Understanding the chemical composition and morphology of interfaces plays a vital role in the development of sensors, drug delivery systems, coatings for biomedical implants, and so forth. In many cases, the interface characterization can be performed by a combination of electrochemical and one of the optical techniques. In this study, we further enhanced capabilities in probing interfaces by combining electrochemical characterization with multiple optical techniques, that is, surface plasmon resonance (SPR) and fluorescence spectroscopy. This new combination was utilized to study the electrochemical proximity assay (ECPA)-a recently developed protein recognition strategy for the point-of-care test. The SPR/fluorescence spectroelectrochemical technique has achieved not only recognition of binding components involved in the ECPA model system, estimation of their thicknesses and surface coverages, but more importantly, highly reliable in situ monitoring of dynamic changes of components involved in interfacial binding via cross-validation and confirmation from three simultaneously generated signals-SPR, fluorescence, and electrochemistry. In addition, the obtained corresponding proportions among magnitudes of three signals provide crucial information for future studies on simultaneous characterization of multiple components in one step and differentiation of nonspecific binding events. Another advantage using this technique is that the excitation of fluorescence is not only confined by surface plasmons, but by photons, so the fluorescence information can be also gained as the distance of fluorophores from the surface exceeds the decay length of surface plasmons.
Keywords: electrochemical proximity assay; fluorescence; spectroelectrochemistry; square wave voltammetry; surface plasmon resonance.