Method and system of microfluidic immunoassay using magnetic beads

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.