In Yee Phang

Janus Particles with Controllable Patchiness and Their Chemical Functionalization and Supramolecular Assembly

Seeing the right face: Mono- and bifunctionalized Janus particles with controllable chemical patchiness are prepared by a general masking/unmasking technique and subsequent chemical functionalization (see picture). Supramolecular host- and guest-functionalized Janus particles were prepared, and specific noncovalent host-guest interactions were used to controllably assemble heterogeneous particles.

The effects of a serine protease, Alcalase®, on the adhesives of barnacle cyprids (Balanus amphitrite)

Barnacles are a persistent fouling problem in the marine environment, although their effects (eg reduced fuel efficiency, increased corrosion) can be reduced through the application of antifouling or fouling-release coatings to marine structures. However, the developments of fouling-resistant coatings that are cost-effective and that are not deleterious to the marine environment are continually being sought. The incorporation of proteolytic enzymes into coatings has been suggested as one potential option. In this study, the efficacy of a commercially available serine endopeptidase, Alcalase® as an antifoulant is assessed and its mode of action on barnacle cypris larvae investigated. In situ atomic force microscopy (AFM) of barnacle cyprid adhesives during exposure to Alcalase supported the hypothesis that Alcalase reduces the effectiveness of the cyprid adhesives, rather than deterring the organisms from settling. Quantitative behavioural tracking of cyprids, using Ethovision™ 3.1, further supported this observation. Alcalase removed cyprid ‘footprint’ deposits from glass surfaces within 26 min, but cyprid permanent cement became resistant to attack by Alcalase within 15 h of expression, acquiring a crystalline appearance in its cured state. It is concluded that Alcalase has antifouling potential on the basis of its effects on cyprid footprints, un-cured permanent cement and its non-toxic mode of action, providing that it can be successfully incorporated into a coating.

Interlaboratory round robin on cantilever calibration for AFM force spectroscopy

Single-molecule force spectroscopy studies performed by Atomic Force Microscopes (AFMs) strongly rely on accurately determined cantilever spring constants. Hence, to calibrate cantilevers, a reliable calibration protocol is essential. Although the thermal noise method and the direct Sader method are frequently used for cantilever calibration, there is no consensus on the optimal calibration of soft and V-shaped cantilevers, especially those used in force spectroscopy. Therefore, in this study we aimed at establishing a commonly accepted approach to accurately calibrate compliant and V-shaped cantilevers. In a round robin experiment involving eight different laboratories we compared the thermal noise and the Sader method on ten commercial and custom-built AFMs. We found that spring constants of both rectangular and V-shaped cantilevers can accurately be determined with both methods, although the Sader method proved to be superior. Furthermore, we observed that simultaneous application of both methods on an AFM proved an accurate consistency check of the instrument and thus provides optimal and highly reproducible calibration. To illustrate the importance of optimal calibration, we show that for biological force spectroscopy studies, an erroneously calibrated cantilever can significantly affect the derived (bio)physical parameters. Taken together, our findings demonstrated that with the pre-established protocol described reliable spring constants can be obtained for different types of cantilevers.

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Abstract Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting ‘curing’ of the adhesive; some force curves also contained a ‘fingerprint’ of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative ‘tanning’ mechanism in barnacle cyprid cement.

Towards a nanomechanical basis for temporary adhesion in barnacle cyprids (Semibalanus balanoides)

Cypris larvae of barnacles are able to use a rapidly reversible temporary adhesion mechanism for exploring immersed surfaces. Despite decades of research interest, the means by which cyprids maintain attachment with surfaces prior to permanent settlement remain poorly understood. Here, we present novel observations on the morphology of ‘footprints’ of a putative adhesive secretion deposited by cyprids during surface exploration. Atomic force microscopy (AFM) was used to image footprints at high resolution and to acquire measurements of interaction forces. R–CH3- and R–NH2-terminated glass surfaces were used for comparison of footprint morphology, and it was noted that on R–NH2 each footprint comprised three times the volume of material deposited for footprints on R–CH3. Direct scaling of adhesion forces derived from AFM measurements did not adequately predict the real attachment tenacity of cyprids, and it is suggested that a mixture of ‘wet’ and ‘dry’ adhesive mechanisms may be at work in cyprid adhesion. High-resolution images of cyprid footprints are presented that correlate well with the known morphology of the attachment structures.

Free-Standing 3 D Supramolecular Hybrid Particle Structures†

Make a stand: The formation of stable and ordered free-standing particle bridges and hollow capsule structures with controllable sizes and geometries is demonstrated by combining the directed assembly of submicrometer particles, transfer printing, and supramolecular layer-by-layer assembly.

Marine biofouling field tests, settlement assay and footprint micromorphology of cyprid larvae of Balanus amphitrite on model surfaces

Atomic force microscopy (AFM), laboratory settlement assays and field tests were used to correlate cyprid footprint (FP) morphology with the behaviour of cyprids on different substrata. AFM imaging under laboratory conditions revealed more porous and larger FPs on glass exposing a CH3-surface than on aminosilane functionalised (NH2-) surfaces. The secreted FP volume was found to be similar on both substrata (2.1–2.6 μm3). Laboratory settlement assays and marine field tests were performed on three substrata, viz. untreated clean glass, NH2-glass, and CH3-glass. The results distinguished settlement preferences for NH2-glass and untreated glass over CH3-terminated surfaces, suggesting that cyprids favour settling on hydrophilic over hydrophobic surfaces. On combining observations from different length scales, it is speculated that the confined FP size on NH2-glass may induce a higher concentration of the settlement inducing protein complex. Settlement may be further facilitated by a stronger adherence of FP adhesives to the NH2-surface via Coulombic interactions.

Freestanding 3D Supramolecular Particle Bridges: Fabrication and Mechanical Behavior

Freestanding particle bridges with controlled composition and macroscopic robustness are demonstrated by the use of supramolecular nanoparticle assembly. Self-assembly of nanoparticles, templating, and supramolecular glue infiltration are combined to form stable and ordered three-dimensional polystyrene particle composites on a polydimethylsiloxane stamp. Freestanding hybrid polystyrene nanoparticle bridges are obtained by transfer printing of the hybrid structures onto topographically patterned substrates via host-guest interactions. The mechanical robustness and rigidity of the particle bridges can be controlled by manipulating the layer-by-layer cycles of supramolecular glues of gold nanoparticles and dendrimers. Atomic force microscopy-based microbending results, in particular the location and force-dependent deflection behavior, confirm that the particle bridge fulfills the classical supported-beam characteristics. As estimated from classical beam theory, the bending moduli of the particle bridges vary between 0.8 and 1.1 GPa, depending on the degree of filling by the supramolecular glues. Failure analysis on the particle structure indicates linear elastic behavior and a plastic deformation upon failure.

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

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 pull-off 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 (LP) was obtained. At high elongation, following repeated stretching up to increasing upper strain limits, footprint proteins detached at total stretched length of 10 µm.

Chemistry-Specific Interfacial Forces Between Barnacle (Semibalanus Balanoides) Cyprid Footprint Proteins and Chemically Functionalised AFM Tips

Cypris larvae of the barnacle Semibalanus balanoides leave proteinaceous footprints on surfaces during pre-settlement exploration. These footprints are considered to mediate temporary adhesion of cyprids to substrata and, as such, represent a crucial first step in the colonization of man-made surfaces by barnacles, a process known as biofouling. Interest in this system also stems from the potential for a synthetic reversible adhesion system, based on the strategy used by cyprids. Cyprid footprints were probed using atomic force microscopy (AFM) and nanomechanical data relating to interfacial adhesion forces were correlated with AFM tip chemistry. Commercial Si3N4-tips and chemically functionalized CH3-tips were chosen to mimic the interactions of cyprid footprints with hydrophilic and hydrophobic surfaces, respectively. Force-extension curves of protein bundles picked up by AFM tips exhibited a characteristic saw-tooth appearance for both types of tip, but demonstrated clear differences relating to pull-off force and pull-off length, based on tip chemistry. Additional (∼6 nN) interfacial adhesion forces between −CH3 functionalized tips and footprints were assigned to hydrophobic interactions. Footprint proteins adhered with greater tenacity to the hydrophobic tip. This may suggest conformational change and denaturing of the protein which would facilitate hydrophobic interaction by enhancing contact forces between −CH3 functionalized tips and hydrophobic groups in the footprint molecule(s). Neither tip removed proteins from the −NH2 substratum suggesting that specific chemical interactions, rather than simple wetting phenomena, govern the adhesion of footprint proteins to that surface.