Impedance Detection of 3‐Phenoxybenzoic Acid with a Noncompetitive Two‐site Phage Anti‐immunocomplex Assay

Abstract

Both direct and non‐competitive two‐site (sandwich) immunoassays are reported for 3‐phenoxybenzoic acid (3‐PBA) utilizing signal transduction by electrochemical impedance spectroscopy (EIS), and the sandwich immunoassay reduces the detection limit for this small molecule analyte by ∼70×. The direct EIS immunoassay utilizes a polyclonal antibody to 3PBA for biomolecular recognition, while the sandwich EIS immunoassay utilizes in addition a previously reported antiimmunocomplex M13 bacteriophage clone. For both immunoassays, the polyclonal antibody film is immobilized atop an Au electrode by amide bond formation. The direct EIS immunoassay exhibits a 3‐PBA sensitivity of 5.4×104 kΩ cm2 M1, and a detection limit of 2.5×10−4 M (54 μg/ml), while the sandwich EIS immunoassay exhibits a 3PBA sensitivity of 4.2×106 kΩ cm2 M−1, and a detection limit of 3.4×10−6 M (0.74 μg/ml). For the sandwich EIS immunoassay, a constant excess (80×109 PFU/ml) of bacteriophage is maintained during all experiments. This is the first report of a bacteriophage‐assisted sandwich EIS assay.