Impedance detection of 3-phenoxybenzoic acid with a noncompetitive two-site phage anti-immunocomplex assay

Authors: PALI, MADHAVI - Southern Illinois University; Bever, Candace; VASYLIEVA, NATALIA - University Of California, Davis; HAMMOCK, BRUCE - University Of California, Davis; SUNI, IAN - Southern Illinois University

Submitted to: Electroanalysis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2018
Publication Date: 9/14/2018
Citation: Pali, M., Bever, C.R., Vasylieva, N., Hammock, B.D., Suni, I.I. 2018. Impedance detection of 3-phenoxybenzoic acid with a noncompetitive two-site phage anti-immunocomplex assay. Electroanalysis. https://doi.org/10.1002/elan.201800457.
DOI: https://doi.org/10.1002/elan.201800457

Interpretive Summary: Immunoassays utilize the exquisite ability of an antibody to detect a particular analyte. These interactions can be studied using electrochemical impedance spectroscopy (EIS) wherein a change in the electron transfer resistance of the circuit is observed relative to the amount of analyte that is present. In this study, a small chemical, 3-phenoxybenzoic acid (3-PBA), a major human metabolite from most commercial pyrethroid insecticides, is detected using two different formats. The first format utilizes an antibody that selectively detects 3-PBA coated onto the electrode surface. As the analyte is introduced, the electrical impedance of the reaction is detected. The second format is performed by using this same antibody-coated electrode surface, but now introducing both the 3-PBA analyte as well as a peptide displayed on a bacteriophage that selectively binds the 3-PBA+antibody immunocomplex in a sandwich type assay. Based on these two formats, the ability to improve the detection limit by 30-fold was observed with the latter format utilizing the larger bacteriophage. This is the first report of a bacteriophage-assisted sandwich EIS assay.

Technical 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 ~30x. The direct EIS immunoassay utilizes a polyclonal antibody to 3-PBA for biomolecular recognition, while the sandwich EIS immunoassay utilizes in addition a previously reported anti-immunocomplex 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 2.62x104 k'-cm2 M-1, and a detection limit of 1.7x10-4 M (36.4 µg/ml), while the sandwich EIS immunoassay exhibits a 3PBA sensitivity of 4.21x106 k'-cm2 M-1, and a detection limit of 5.5x10-6 M (1.2 µg/ml). For the sandwich EIS immunoassay, a constant excess (80x109 PFU/ml) of bacteriophage is maintained during all experiments. This is the first report of a bacteriophage-assisted sandwich EIS assay.