Monoclonal antibody--gold biosensor chips for detection of depurinating carcinogen--DNA adducts by fluorescence line-narrowing spectroscopy.
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.
Duhachek
S D
SD
Ames Laboratory-USDOE and Department of Chemistry, Microanalytical Instrumentation Center, Iowa State University, 50011, USA.
Kenseth
J R
JR
Casale
G P
GP
Small
G J
GJ
Porter
M D
MD
Jankowiak
R
R
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D015374:Biosensing Techniques; D002273:Carcinogens / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis* / Q000276:immunology; D006046:Gold / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000911:Antibodies, Monoclonal; D002273:Carcinogens; D018736:DNA Adducts; D006046:Gold