Microcapillary reversed-displacement immunosensor for trace level detection of TNT in seawater

A reversed-displacement immunosensor to detect the explosive, 2,4,6-trinitrotoluene (TNT) using a chemically modified borosilicate glass microcapillary is described. The inner core of a glass microcapillary was modified with 3-aminopropyltriethoxysilane and functionalized with the TNT analog, trinitrobenzene sulfonic acid (TNBSA). A Cy5-labeled anti-TNT antibody was incubated within the trinitrobenzene (TNB) modified microcapillary until binding equilibrium, forming the TNB-antiTNT antibody complex. The TNB-microcapillary was then subjected to a constant flow of buffer as samples were applied. As samples containing TNT were introduced into the microcapillary flow stream, the Cy5-labeled antibody bound to the immobilized TNB was displaced and fluorescence measured by a fluorometer. Using the microcapillary-based reversed-displacement immunosensor (MRDI) format, parts-per-trillion (pptr) detection limits for TNT in seawater (0.25 ng/mL or 250 pptr) was achieved with an analysis time under 5 min. Reported also are experimental results comparing silane deposition conditions and the effect on immunoassay performance and the comparison of fluorescence signal response with the MRDI using four different monoclonal anti-TNT antibodies.