Devices, systems, and methods for conducting sandwich assays using sedimentation
Inventors
SCHAFF, Ulrich Y. • Sommer, Gregory J. • Singh, Anup K. • Hatch, Anson V.
Assignees
National Technology and Engineering Solutions of Sandia LLC • Sandia National Laboratories
Publication Number
US-8945914-B1
Publication Date
2015-02-03
Expiration Date
2030-09-28
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Abstract
Embodiments of the present invention are directed toward devices, systems, and method for conducting sandwich assays using sedimentation. In one example, a method includes generating complexes on a plurality of beads in a fluid sample, individual ones of the complexes comprising a capture agent, a target analyte, and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.
Core Innovation
Embodiments of the present invention are directed toward devices, systems, and methods for conducting sandwich assays using sedimentation. The invention involves generating complexes on a plurality of beads suspended in a fluid sample, where each complex includes at least a capture agent, a target analyte, and a labeling agent. The beads containing these complexes are transported through a density media that has a density lower than the beads but higher than the fluid sample. This transporting occurs at least in part by sedimentation, such as by applying centrifugal force.
This invention addresses issues present in conventional sandwich assays where free labeling agents remain unbound in the fluid sample and can produce false positive signals, which obscure accurate detection of the target analyte. Traditional methods require multiple wash steps to remove these free agents and amplification steps to increase the detectable signal. The invention uses sedimentation to separate and concentrate the complexed beads from the free labeling agents, reducing or eliminating the need for wash and amplification steps.
Claims Coverage
The patent includes three independent claims directed to a method, an apparatus, and a system for conducting sandwich assays using sedimentation and density media separation.
Method of conducting a sandwich assay using sedimentation and density media separation
Providing a fluid sample on a microfluidic disk comprising beads with complexes of capture agent, target analyte, and labeling agent, and free labeling agent; supplying a density media from a reservoir to a detection region with controlled centrifugal forces such that density media has density between beads and fluid sample; spinning the disk with different centrifugal forces to transport beads through the density media by sedimentation while restricting free labeling agents; separating different bead populations based on differing properties to distinct detection locations; detecting signals from labeling agents; and generating electronic detection signals based on detected labeling agent signals.
Apparatus configured for bead sedimentation and density media transport within a microfluidic disk
A substrate defining a channel containing fluid sample with beads and free labeling agents; a detection region coupled to the channel; a media reservoir in fluid communication with the detection region operable to transport density media responsive to a first centrifugal force; density media having density between the beads and fluid sample; and the channel and detection region configured so beads with different properties sediment through density media to distinct detection locations while restricting free labeling agents' transport.
System comprising a microfluidic disk, motor, detection module, and processing device for sedimentation-based sandwich assays
A microfluidic disk with substrate defining a channel and detection region containing beads with complexes and free labeling agents; a reservoir with density media having density between fluid sample and bead densities; the disk structured so density media flows into detection region responsive to a first centrifugal force and beads are transported through density media by a second centrifugal force; a motor to spin the disk; a detection module for detecting label agent signals and generating electronic signals; and a processing device controlling motor and detection module and receiving electronic signals.
These independent claims collectively cover the process, physical microfluidic apparatus, and integrated system components involved in forming, separating, and detecting bead-based sandwich assay complexes using sedimentation through a density media on a microfluidic disk platform employing controlled centrifugal forces.
Stated Advantages
Reduction or elimination of wash steps traditionally needed to remove free labeling agents by using sedimentation to separate complexed beads from unbound agents.
Concentration of complexed beads through sedimentation increases detectable signal, reducing or eliminating the need for labeling agent amplification.
Ability to multiplex assays by using beads of different sizes and/or densities that sediment at different rates, enabling simultaneous detection of multiple target analytes.
Documented Applications
Immunoassays including detection of proteins using beads coated with antibodies and fluorescently labeled detection antibodies.
Gene expression assays involving hybridization assays using beads functionalized with mRNA complementary probes and labeled nucleotide probes for mRNA detection.
Whole blood assays involving separation of blood cells from beads suspended in plasma using sedimentation within a microfluidic disk.
Conducting pregnancy test assays by sedimentation using gravitational field without powered centrifugation, with silica beads sedimenting through a density media to allow detection.
Assays employing biological organism beads that produce detectable indicators responsive to analyte presence, such as genetically engineered B-cells producing luminescent signals, with signal enhancement by enzyme substrates in density media.
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