Imaging Active Surface Processes in Barnacle Adhesive Interfaces

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces.