Devices and methods for detection and quantification of immunological proteins, pathogenic and microbial agents and cells

A microfluidic immunoassay device for detecting and measuring concentration of an analyte in a liquid sample, comprising a liquid sample inlet, a plurality of reaction chambers, each of the reaction chambers having an outlet manifold micro-channel where the liquid sample exits, wherein when the liquid sample flows through the plurality of reaction chambers, a plurality of antibody-coated magnetic-responsive micro-beads deposited on the surfaces of the reactions chambers disperses in the liquid sample and binds to any analyte present in the liquid sample, wherein any analyte present in the liquid sample also binds to antigen-specific antibodies immobilized on the surfaces of the plurality of reaction chambers to form Ab1-analyte-Ab2-coated magnetic-responsive micro-bead complexes, wherein any unbound antibody-coated magnetic-responsive micro-beads exit each of the plurality of reaction chambers via the outlet manifold micro-channels, each of said outlet manifold micro-channels terminating in a passive valve, wherein the liquid sample flow in the reaction chambers is substantially stopped for a period of time at each of the passive valves, wherein the substantial stoppage of flow allows the analyte to incubate with the Ab1 and Ab2 antibodies in the plurality of reaction chambers, wherein a primary flow splitter micro-channel is in continuous fluid connection with one of the outlet manifold micro-channels, said primary flow splitter micro-channel in continuous fluid connection with a delay micro-channel, said delay micro-channel in continuous fluid connection with a connector micro-channel, wherein said passive valves also are in continuous fluid connection with the connector micro-channel, said connector micro-channel in continuous fluid connection with a terminal flow splitter micro-channel which bifurcates to form a calibration micro-channel (Co) and at least one test micro-channel (Cm), said calibration micro-channel in continuous fluid connection with one or more calibration graduated columns, said at least one test micro-channel in continuous fluid connection with one or more test graduated columns, wherein said at least one test micro-channel is exposed to a magnetic field gradient which causes flocculation of the magnetic-responsive micro-beads in the at least one test micro-channel, wherein the flocculation reduces the flow rate (Qm) of the liquid sample in the at least one test micro-channel compared to the flow rate (Qo) of the liquid sample in the calibration micro-channel, wherein the volume of liquid collected in the one or more calibration graduated columns, Vo, and the volume collected in the one or more test graduated columns, Vm, are a proxy for the concentration of the analyte in the liquid sample.