For the flow rate dependence study, 50 ng/mL of fluorescently-labeled CRP antigen was delivered using a programmable NE-1000 syringe pump from New Era Pump systems Inc. (Farmingdale, NY) at flow rates of 10, 25, 100 and 200 μL/min for 7 min. Sensor beads labeled with 6 mg/mL of anti-CRP monoclonal antibodies were used.
For the penetration depth study, 36 mL of 50 ng/mL fluorescently-labeled CRP was re-circulated for 2 h using a peristaltic pump (FiaLAB, Bellevue, WA) at 200 μL/min. Sensor beads labeled with 2 mg/mL of anti-CRP polyclonal antibodies were used.
For the sandwich immunoassay standard curves, PSA antigen was purchased from Fitzgerald (Concord, MA) and concentrations of 5, 2, 1, 0.5, 0.2, and 0.1 ng/mL were prepared by serial dilutions. A volume of 100 μL of sample and 8 μL of the detection antibody was loaded into the fully integrated p-BNC card as previously described.39 (link) The sample was delivered at 5 μL/min for 20 min. A PBS buffer wash was delivered at 100 μL/min for 1 min. The detection antibody was delivered at 20 μL/min for 3 min. A final PBS buffer wash was delivered at 100 μL/min for 4 min. The total assay time was 28 min and the total assay volume used was 660 μL. Sensor beads labeled with 2.53 mg/mL of anti-free PSA monoclonal antibodies were used.
For the penetration depth study, 36 mL of 50 ng/mL fluorescently-labeled CRP was re-circulated for 2 h using a peristaltic pump (FiaLAB, Bellevue, WA) at 200 μL/min. Sensor beads labeled with 2 mg/mL of anti-CRP polyclonal antibodies were used.
For the sandwich immunoassay standard curves, PSA antigen was purchased from Fitzgerald (Concord, MA) and concentrations of 5, 2, 1, 0.5, 0.2, and 0.1 ng/mL were prepared by serial dilutions. A volume of 100 μL of sample and 8 μL of the detection antibody was loaded into the fully integrated p-BNC card as previously described.39 (link) The sample was delivered at 5 μL/min for 20 min. A PBS buffer wash was delivered at 100 μL/min for 1 min. The detection antibody was delivered at 20 μL/min for 3 min. A final PBS buffer wash was delivered at 100 μL/min for 4 min. The total assay time was 28 min and the total assay volume used was 660 μL. Sensor beads labeled with 2.53 mg/mL of anti-free PSA monoclonal antibodies were used.
