Surface plasmon resonance-enhanced fluorescence implementation of a single-step competition assay: demonstration of fatty acid measurement using an anti-fatty acid monoclonal antibody and a Cy5-labeled fatty acid.

The development of a single-step, separation-free method for measurement of low concentrations of fatty acid using a surface plasmon resonance-enhanced fluorescence competition assay with a surface-bound antibody is described. The assay behavior was unexpectedly complex. A nonlinear coverage-dependent self-quenching of emission from surface-bound fluorescent label was deduced from the response kinetics and attributed to a surface plasmon-mediated energy transfer between adsorbed fluorophores, modified by the effects of plasmon interference. Principles of assay design to avoid complications from such effects are discussed. An anti-fatty acid mouse monoclonal antibody reacting to the alkyl chain was prepared and supported on a gold chip at a spacing appropriate for surface-plasmon field-enhanced fluorescence spectroscopy (SPEFS), by applying successively a self-assembled biotinylated monolayer, then streptavidin, then biotinylated protein A, and then the antibody, which was crosslinked to the protein A. Synthesis of a fluorescently (Cy5) tagged C-11 fatty acid is reported. SPEFS was used to follow the kinetics of the binding of the labeled fatty acid to the antibody, and to implement a competition assay with free fatty acid (undecanoic acid), sensitive at the 1 microM scale, a sensitivity limit caused by the low affinity of antibodies for free fatty acids, rather than the SPEFS technique itself. Free fatty acid concentration in human serum is in the range 0.1-1mM, suggesting that this measurement approach could be applied in a clinical diagnostic context. Finally, a predictive, theoretical model of fatty acid binding was developed that accounted for the observed "overshoot" kinetics.

Applications of tailored ferrocenyl molecules as electrochemical probes of biochemical interactions.

The development of electrochemical probes useful for investigating the occupancy by other molecules of sites on complex proteins such as human serum albumin (HSA) is described. Ferrocenyl-(oxoethylene)-fatty acid compounds of different fatty acid chain length probed different binding sites on HSA. The interaction could be changed from one primarily with a drug binding site, when the probe was ferrocene methanol, to one predominantly with medium-chain fatty acid binding sites, by adding an (oxoethylene)-fatty acid substituents. Finally, the interaction could be changed to one interacting primarily with high-affinity long-chain fatty acid binding sites, as the fatty acid chain length in ferrocene-(oxoethylene)-fatty acid molecules increased. These results strongly implied that the binding could be further tailored by relatively simple modifications to the probe, for example, by changing the balance of hydrophobicity and hydrophilicity. The possibility of a procedure using mass-produced electrochemical cells to determine the fractional occupancy of different sites on HSA is demonstrated.

Design and synthesis of ferrocene probe molecules for detection by electrochemical methods.

A series of ferrocenyl conjugates to fatty acids have been designed and synthesized to establish the key properties required for use in biomolecular binding studies. Amperometric detection of the ferrocene conjugates was sought in the region of 0.3 V (vs Ag/AgCl) for use in protein/blood solutions. Different linkers and solubilizing moieties were incorporated to produce a conjugate with optimal electrochemical properties. In electrochemical studies, the linker directly attached to the ferrocene was found to affect significantly the E(1/2) value and the stability of the ferrocenium cation. Ester-linked ferrocene conjugates had E(1/2) ranging from +400 to +410 mV, while amide-linked compounds ranged from +350 to +370 mV and the amines +260 to +270 mV. Folding of long-chain substituents around the ferrocene, also significantly affected by the choice of linker, was inferred as a secondary effect that increased E(1/2). The stability of the ferrocenium cation decreased systematically as E(1/2) increased. Disubstituted ferrocene ester and amide conjugates, with oxidation potentials of +640 and +570 mV, respectively, showed only a barely discernible reduction wave in cyclic voltammetry at 50 mV/s. Electrochemical measurements identified two lead compounds with the common structural characteristics of an amide and carbamate linker (compounds 17 and 21) with a C(11) fatty acid chain attached. It is envisaged that such molecules can be used to mimic and study the biomolecular binding interaction between fatty acids and molecules such as human serum albumin.

Disk-generation/ring-collection scanning electrochemical microscopy: theory and application.

The potential of ring-disk ultramicroelectrodes (RD UMEs) as probes for scanning electrochemical microscopy (SECM) was investigated both theoretically and experimentally. In particular, the disk-generation/ring-collection (DG/RC) mode of operation was considered. In this case, the interaction of two species with the substrate under investigation can be followed simultaneously from single tip current-distance measurement (approach curve) to the substrate. Theoretical approach curves for DG/RC were calculated by numerical methods. Such approach curves to both insulating and conducting substrates indicate a strong tip response dependence on the ring radius while the response was relatively insensitive to ring thickness and overall tip radius. The RD tip was characterized by fitting experimental approach curves recorded at insulating and conducting substrates to simulated curves for a given tip geometry. DG/RC SECM was then applied to investigate the partitioning of iodine across a liquid-liquid interface.