divide the droplet. This division yields a set of droplets, wherein at least one resulting droplet contains the immobilized bead(s) and another droplet is substantially free of these bead(s).
The background section states that current assay and diagnostic methods suffer from issues such as high reagent costs, labor intensity, lack of automation, large sample consumption, and susceptibility to operator error and contamination. In addition, existing continuous-flow microfluidic systems are hampered by fixed channels and complicated supporting infrastructure, making it difficult to achieve point-of-care usability and highly integrated multi-functionality in a portable format.
To solve these problems, the invention provides systems, apparatuses, and methods utilizing droplet-based manipulations—such as loading, dispensing, splitting, merging, washing, and incubating—on microactuators controlled by electronically-addressable electrodes. Washing protocols are implemented by manipulating discrete droplets to contact, merge, and split at surfaces (including beads), enabling highly efficient and automatable surface processing steps such as washing excess and unbound materials. These innovations enable miniaturization, automation, and multiplexing of biochemical and immunoassay protocols with reduced sample and reagent consumption.
Claims Coverage
The patent includes two independent claims, each describing inventive features involving electrowetting droplet-based surface assays, particularly immunoassays, executed using substrates with electronically-controlled electrodes.
Electrowetting-based immunoassay using bead surfaces and automated washing
The claimed method comprises: - Immobilizing an antibody specific for a target analyte on the surface of one or more beads. - Washing the immobilized antibody to remove excess, using droplet-based operations. - Using electrowetting droplet operations with two parallel substrates separated by a gap, where one or both substrates have electrodes electronically coupled to and controlled by a controller, to: - Expose the immobilized antibody to a sample droplet. - Wash the immobilized antibody to remove unbound components of the sample droplet. - Expose the immobilized antibody to a droplet that includes a reporter antibody. - Wash away excess reporter antibody using further droplet-based washing operations. - Measuring a measurable parameter or signal facilitated by the reporter antibody. This inventive feature uses automatable, precise droplet manipulation enabled by electrowetting on microactuator substrates to conduct all steps of a surface-based immunoassay (including washing) in a controlled, programmable manner.
Electrowetting-based immunoassay protocol controllable by a single substrate
This claim covers: - Immobilizing an antibody specific for a target analyte on bead surfaces. - Washing the immobilized antibody to remove excess antibody using droplet-based protocols. - Using electrowetting droplet operations with at least one substrate comprising electrodes (electronically coupled to and controlled by a controller) to: - Expose the immobilized antibody to a sample droplet. - Wash the immobilized antibody to remove unbound sample components. - Expose the immobilized antibody to a droplet including a reporter antibody. - Wash away excess reporter antibody using droplet-based washing protocols. - Optionally, measuring a signal facilitated by the reporter antibody and providing a corresponding output. This inventive feature specifies that electrowetting droplet operations—including automated washing and analyte/reporting steps—can be achieved with only one actuated substrate, still using electrode-based control and automation.
In summary, the independent claims protect droplet-based, electronically-controlled execution of immunoassay protocols on bead surfaces using electrowetting-enabled substrates, covering both designs with dual substrates and with single actuated substrates, and encompassing fully-automated washing, assay, and readout steps.
Stated Advantages
Enables small sample and reagent consumption, with assays performed using droplets of 1 μL or less, or 100 nanoliters or less.
Provides faster analysis and higher throughput due to miniaturized format and multiplexing capability.
Allows complete automation and integration, reducing operator dependence and human error compared to manual or less-integrated platforms.
Reduces variability in immunoassay performance, enhancing standardization and reproducibility within and between laboratories.
Enables rapid, programmable execution of surface washing steps with high efficiency, achieving substantial removal of unbound substances from surfaces or beads.
Supports point-of-care or point-of-sample collection applications due to the portable, compact design of the system.
Facilitates multiplexed detection of many analytes (2 up to 50 or more) in a single small-volume sample, supporting both high and low throughput research settings.
Reduces equipment size and cost by miniaturizing liquid handling functions and utilizing low-cost, disposable microactuators.
Improves reliability of washing protocols, retaining >99% of beads and enabling efficient removal of unwanted assay components.
Permits independent and simultaneous manipulation of multiple droplets for parallel processing of numerous assays on a single platform.
Documented Applications
Clinical diagnostic immunoassays, including detection and quantification of antigens and antibodies for cardiac markers, tumor markers, drugs, hormones, and infectious diseases.
Nucleic acid amplification and detection assays, such as PCR-based DNA and RNA assays, including quantitative PCR and real-time detection.
Nucleic acid sequencing, including pyrosequencing, sequencing for diagnostics, forensic analysis, pharmacogenomics, genome sequencing, expression profiling, and detection of sequence variation (SNPs, insertions/deletions, rearrangements).
Affinity-based assays, including direct, indirect, sandwich, and competitive immunoassays, for detection and quantification of various biological or synthetic analytes.
Droplet-based immuno-PCR for sensitive detection and quantification of analytes at trace levels.
Analysis of blood and other biological fluids, comprising detection of metabolites, electrolytes, proteins, enzymes, gases, and hematocrit.
Environmental or bioterrorism-related detection, such as surveillance for biological, chemical, or explosive agents including anthrax, botulism, influenza, and other pathogens or toxins.
Forensic DNA or protein analyses, including identification, paternity testing, and detection of genetically modified organisms.
Applications in clinical trials, supporting genomically specific diagnostics and treatment by detecting genetic variants related to efficacy, safety, or disease forecasting.
Point-of-care or point-of-sample collection testing for rapid diagnostics in medical settings, field situations, or resource-limited environments.
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