A new biosensor platform that provides simultaneous fluorescence detection and electrochemical control of biospecific binding has been developed and investigated using antibody-antigen and streptavidin-biotin interactions. Specifically, biotin was covalently bound to a transparent indium-tin oxide (ITO) working electrode, which also served as an integral part of a total internal reflection fluorescence (TIRF) flow cell. TIRF was used to monitor biospecific interactions, while electrochemical polarization was employed to control interactions between biotin and streptavidin or between biotin and anti-biotin antibodies. Both streptavidin and polyclonal anti-biotin antibodies bound kinetically irreversibly to the biotinylated surface. In the absence of electrochemical control, the assay exhibited an extremely slow release of the bound analytes, causing poor regeneration ability of the biosensor surface. However, electrochemical polarization was found to stimulate dissociation of kinetically irreversibly bound biotin-streptavidin and antibody-antigen complexes. A "square wave" polarization function stimulated dissociation more effectively than a "saw tooth" function over the same voltage range. Application of the square wave polarization resulted in regeneration of an active biotinylated surface. Electrochemical polarization also modified affinity and kinetics of protein adsorption, which could likely be used to promote biospecific interactions and/or to suppress nonspecific adsorption.
In conventional fluorescence spectroscopy, fluorescence intensity at high fluorophore concentration is oRen not proportional to fluorophore concentration, owing to primary and secondary absorption (inner-filter effects). In this paper, fluorescence calibration curves for anthracene solutions were obtained using a conventional right angle cell, a frontal reflection cell, a short pass cell, and a total internal reflection fluorescence (TIRF) cell for comparing the observed primary inner-filter effects. Measurements were also made of a two-component mixture using the nonfluorescent 94trofluorene with anthracene for comparing primary and secondary inner-filter effects. A conventional right angle cell exhibited the widest linear dynamic range and lowest detectable anthracene concentration, whereas the TIRF cell provided the best linearity at high concentrations. The TIRF cell was determined to have significant potential for quantitative analysis of hghly concentrated and/or turbid solutions.
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