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Quantitative Evaluation of Sensitivity and Selectivity of Multiplex NanoSPR Biosensor Assays

Department of Agricultural and Biological Engineering, Bindley Biosciences Center, Purdue University, West Lafayette, Indiana

Correspondence: Address reprint requests to Dr. J. Irudayaraj, E-mail: josephi{at}purdue.edu.

A new functionalization procedure was developed to replace cyltrimethylammoniumbromide coating on gold nanorods (GNRs) fabricated through seed-mediated growth with chemically active alkanethiols; antibodies were then attached to the GNRs to yield gold nanorod molecular probes (GNrMPs). The functionalization procedure was shown to minimize nonspecific binding. Multiplex sensing was demonstrated for three targets (goat anti-human IgG, goat anti-rabbit IgG, and goat anti-mouse IgG) through the distinct response of the plasmon spectra of GNrMPs to binding events. Quantification of the plasmonic binding events and estimation of ligand binding kinetics tethered to these nanoscale structures was also demonstrated through a mathematical approach. Evaluation of the experimental and theoretical data yields an affinity constant K_a = 1.34 x 10⁷ M^–1, which was in agreement with the IgG-antiIgG binding affinity reported in the literature. The GNrMP sensors were found to be highly specific and sensitive with the dynamic response in the range between 10^–9 M and 10^–6 M. The limit of detection of GNrMPs was found to be in the low nanomolar range, and is a function of the binding affinity: for a higher probe-target affinity pair, the limit of detection can be expected to reach femto molar levels. This technique can play a key role in developing tunable sensors for sensitive and precise monitoring of biological interactions.