Microfluidic devices, systems, and methods for quantifying particles using centrifugal force
Inventors
SCHAFF, Ulrich Y. • Sommer, Gregory J. • Singh, Anup K.
Assignees
National Technology and Engineering Solutions of Sandia LLC • Sandia National Laboratories
Publication Number
US-9186668-B1
Publication Date
2015-11-17
Expiration Date
2030-09-28
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Abstract
Embodiments of the present invention are directed toward microfluidic systems, apparatus, and methods for measuring a quantity of cells in a fluid. Examples include a differential white blood cell measurement using a centrifugal microfluidic system. A method may include introducing a fluid sample containing a quantity of cells into a microfluidic channel defined in part by a substrate. The quantity of cells may be transported toward a detection region defined in part by the substrate, wherein the detection region contains a density media, and wherein the density media has a density lower than a density of the cells and higher than a density of the fluid sample. The substrate may be spun such that at least a portion of the quantity of cells are transported through the density media. Signals may be detected from label moieties affixed to the cells.
Core Innovation
Embodiments of the present invention are directed toward microfluidic systems, apparatus, and methods for measuring a quantity of cells in a fluid using centrifugal forces within microfluidic channels defined at least in part by a substrate. The invention includes differential white blood cell measurement using a centrifugal microfluidic system by introducing a fluid sample containing cells into a microfluidic channel, transporting the cells toward a detection region containing a density media with a density lower than the cells but higher than the fluid sample, and spinning the substrate to transport cells through the density media. Signals from label moieties affixed to the cells are detected to quantify the cells.
The problem being solved concerns the limitations of common white blood cell measurement techniques such as flow cytometry, electrical impedance counting, and microscopic visual counting, which typically require larger fluid samples, skilled technicians, and stand-alone diagnostic procedures. Furthermore, existing microfluidic systems are still under development for fully automated and integrated cell quantification. The invention addresses the challenge of quantifying cells in small fluid samples efficiently using centrifugal forces and density media to separate and detect labeled cells in a microfluidic system.
The invention provides a microfluidic disk including a substrate defining microfluidic channels and chambers, such as cell quantification areas containing reservoirs for sample, labeling or lysing agents, and one or more density media. The disk is spun to create centrifugal forces that transport fluid samples and density media through channels to a detection region where cells are separated based on size and density by traveling through layered density media. Detection modules detect signals from label moieties affixed to cells as they pass or accumulate in the detection region, enabling quantification of different cell types such as lymphocytes, monocytes, and granulocytes. The approach allows separating and quantifying various cells including white blood cells and potentially other particles based on their physical properties in a controlled microfluidic environment.
Claims Coverage
The patent includes one independent claim outlining a comprehensive method for quantifying white blood cells using layered density media and centrifugal microfluidics. The main inventive features cover sample introduction, cell labeling, density media layering, centrifugal transport, detection, and quantification steps.
Layered density media with differing densities
Providing first and second density media in separate chambers within the substrate, where the two media have different densities lower than white blood cells but higher than the fluid sample, and transporting these media into a detection region as layered density media by spinning the substrate.
Centrifugal transport of labeled white blood cells through layered density media
Spinning the substrate to move the quantity of white blood cells toward and through at least a portion of the first or second density media in the detection region, enabling separation based on density differences and size.
Formation of complexes including label moieties affixed to white blood cells
Forming complexes by affixing label moieties such as DNA dyes, lipid dyes, or antibodies with affinity for specific cell surface proteins to at least a portion of white blood cells prior to or during transport.
Detection of signals near the interface of layered density media
Detecting emission signals from label moieties near an interface between the first and second density media within the detection region to identify and quantify different cell subpopulations.
Selective fluid flow control using microfluidic spin-rate dependent valves
Employing microfluidic channels configured to serve as valves that restrict transport of fluid samples and enable controlled transport of density media into the detection region based on substrate spin rate thresholds.
Diverting cells to separate chambers via timed valve opening during sedimentation
Diverting at least a portion of the labeled white blood cells to a separate chamber in fluid communication with the channel prior to complete sedimentation by opening a valve at selected timing when cells reach certain locations.
Quantification of cell numbers based on detected signals and pellet dimensions
Calculating cell counts by integrating signals received from label moieties and, where applicable, measuring heights of cell pellets formed at the end of the detection region after sedimentation.
Use of centrifugal force and microfluidics to separate multiple white blood cell subtypes
Separating lymphocytes, monocytes, and granulocytes along the detection region by layered density media with densities selected between cell densities to form distinct or combined bands for detection and quantification.
Together, these inventive features describe a method leveraging microfluidic centrifugal forces and layered density media to transport, separate, label, detect, and quantify white blood cells and subpopulations within a fluid sample efficiently and accurately using integrated microfluidic structures, fluid density control, and optical detection of label moieties.
Stated Advantages
Enables quantification of cells using small fluid samples.
Facilitates automated, integrated, and microscale cell counting without requiring skilled technicians.
Separates cells based on density and size using layered density media and centrifugal force for improved differentiation of cell subtypes.
Allows simultaneous measurement of multiple cell types including lymphocytes, monocytes, granulocytes, red blood cells, bacteria, and viruses.
Supports post-processing steps on separated cell populations for further analysis such as PCR, immunoassays, or biochemical assays.
Documented Applications
Differential white blood cell counting in whole blood samples for medical diagnostics of conditions like sepsis, leukemia, AIDS, and radiation exposure.
Quantifying bacterial and viral load in fluid samples by labeling and separating smaller particles near the top of density media.
Selective isolation and further analysis of lymphocytes from blood samples using flow diversion to post-processing areas.
Separation, counting, and analysis of tumor cells from biopsy homogenates by utilizing differences in size and density.
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