Monoclonal Antibody−Gold Biosensor Chips for Detection of Depurinating Carcinogen−DNA Adducts by Fluorescence Line-Narrowing Spectroscopy

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Abstract
A new direct readout methodology for detection and quantitation of fluorescent carcinogen−DNA adducts is described. It combines the binding specificity of an immobilized monoclonal antibody (MAb) with high-resolution, low-temperature fluorescence spectroscopy. The MAb, which is covalently bound to a gold surface via a chemisorbed disulfide coupling agent, binds the adduct of interest in an aqueous sample. Laser-induced fluorescence under nonline narrowing (FNLN) and line-narrowing (FLN) conditions was used to detect (benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) bound to immobilized MAb. At room temperature, the BP-6-N7Gua fluorescence was not detected, most likely because of quenching by the gold surface and/or efficient dynamical quenching. However, fluorescence was observed at room temperature when the surface was covered with a thin layer of glycerol, and possible reasons for the fluorescence enhancement are considered. Lowering of the temperature to 77 K led to nearly an order of magnitude increase in fluorescence intensity. Highly structured FLN spectra obtained at 4.2 K allowed for definitive adduct identification. The potential of this methodology for risk assessments of individuals exposed to polycyclic aromatic hydrocarbons is discussed.