Atomic force microscopy of the morphology and mechanical behaviour of barnacle cyprid footprint proteins at the nanoscale

Abstract

Barnacles are a major biofouler of man-made underwater structures. Prior to settlement, cypris larvae explore surfaces by reversible attachment effected by a 'temporary adhesive'. During this exploratory behaviour, cyprids deposit proteinaceous 'footprints' of a putatively adhesive material. In this study, footprints deposited by Balanus amphitrite cyprids were probed by atomic force microscopy (AFM) in artificial sea water (ASW) on silane-modified glass surfaces. AFM images obtained in air yielded better resolution than in ASW and revealed the fibrillar nature of the secretion, suggesting that the deposits were composed of single proteinaceous nanofibrils, or bundles of fibrils. The force curves generated in pulloff force experiments in sea water consisted of regions of gradually increasing force, separated by sharp drops in extension force manifesting a characteristic saw-tooth appearance. Following the relaxation of fibrils stretched to high strains, force -distance curves in reverse stretching experiments could be described by the entropic elasticity model of a polymer chain. When subjected to relaxation exceeding 500 ms, extended footprint proteins refolded, and again showed saw-tooth unfolding peaks in subsequent force cycles. Observed rupture and hysteresis behaviour were explained by the 'sacrificial bond' model. Longer durations of relaxation (.5 s) allowed more sacrificial bond reformation and contributed to enhanced energy dissipation (higher toughness). The persistence length for the protein chains (L P ) was obtained. At high elongation, following repeated stretching up to increasing upper strain limits, footprint proteins detached at total stretched length of 10 mm.

Antifouling properties of hydrogels

“…This walking is called 'exploring behavior', aiming to decide whether the substrate is suitable for settling [14,15]. After the exploring behavior, a cypris attaches firmly and permanently to the substrate by secreting the adhesive proteins [16,17] and then transforms into a juvenile. In the post-settlement stage, barnacles secrete cement proteins to the substrate and broaden their adhesion area [18].…”

Matching the nano- to the meso-scale: Measuring deposit–surface interactions with atomic force microscopy and micromanipulation

“…Previous work on different fouling deposits and surfaces have used atomic force microscopy (AFM) to study surfaces, such as Parbhu et al ,(2006) Santos et al (2004, Verran and Whitehead (2006) and Whitehead et al (2006). AFM is able both to measure surface topography and energetics over a nanometre to micron scale (such as in marine biofilms by Phang et al, 2006Phang et al, , 2010 However there is very little work done in which AFM has been used to study food fouling problems.…”

Analysis of the Behaviours Mediating Barnacle Cyprid Reversible Adhesion

“…Phang et al [20] used atomic force microscopy (AFM) to characterise nanoscopically the mechanical behaviour of this material, reporting an absence of strain-rate dependence in the force required to rupture protein aggregates, therefore implying viscous, rather than viscoelastic character for subunits of the footprint material. However, caution must always be exercised when extrapolating such data to the whole-organism and it is clear that in its natural application the footprint material is highly elastic and not simply viscous (Movie S10).…”

“…However, caution must always be exercised when extrapolating such data to the whole-organism and it is clear that in its natural application the footprint material is highly elastic and not simply viscous (Movie S10). In fact, when testing droplets of spider adhesive, Sahni et al [35] only observed viscoelastic character at strain rates above 10 µm/second, whereas Phang et al [20] performed experiments between 0.5 and 11 µm/second. It is perhaps not surprising, therefore, that only a gradual increase in rupture force was observed in the range of strain rates tested by Phang et al [20].…”

“…Although they found no direct evidence for a mechanism of action, Phang et al [18] proposed the possibility that the cuticular villi or ‘hairs’ covering the attachment disc may be involved in adhesion to surfaces (first proposed by Crisp [26] in a similar way to the cuticular hairs covering the pulvilli of dipteran flies [9], [31]. Phang et al [20] also identified common characteristics of adhesive proteins in the cyprid footprint material, such as intermolecular sacrificial bonds, supporting the involvement of this material in temporary adhesion. Despite these studies and several other attempts to use advanced biophysical methods to determine the relative adhesive contributions of the footprint material, the morphology of the disc and the behaviour of the cyprid during adhesion, the entire process of temporary adhesion remains poorly understood.…”