Selection of phage displayed peptides for the detection of 2,4,6-trinitrotoluene in seawater Original Research Article

We have selected for phage displayed peptides that showed specific binding to a 2,4,6-trinitrotoluene (TNT) derivative, 2,4,6-trinitrobenzene (TNB) in environmentally relevant conditions, and have integrated the selected phage into a continuous flow immunosensor platform for the detection of TNT. A library of 12 random amino acid peptides (12-mers) displayed on phage was panned against TNB coupled to the protein bovine serum albumin (BSA) in a solution of artificial seawater. Eight phage clones, seven of which share an identical amino acid sequence, bound selectively to TNB–BSA in artificial seawater as judged by enzyme-linked immunosorbent assay (ELISA). Addition of TNT, inhibited binding of the phage. Whole phage were labeled with the dye cyanine 5 (Cy5), and incorporated into a flow sensor platform. Labeled phage were loaded onto a TNB–affi-gel packed column, and a reproducible signal, at least five times greater than background, was observed on repeat injections of 10 mg/l TNT dissolved in seawater. This study presents one of the first examples of phage selection in a non-physiological medium, and the first demonstration that dye-labeled phage can be integrated into a continuous flow sensor.