A Displacement Flow Immunosensor for Explosive Detection Using Microcapillaries

In this paper, we report on the characterization of a capillary-based displacement flow immunosensor for the explosive, 2,4,6-trinitrotoluene (TNT). Anti-TNT antibody is immobilized onto the silanized inner walls of a fused-silica capillary using a heterobifunctional cross-linker followed by saturating the capillary with fluorophore-labeled antigen. To perform the assay, an aliquot of target analyte solution is injected into the capillary column. The target analyte competes for the binding pocket of the fluorophore-labeled antigen, which is detected downstream. We have demonstrated that the sensitivity of the capillary-based immunosensor is improved by at least 3 orders of magnitude compared to packed-column or membrane-based displacement flow immunosensors. We have investigated the effect of flow rate and length of the capillary on the performance of the capillary immunosensor. We have demonstrated that, by the appropriate choice of an antibody, one can prepare a capillary immunosensor for the specific detection of TNT or as a detector for both TNT and its major metabolites. The limit of detection for TNT using the capillary immunosensor is 440 amol (100 μL injection of 1 pg/mL TNT solution). The relative standard deviation (RSD) of the area under the peaks resulting from 10 back-to-back 100 μL injections of 5 ng/mL TNT to the capillary immunosensor is 4%. The RSD for column-to-column variation is observed to be 5% ( n = 5). The entire assay can be performed in less than 3 min.