Extended range immunoassay devices with immunosensor and magnetic immunosensor

A method of determining a concentration of an analyte in a fluid sample utilizing a device comprising a conduit and a housing, the conduit comprising a first sensor and a second sensor,

• the method comprising:
• in the conduit, contacting with the fluid sample a signal antibody that is specific for the analyte and is conjugated to an enzyme thereby producing a first mixture and allowing the signal antibody to dissolve in the fluid sample
• and form a first complex of the signal antibody and the analyte;
• contacting the first mixture of the fluid sample and the signal antibody with magnetic beads coated with first capture antibodies specific for the analyte thereby producing a second mixture and allowing the magnetic beads to dissolve in the first mixture and form a second complex comprising the first complex and the first capture antibodies coated on the magnetic beads;
• contacting the first mixture of the fluid sample and the signal antibody specific for the analyte with the first sensor, the first sensor comprising immobilized on its surface second capture antibodies specific for the analyte, thereby forming a third complex comprising the first complex and second capture antibodies specific for the analyte;
• contacting the second mixture comprising the second complex with the second sensor and attracting the second complex to the second sensor by a magnetic field generated by a high-field magnet located within a trench that extends to a region in the housing below the second sensor;
• applying to the first sensor and the second sensor via the conduit a fluid comprising a substrate for the enzyme;
• detecting by the first sensor a first signal from a reaction of the substrate with the third complex, wherein the first signal is indicative of a first concentration of the analyte in an upper concentration range;
• detecting by the second sensor a second signal from a reaction of the substrate with the second complex attracted by the magnetic field to the second sensor, wherein the second signal is indicative of a second concentration of the analyte in a lower concentration range and determining the concentration of the analyte in the fluid sample based on the first concentration, the second concentration, and a predetermined crossover concentration.