Evaporative Drying Induced Self-Assembly of Epicuticular Wax: A Biomimetic Approach in Tuning Surface Roughness.

Epicuticular wax is an example of a naturally created functional material that forms a layer on the outermost surface of plants with the objective to protect them from adverse environmental conditions, such as UV-solar radiation, uncontrolled water loss, microbial attacks, and so forth. Their functionalities are often attributed to the chemical composition of the wax as well as the physical structuration formed by the wax crystals on the surface. With this work, we present a simple, one-step biomimetic approach to replicate similar surface structures, on model substrate, using wax extracted from Euphorbia Cerifera (Candelilla wax). First, we describe formation of structured wax due to self-assembly induced by evaporative drying on quartz plates. Subsequently, we highlight the fundamental physical parameters required to tune the surface morphology. Our experiments reveal that it is possible to achieve considerably diverse surface morphologies depending on the solvent properties and deposition temperature. This diversity is due to the kinetics of recrystallization of wax during evaporation of solvent which, in turn, is primarily driven by the solubility of wax as well as evaporation rate of the solvent. Thus, the final morphology that we obtain is an interplay between recrystallization kinetics and solvent evaporation. Additionally, the degree of crystallinity of the structured films could also be tuned by solvent polarity. Surprisingly, X-ray diffraction indicates that the crystalline structure at the molecular level remains similar to that of bulk Candelilla wax. Our results provide fundamental insights into the replication of epicuticular wax films and identification of tuning parameters to obtain different surface morphologies with the same wax material for potential bioinspired multifunctional coatings in cosmetic applications.

In situ mapping of biomineral skeletal proteins by molecular recognition imaging with antibody-functionalized AFM tips.

Spatial localizing of skeletal proteins in biogenic minerals remains a challenge in biomineralization research. To address this goal, we developed a novel in situ mapping technique based on molecular recognition measurements via atomic force microscopy (AFM), which requires three steps: (1) the development and purification of a polyclonal antibody elicited against the target protein, (2) its covalent coupling to a silicon nitride AFM tip ('functionalization'), and (3) scanning of an appropriately prepared biomineral surface. We applied this approach to a soluble shell protein - accripin11 - recently identified as a major component of the calcitic prisms of the fan mussel Pinna nobilis [1]. Multiple tests reveal that accripin11 is evenly distributed at the surface of the prisms and also present in the organic sheaths surrounding the calcitic prisms, indicating that this protein is both intra- and inter-crystalline. We observed that the adhesion force in transverse sections is about twice higher than in longitudinal sections, suggesting that accripin11 may exhibit preferred orientation in the biomineral. To our knowledge, this is the first time that a protein is localized by molecular recognition atomic force microscopy with antibody-functionalized tips in a biogenic mineral. The 'pros' and 'cons' of this methodology are discussed in comparison with more 'classical' approaches like immunogold. This technique, which leaves the surface to analyze clean, might prove useful for clinical tests on non-pathological (bone, teeth) or pathological (kidney stone) biomineralizations. Studies using implants with protein-doped calcium phosphate coating can also benefit from this technology. STATEMENT OF SIGNIFICANCE: Our paper deals with an unconventional technical approach for localizing proteins that are occluded in biominerals. This technique relies on the use of molecular recognition atomic force microscopy with antibody-functionalized tips. Although such approach has been employed in other system, this is the very first time that it is developed for biominerals. In comparison to more classical approaches (such as immunogold), AFM microscopy with antibody-functionalized tips allows higher magnification and keeps the scanned surface clean for other biophysical characterizations. Our method has a general scope as it can be applied in human health, for non-pathological (bone, teeth) and pathological (kidney stone) biomineralizations as well as for bone implants coated with protein-doped calcium phosphate.

Polymerization of tetrazine-substituted diacetylenes as aggregates in suspension.

Polydiacetylenes (PDAs) are conjugated polymers that have been widely exploited for their chromogenic and fluorogenic transitions upon exposure to external stimuli and biomolecules of interest. Herein, we propose a comparative study of the polymerization dynamics of two diacetylene derivatives, TzDA1 and TzDA2, in the form of aggregates in suspension prepared by reprecipitation method from organic solvents in water, varying the diacetylene concentration and solvent proportions, and sonication in water, varying the time and temperature. Both derivatives bear a tetrazine fluorophore, which serves both to increase the fluorescence quantum yield of the system and to track the polymerization by fluorescence quenching exclusively by the blue-PDA, and differ by the chain termination. It was shown that adding a butyl ester function in TzDA2 to a simple urethane (TzDA1) influences the polymerizability and kinetics of polymerization of the aggregates in suspension. In addition, we showed that also the preparation method and preparation conditions do have an influence on the polymerization dynamics, suggesting that a careful study of these properties should be carried out before investigating the applications of such objects.

Enhanced mechano-responsive fluorescence in polydiacetylene thin films through functionalization with tetrazine dyes: photopolymerization, energy transfer and AFM coupled to fluorescence microscopy studies.

The development of mechano-responsive fluorescent materials is essential for the design and construction of reliable and versatile sensors for mechanical stress. Herein, novel energy transfer-based systems with tetrazine fluorophore and a polydiacetylene (PDA) backbone are synthesized and studied comparatively to a simple polydiacetylene in the form of thin films. Their photopolymerization properties, energy transfer efficiencies and fluorescent response to nanoscale mechanical stimulation are assessed. It is pointed out that the self-assembling group on the PDA chain influences the geometrical arrangement of the chains and the film morphology and, as a consequence, the efficiency and kinetics of polymerization and the energy transfer efficiency. Moreover, we show that the strategy of introducing tetrazine fluorophore provides a new effective route of improving force detectability by fluorescence using polydiacetylenes as mechano-responsive units.

Mechano-responsive fluorescent polydiacetylene-based materials: towards quantification of shearing stress at the nanoscale.

An OFF to ON mechano-responsive fluorescent polydiacetylene derivative has been prepared by photopolymerisation of a diacetylene covalently linked to a tetrazine fluorophore. This system has been studied at the nanoscale and shows a fluorescence emission intensity that increases with the intensity of the mechanical stimulation in the 20-200 nN range.