US-12618836-B2 - Method and system of microfluidic immunoassay using magnetic beads

Abstract

A microfluidic Western blot method and system including a microfluidic western blot method for immunoassay of proteins, the method including introducing a sample including the proteins onto a chip; electrophoretically separating the proteins; binding the separated proteins to beads to form protein-attached beads, the beads being magnetic; flowing the protein-attached beads into a magnetic holding region; applying a magnetic field to the magnetic holding region to fix the protein-attached beads in place within the magnetic holding region; binding primary antibodies to target proteins on the protein-attached beads; binding secondary antibodies to the bound primary antibodies; and detecting the bound secondary antibodies.

Inventors

• Andrew Barry
• Laurel Provencher
• Seth Cohen
• I-Jane Chen
• Jun Yan
• Jingjing Wang

Assignees

• CALIPER LIFE SCIENCES, INC.

Dates

Publication Date

20260505

Application Date

20201125

Claims (18)

1. 1 . A microfluidic Western blot system for immunoassay of analytes in a sample, the system including a microfluidic chip comprising: a sample well configured to hold the sample; a sample channel in fluid communication with the sample well and configured to supply the sample to the system; an injection channel in fluid communication with the sample channel and configured to draw the sample from the sample well through the sample channel into the injection channel by applying a differential voltage between a sample well electrode and an injection electrode disposed downstream of the injection channel; a first separation electrode channel in fluid communication with the sample channel and the injection channel, the first separation electrode channel is configured to draw the sample from the injection channel into the first separation electrode channel by applying a differential voltage between the injection electrode and a first separation electrode disposed downstream of the first separation electrode channel; a separation region in fluid communication with, and downstream of, the sample well, the sample channel, the injection channel, and the first separation electrode channel, wherein the sample channel, the injection channel, and the first separation electrode channel are all operably connected together at an upstream end of the separation region at a common, non-linear junction, the channels being electrically discrete and individually addressable by the sample-well electrode, the injection electrode, and the first separation electrode, such that independent voltage control at the electrodes selectively routes a discrete plug of the sample among the channels without electrical cross-talk, the injection electrode being positioned intermediate the sample well and the first separation electrode to establish a progressive voltage gradient through the junction during sample transfer the separation region is configured to draw the sample from the first separation electrode channel into the separation region and electrophoretically separate analytes in the sample within the separation region based on size and charge by applying a differential voltage between the first separation electrode and a second separation electrode disposed downstream of the separation region; a peak detection microfluidic channel disposed downstream of the separation region and upstream of a bead well, the peak detection microfluidic channel defining an optically accessible portion of the microfluidic chip configured to permit detection of fluorescent or luminescent optical signals from the separated analytes migrating from the separation region, the peak detection microfluidic channel enabling measurement of migration times and signal intensities of the separated analytes corresponding to analyte size and concentration and enabling generation of data for quantitative analysis of the separated analytes and for timing control of subsequent electrophoretic transfer toward the bead well; a bead well in fluid communication with, and downstream of, the separation region, the bead well disposed upstream of the second separation electrode, the bead well is configured to supply magnetic beads for attachment to the separated analytes to form protein-attached magnetic beads, the bead well is configured to supply the magnetic beads for attachment by applying a differential voltage between an electrode associated with the bead well and the second separation electrode; a magnetic holding region in fluid communication with, and downstream of, the separation region and the bead well, the magnetic holding region being electrically isolated from the separation region to prevent electrophoretic migration during immunoassay incubation, and operably coupled to an electromagnet oriented to induce a magnetic field orthogonal to the electrophoretic flow direction within the magnetic holding region, the electromagnet is configured to fix the protein-attached magnetic beads in place within the magnetic holding region, the first and second separation electrodes are configured to move the separated analytes from the separation region to the magnetic holding region by applying a differential voltage between the first and second separation electrodes; and an antibody well in fluid communication with, and upstream of, the magnetic holding region and second separation electrode, the antibody well is configured to flow antibodies into the magnetic holding region via an immunoassay channel for binding to the protein-attached beads by applying a differential voltage between an electrode associated with the antibody well and the second separation electrode.
2. 2 . The system of claim 1 , wherein the antibody well is configured to hold either primary or secondary antibodies, and further comprising: a second antibody well configured to hold the other of primary or secondary antibodies, wherein the immunoassay channel is configured to supply primary antibodies or secondary antibodies to the magnetic holding channel for binding to the protein-attached magnetic beads.
3. 3 . The system of claim 1 , further comprising a blocking buffer well and a detection reagent well, wherein the immunoassay channel is configured to supply blocking buffer or detection reagent to the magnetic holding channel for binding to the protein-attached magnetic beads.
4. 4 . The system of claim 1 , further comprising a destain well operably coupled to, and downstream of, the separation region, the destaining well is configured to add destaining solution to the separated analytes in sample to remove detergent micelles, allowing visualization of protein peaks in the sample and reducing signal background upon detection, the destain well is configured to add destaining solution by applying a differential voltage between an electrode associated with the destain well and the second separation electrode.
5. 5 . The system of claim 1 , further comprising a heating element operably coupled to the magnetic holding region to control temperature for incubation within the magnetic holding region.
6. 6 . The system of claim 1 , further comprising a sipper port operably coupled to the sample well for automating liquid sampling.
7. 7 . The system of claim 1 , wherein the upstream end of the separation region connecting together all of the sample channel, the injection channel, and the first separation electrode channel is configured to draw the sample non-linearly (i) from the sample channel into the injection channel, (ii) from the injection channel into the separation electrode channel; and (iii) from the separation electrode channel into the separation region.
8. 8 . The system of claim 1 , wherein the first separation electrode, second separation electrode, and injection electrode are positioned non-linearly such that: the injection channel is configured to draw the sample from the sample well, through the sample channel, to the upstream end of the separation region, and subsequently into the injection channel, the first separation electrode channel is configured to draw the sample from the injection channel, back to the upstream end of the separation region, and subsequently into the first separation electrode channel, and the separation region is configured to draw the sample from the first separation electrode channel, back to the upstream end of the separation region, and subsequently into the separation region.
9. 9 . The system of claim 1 , further comprising a control circuit operably coupled to the electrodes associated with the sample well, injection channel, first separation electrode channel, and separation region, the control circuit is configured to receive data from the peak detection microfluidic channel and to automatically adjust the applied voltages to maintain uniform electrophoretic separation and timed transfer of the separated analytes toward the bead well.
10. 10 . The system of claim 1 , further comprising a wash-buffer well in fluid communication with the magnetic holding region, the wash-buffer well is configured to deliver buffer solution by electrophoretic or electroosmotic flow between an electrode associated with the wash-buffer well and the second separation electrode to remove unbound reagents from the protein-attached magnetic beads before antibody incubation.
11. 11 . The system of claim 1 , wherein the magnetic holding region comprises a plurality of discrete sub-regions, each associated with a different antibody well and electromagnet zone, such that multiple analytes are simultaneously immunoassayed on spatially separated groups of magnetic beads within the microfluidic chip.
12. 12 . A microfluidic Western blot system including a microfluidic chip for immunoassay of analytes in a sample, the microfluidic chip comprising: a sample well configured to hold the sample; a sample channel in fluid communication with the sample well and configured to supply the sample to the system; an injection channel in fluid communication with the sample channel, and the injection channel is configured to draw the sample from the sample well through the sample channel into the injection channel by applying a differential voltage between a sample well electrode corresponding to the sample well and an injection electrode disposed downstream of the injection channel; a first separation electrode channel in fluid communication with the sample channel and the injection channel, the first separation electrode channel is configured to draw the sample from the injection channel into the first separation electrode channel by applying a differential voltage between the injection electrode and a first separation electrode disposed downstream of the first separation electrode channel; a separation region in fluid communication with, and downstream of, the sample well, the sample channel, the injection channel, and the first separation electrode channel, wherein the sample channel, the injection channel, and the first separation electrode channel each terminate at a common, non-linear junction disposed at an upstream end of the separation region, the junction being spatially offset such that none of the three channels are collinear, the channels being electrically discrete and individually addressable by the sample-well electrode, the injection electrode, and the first separation electrode such that independent voltage control at the electrodes selectively routes a discrete plug of the sample among the channels without electrical cross-talk or unintended flow, and the injection electrode being positioned intermediate the sample well and the first separation electrode to establish a progressive voltage gradient through the junction during sample transfer; a peak detection microfluidic channel disposed downstream of the separation region and upstream of a bead well, the peak detection microfluidic channel defining an optically accessible portion of the microfluidic chip configured to permit detection of fluorescent or luminescent optical signals from the separated analytes migrating from the separation region, the peak detection microfluidic channel enabling measurement of migration times and signal intensities of the separated analytes corresponding to analyte size and concentration and enabling generation of data for quantitative analysis of the separated analytes and for timing control of subsequent electrophoretic transfer toward the bead well; a bead well in fluid communication with, and downstream of, the separation region, the bead well is configured to supply magnetic beads via a bead channel for attachment to the separated analytes to form protein-attached magnetic beads; a magnetic holding region in fluid communication with, and downstream of, the separation region, the bead well, and the first separation electrode, the magnetic holding region being electrically isolated from the separation region to prevent electrophoretic migration during immunoassay incubation, and operably coupled to an electromagnet oriented to induce a magnetic field orthogonal to the electrophoretic flow direction within the magnetic holding region, the electromagnet is configured to fix the protein-attached magnetic beads in place within the magnetic holding region; an antibody well in fluid communication with, and upstream of, the magnetic holding region, the antibody well is configured for flowing antibodies into the magnetic holding region via an immunoassay channel for binding to the protein-attached beads; and wherein: the separation region is configured to draw the sample from the first separation electrode channel into the separation region and electrophoretically separate analytes in the sample within the separation region based on size and charge by applying a differential voltage between the first separation electrode and a second separation electrode disposed downstream of the separation region, the bead well is disposed upstream of the second separation electrode, the bead well is configured to supply the magnetic beads for attachment by applying a differential voltage between an electrode associated with the bead well and the second separation electrode, the magnetic holding region is disposed upstream of the second separation electrode such that the first and second separation electrodes are configured to move the sample from the separation region to the magnetic holding region by applying a differential voltage between the first and second separation electrode, and the antibody well is disposed upstream of the second separation electrode, the antibody well is configured to flow antibodies into the magnetic holding region by applying a differential voltage between an electrode associated with the antibody well and the second separation electrode.
13. 13 . The microfluidic chip of claim 12 , wherein the antibody well is configured to hold either primary or secondary antibodies, and further comprising: a second antibody well configured to hold the other of primary or secondary antibodies, wherein the immunoassay channel is configured to supply primary antibodies or secondary antibodies to the magnetic holding channel for binding to the protein-attached magnetic beads.
14. 14 . The microfluidic chip of claim 12 , further comprising a blocking buffer well and a detection reagent well, wherein the immunoassay channel is configured to supply blocking buffer or detection reagent to the magnetic holding channel for binding to the protein-attached magnetic beads.
15. 15 . The microfluidic chip of claim 12 , further comprising a destain well operably coupled to, and downstream of, the separation region, the destaining well is configured to add destaining solution to the separated analytes in sample to remove detergent micelles, allowing visualization of protein peaks in the sample and reducing signal background upon detection.
16. 16 . The microfluidic chip of claim 12 , further comprising a heating element operably coupled to the magnetic holding channel to control temperature for incubation within the magnetic holding channel.
17. 17 . The microfluidic chip of claim 12 , wherein at least one internal surface of the separation region or the sample channel is coated with a hydrophilic polymer or zwitterionic surface layer to reduce protein adsorption and improve analyte migration uniformity during electrophoresis.
18. 18 . The microfluidic chip of claim 12 , further comprising a calibration mode in which a reference sample containing molecular-weight standards is electrophoretically separated within the separation region to generate calibration peaks that establish migration-time and distance correlations for quantitative determination of analyte molecular weight.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a divisional of and claims the benefit of priority to U.S. patent application Ser. No. 15/830,777 filed Dec. 4, 2017, which is a continuation of U.S. Ser. No. 14/597,999, issued U.S. Pat. No. 9,835,623, filed on Jan. 15, 2015, which claims the benefit of priority to U.S. Provisional Patent Application No. 61/927,960, filed Jan. 15, 2014. The entire disclosures of which are incorporated herein in their entirety, for any purpose whatsoever. TECHNICAL FIELD The technical field of this disclosure is assay systems, particularly, methods and systems of microfluidic immunoassay using magnetic beads. BACKGROUND OF THE INVENTION The use of microfluidic technology has been proposed for a number of analytical chemical and biochemical operations. This technology allows one to perform chemical and biochemical reactions, macromolecular separations, and the like, that range from the simple to the relatively complex, in easily automated, high-throughput, low-volume systems. Further information about microfluidic devices and systems is presented in U.S. Pat. No. 6,534,013 to Kennedy, issued Mar. 18, 2003, and incorporated in its entirety herein by reference. As used herein, the term “microfluidic,” or the term “microscale” when used to describe a fluidic element, such as a passage, chamber or conduit, generally refers to one or more fluid passages, chambers or conduits which have at least one internal cross-sectional dimension, e.g., depth or width, of between about 0.1 pm and 500 pm. In the devices of the present invention, the microscale channels preferably have at least one cross-sectional dimension between about 0.1 pm and 200 pm, more preferably between about 0.1 pm and 100 pm, and often between about 0.1 pm and 20 pm. In general, microfluidic systems include a microfluidic device, or chip, that has networks of integrated submicron channels in which materials are transported, mixed, separated, and detected. Microfluidic systems typically also contain components that provide fluid driving forces to the chip and that detect signals emanating from the chip. Microfluidic chips may be fabricated from a number of different materials, including glass or polymeric materials. An example of a commercially available microfluidic chip is the DNA LabChip® manufactured by Caliper Life Sciences, Inc. of Hopkinton, Massachusetts, and used with the Agilent 2100 Bioanalyzer system manufactured by Agilent Technologies, Inc. of Palo Alto, California. The chip has two major components: a working part made of glass, and a plastic caddy or mount bonded to the working part. The working part contains microfluidic channels in its interior, and wells on its exterior that provide access to the microfluidic channels. The working part is typically fabricated by bonding together two or more planar substrate layers. The microfluidic channels in the working part are formed when one planar substrate encloses grooves formed on another planar substrate. The mount protects the working part of the chip, and provides for easier handling of the chip by a user. The increased ease of handling partially results from the fact that the mount is larger than the working part of the device, which in many cases is too small and thin to be easily handled. The mount may be fabricated from any suitable polymeric material, such as an acrylic or thermoplastic. The glass working part is typically bonded to the polymeric mount using a UV-cured adhesive. Reservoirs in the mount provide access to the wells on the working part of the chip. The reservoirs hold much greater volumes of material than the wells in the working part, thus providing an interface between the macro-environment of the user and the microenvironment of the wells and channels of the microfluidic device. This type of microfluidic chip is a “planar” chip. In a planar chip, the only access to the microchannels in the chip is through the reservoirs in the caddy and in-turn through the wells in the working part. Another type of microfluidic chip is a “sipper” chip, which has a small tube or capillary (the “sipper”) extending from the chip through which fluids stored outside the chip can be directed into the microfluidic channels in the chip. Typical sipper chips have between one and twelve sippers. In use, the sipper is placed in a receptacle having sample material and minute quantities of the sample material are introduced, or “sipped” through the capillary tube to the microfluidic channels of the chip. This sipping process can be repeated to introduce any number of different sample materials into the chip. Sippers make it easier to carry out high-throughput analysis of numerous samples on a single microfluidic chip. Western blot electrophoresis assays have been developed to detect specific proteins in a sample. The process can be divided into three parts: protein separation, sample transfer, and immunoassay. In protein separation, mechanical and/or ch