2053-1591%2Faa9940

Materials Research Express
ACCEPTED MANUSCRIPT
Extracellular micro and nanostructures forming the velvet worm solidified
adhesive secretion
To cite this article before publication: Yendry Corrales et al 2017 Mater. Res. Express in press https://doi.org/10.1088/2053-1591/aa9940
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Yendry Regina Corrales-Ureña1, Angie Sanchéz1, Reinaldo Pereira1, Klaus Rischka2, Thomas.
Kowalik2and José Vega-Baudrit1,3.
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1. National Laboratory of Nanotechnology, Costa Rica, LANOTEC-CENAT-CONARE. 1.3 km north
from the USA embassy, San José, Costa Rica.
2. Fraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM, Adhesive and
Polymer Chemistry. Wiener Straße 12, 28359 Bremen, Germany.
3. National University of Costa Rica, UNA,Heredia, Costa Rica.
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Corresponding author: [email protected]
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Extracellular micro and nanostructures forming the velvet worm solidified
adhesive secretion
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Abstract
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The onycophoran Epiperipatus hilkae secrets a sticky slime that solidifies almost immediately upon
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contact with air and under high humidy environmental condition forming a glassy like material. The
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general adhesive biochemical composition, the releasing and hardening mechanism have been
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partially described. In this study, the structural characterization of the extracellular microstructures
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and nanostructures forming the solid adhesive of the secretion from Epiperipatus hilkae velvet worm
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is presented. The adhesive secretion is formed by macro-threads, which, in their solid state, are
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composed of globular particles approximately 700 nm in diameter that are distributed homogeneously
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throughout the matrix surface, and nanoparticles approximately 70 nm in diameter that self-assemble
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forming fiber-like structures. Nanoparticles with non roundish forms are also observed. These 70 nm
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particles could be associated to proteins that form high density coverage films with low roughness;
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suggesting the formation of two dimensional ordered films. A crystalline and an amorphous phase
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composes the solidified secretion. The glassy or viscoelastic properties depend on the time in contact
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with air before being adhered to a solid surface and/or the mechanical stimulus; suggesting a key role
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of the drying on the hardening process.
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Keywords: adhesive, natural, onycophoran, velvet worms, nanoparticules, protein, crystals.
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Introduction
Advances in nanotechnology and molecular biology have promoted material development using bioinspired approaches [1]. Nano-defined self-assemblies derived from biological systems have been
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used as an inspiration for the innovative development of materials, such as bio-adhesives that could
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work efficiently in water using cross-linked non-toxic components. Some animals and plants produce
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adhesive secretions for prey capture, defence, prevention of dehydration, and camouflage, among
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other things; and have been used as the inspiration for the design of new adhesives to be applied in
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the medical, bio-electronical, textile and cosmetics industries [2,3]. Recent examples include mussels,
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frogs, ivy plants, sandcastle worms, geckos, sea cucumbers and tubeworms [4-12]. Each organism
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has its own features and the physicochemical characterization of biological adhesives is challenging.
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In general, these secretions are composed mainly of mixtures of proteins, carbohydrates, surfactants,
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peptides, water and some ions like Ca2+. Natural adhesives usually consist of complex biopolymer
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blends, forming in many cases extracellular nanometric structures that play a key role in the adhesion
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mechanism. Some of the functions of the extracellular nanostructures are attributed to the
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enhancement of energy dissipation, as it is frequently found in climbing animals that produce
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fibrillary structures. These structures are thought to be responsible for a mechanism analogous to the
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molecular stretching of polymeric chains and also, through their nanostructures, to influence the
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contact points with the target surfaces to minimize crack length and propagation [10].
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The onychophorans, or velvet worms, live in tropical areas such as Costa Rica. These are small
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terrestrial invertebrates, similar in appearance to caterpillars, which capture their prey by means of a
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sticky secretion that is launched through passive oscillation. The viscous slime is expelled by dual
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high-speed, and force flexible, tubes or cannons called oral papillae; the muscular action produces a
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swinging movement of the adhesive-spelling organs. In these cannons the slime is squirted and under
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an oscillation process the material is expelled like a jet, in small drops, some inches away [13].
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The velvet worms in Costa Rica belong to the family of Peripatidae, which has a circumtropical and
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subtropical distribution, including the African continent (tropical West Africa), Southeast Asia and
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tropical America. Their liquid ejection mechanism operates on a micrometric scale and is a model for
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the micro and nanofabrication of fibres. The secretion contains several substances of high commercial
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and biotechnological potential: low-structure adhesive proteins, molecules that can recognize sugars
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(lectins), surfactants, polyphenols, and peptides with potential antimicrobial properties. The general
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composition of the slime has been characterized. It is composed mainly by 90 wt. % of water and
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10 wt. % of proteins, carbohydrates and lipids. Small peptides are also present in the adhesive
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secretion [17]. Despite having been studied, by different authors [13-21], only recently the
mechanical and morphological properties of the velvet worm secretion was partially described;
suggesting a mechanically responsive material that can be drawn into stiff fibers that can be dissolved
and regenerated [22].
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For understanding the hardening mechanism, in this contribution we describe the morphologies of
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the extracellular structures in the solidified secretion by atomic force microscopy (AFM), optical and
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polarized microscopy, transmission electron microscopy (TEM), contact angle and the chemical
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composition using through Fourier transform infrared spectroscopy (FT-IR), energy dissipation X-
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ray diffraction (EDX) characterization techniques.
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Materials and methods
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Sample collection: specimens were collected in San Ramón Costa Rica in their natural habitat.
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Autoclave silica sample holders were used for collecting the samples in situ. Five samples of two
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specimens were analyzed. The procedure for collecting the adhesive used was previously described
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by Concha et al. [13]. The pristine sample without any treatment was analyzed by an optical
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microscope Motic BA 410, a polarized microscope BA 300Pol and a confocal microscope Leica
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DCM 3D. The environmental conditions during the measurements not made in vacuum were:
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temperatures of 22°C and an average of 85 % relative humidity. The samples were storage under
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aseptic conditions and imaged by AFM the same day for avoiding degradation. However, the material
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maintained their aspect and size during several days under storage at ambient conditions. To
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determinate the influence of the drying, the samples were put in an oven at 95°C for 30 min. The
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surface topography was analyzed using an AFM Asylum Research operated in the tapping mode in
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air. Silicon probes (model Tap150Al-G, back side of the cantilever covered Al) with resonance
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frequencies of 150 kHz and force constant of 5 N/m were used. Slices of the solidified adhesive were
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cut using a new and clean glass knife forming a powder and mounted onto a 400 mesh
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copper/palladium carbon coated grid.The TEM images were obtained on a JEOL JEM2010
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Transmission Electron Microscope/EDX set at 120 kV. Several samples were prepared and analyzed:
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A1: sections of the solidified secretion, A2) A1 after adding a drop of 5 μl miliQ water on top of
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surface and let it dry. Next, they were fixed with a solution of 2.5 wt.% glutaraldehyde in 1mM
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phosphate buffer to preserve the ultrastructure, B1 and B2 were prepared as sample A1 and A2
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without fixative. The purpose of A and B preparations was to have a clean EDX analysis to determine
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the possible composition of the structures forming the secretion and avoiding changes due to drying
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under the TEM vacuum chamber. FT-IR spectra were measured on a Bruker Vertex 70 with an IR
Scope II extension. The measurements were performed in ATR technique (Attenuated Total
Absorption in the IR Scope II extension, standard Bruker ATR-lens with Germanium crystal, one
reflection, 32 µm tip diameter). ATR technique allows surface sensitive measurements with
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information depths up to 1.5 µm. The information depth depends on the surface roughness, refraction
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index of the used crystal and samples; among other variables and the result is an average value. Single
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measurement parameters were a scan number of 64, a resolution of 2 cm-1 and a background
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measurement against air. Mapping mode was performed with the same parameters as automated
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measurement. Data search was performed with the Bruker own software OPUS on base of own,
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Merck and Sadtler data bases. The apparent contact angles were measured using a goniometer
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(OCA15 Plus, Dataphysics Instruments, Germany) by sessile drop technique and MiliQ grade water
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was used as probe liquid; the volume of the drops was constant (10 μl) for each measurement at a
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temperature of 22˚C. The contact angle values reported are an average value of at least three separate
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drops on different substrates area. The recorded images were analyzed by SCAN 20 Dataphysics
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software.
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Results and discussions
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In order to understand the adhesion principle of the onychophoran slime, a morphological
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characterization was performed. Secreted material was deposited by the specimen directly onto a
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clean silica substrate; possible external residues of soil could be correlated to the residues in the
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papillae. The samples were collected in San Ramón Alajuela, Costa Rica, in their natural habitat
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inside the forest. The specimen was a Epiperipatus hilkae [14], figure 1 A. Figure 1 B shows a light
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microscopy image of the biofilm secreted. The solidified adhesive formed macro-meter linear threads.
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Figure S1 shows fiber like formation inside the biofilm and in other cases some particles like
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structures. The adhesive could be described as translucent material which spontaneously adhered to
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glass, figure 1 B, and it is a glassy-like solid. The secretion solidified in approximately 10 seconds
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after expelling and being in contact with the substrate surface. A whitish viscoelastic material was
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obtained when the material was accumulated at the bottom of the substrate; not having time to expand
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through the surface, as it can be seen in figure 1 C. In that case, it remained in a viscoelastic state
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even for 15 days until it was mechanically mixed and stretched with the metallic tweezers several
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times; producing the hardening of the material.
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Figure 1. A) Photograph of the onychophoran specimen. B) Light microscopy image of the secretion
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on a silica substrate, 10 X C) Photograph of the whitish viscoelastic material.
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For determining the role of the drying in the hardening mechanism, a piece of the material was cutted
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with a metallic blazer and placed on top of a glass substrate. A drop of water was added on top of
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the solid material and this was immediately contracted in a roundish form. A plastic pipette was used
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to apply force and deform it, but this could not be easily elongated. Next, the water was removed by
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capillarity using a paper towel and the material was in a semi-dried state. In that moment the material
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could be deformed preferentially in a fiber like structure; more in the Y edge than in the X edge, as
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it can be seen in figure 2 A and B.
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viscoelastic a glassy like material. This cycle could be repeated several times (supplementary video
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V1). It suggests that the material could be contracted inside the reservoir when is in a viscous aqueous
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suspension (90 wt.% water). After a mechanical stimulus by the papillae oscillation and being
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expelled it can be elongated when it is in a semi-dried state; molding the form of the prey before
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changing to a high strength glassy material. Bauer et al reported stiffness of the solidified secretion
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in the range of silkworm silk and nylon [22]. This results suggested the key role of the drying in the
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assembly of the material. However, further studies are necessary to determine the role of a possible
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chemical reaction, complex formation, etc.
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After few seconds or mixturing, it changed their state from
Figure 2. Solidified secretion A) under water B) stretched after drying.
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The threads were analyzed by a confocal microscope to determine their thickness. Figure S2 and S3
shows a broad difference between the borders and center of the thread; showing features of lamellar
structural layers. The thickness of the threads was in the µm range. An example of the cross-section
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is shown in figure S2 and S3 C; an average of 6.5 µm height was determined. The glassy coating
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formed with the secretion changed the silica surface to more hydrophobic domains; the value of the
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contact angle between the surface and a drop of water was 80±5°; figure S4.
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Figure 3. Optical microscope images A) 10 X B) 40 X of the solidified secretion on glass substrates.
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Figure 3 A shows a fractal like structure inside the matrix. Figure 3 B shows fibers like structures (e.g
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red arrows) decorated by drops (blue arrow). Figure 4 A to D shows the polarized microscopy images
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of the material. Figure 4 shows the presence of birefringent phase; showing molecular order [23].
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Two kinds of structures were observed by the polarized light microscope: fibers, figure 4 A and B,
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and some crystal like structures, figure 4 C and D. However, not all the threads analyzed presented
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a birefringent phase, as it can be seen in figure S 5 A and C. Further studies are necessary to determine
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if the birefringent material is due to crystallization of the proteins, salts or material contained
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previously in the reservoir as liquid crystals [22]. The proteins could self-assemble into a variety of
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states: crystals, dense liquid phases, gels, fibers, and amorphous aggregates, two different states:
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crystals and aggregates and even two different crystals forms [25].
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Figure 4. Polarized optical microscope images of the solidified secretion A) 10 X B)40X C) 10 X
and D)40 X.
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The solidified secretion was characterized by FT-IR spectroscopy without any disturbance of the
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original secretion after settling, as it is shown in figure S6. Figure S6 shows some examples of the
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areas under analysis highlighted with different colors. The spectrum obtained for each area was
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plotted with the same color as the one assigned for the area. All the areas presented similar absorbance
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peaks. The spectrum shows a broad band between 3000 and 3800 cm-1 from the OH symmetric and
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asymmetric stretching modes and a N–H stretching of the protein backbone. The peak at 2918 cm-1
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corresponds to CH stretching. The peak at 1630 cm-1 is assigned to the amide I region and the
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absorption at this wavelength is correlated with a β-sheet secondary structure. The peak at 1447 cm-1
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corresponds to CH2/CH3 deformation. The peak at 1540 cm-1 corresponds to amide II and that at 1240
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cm-1 to amide III [25]. The peak at 1050 cm-1 is associated with polysaccharides [17]. The results
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agreed with the composition reported previously by Benkendorff et al. for the Euperipatoides
kanangrensis. They described the secretion as a proteinaceous-based slime that also contains
carbohydrates [17]. Amyloids β-sheet fibrillary proteins show a maxima absorption between 1611
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and 1630 cm-1 [25-26]. The possible fibrillary protein structure is consistent with the microscopy
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studies, which also identified fibrillary networks. Unstructured proteins forming the slime of the
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Australian onychophoran Euperipatoides rowelli and β-sheet structures of the onychophoran
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Macroperipatus geagy from Colombia have been reported [27-28].The proteins may have changed
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their conformation due to drying, and in further studies the FT-IR results will be compared with the
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non-solidified adhesives [29-30]. Furthermore, Baer et al. showed clear differences in protein
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molecular weight and patterns between families, which could influence in the secondary structures
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formed [8].
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TEM images of the solid material are shown in figures 5, sample prepared as described for A1. These
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material contains a fiber-like structures with diameters of 51±10 nm, as it can be seen in figures 5 A.
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Figure 5 B shows a material with a non define structure and apparent high roughness. Figure 5 C
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shows a micrometer fiber formed by smaller fibers with an average diameter of 68±8 nm. Figures 5
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D and E show more electro-dense globular nanostructures with diameters of 118 ±28 nm. The
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difference in electro density suggests a different chemical composition in comparison to the
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surrounding matrix. Figure 5 F shows material forming patterns and an amorphous material. This
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molecular order could be correlated with the polarized microscope images showing a birefringent
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phase.
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Figure 5. TEM images of the solid slime secretion A)8000 B) 8000 X C) 800 X D) 6000X E)
40000X F)40000X.
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Figure 6 shows a more detailed images of the nanofibers. These fibers are organized in regions with
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different assembly directions; showing a conformation that could be correlated to a grain boundary;
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giving more resistance to applied forces in different angles. A more electrodense material with
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roundish and non-roundish morphology is imaged on top of the fibers, Figure 6 A, B, D and S6.
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Figure 6. TEM images of the solid slime secretion A)1500 X B) 2500 X C) 4000X D) 5000 X
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The substrate material was submerged in water for 4 hours to determine the stability and to elucidate
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the non soluble structures inside the matrix. Figure S7 A shows the thread which was partially break
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it in pieces and/or dissolved; the fractal like structure and globules with similar sizes were observed
over the silica surface, figure S 7 B. Figure 7 was prepared as described for A 2. Figure 7 A shows
fractal structures bounded to a fibre like structure. Figure 7 B shows a crystal like particles, that could
be correlated to the material imaged by polarize light microscopy. Figure 7 C shows globular particles
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in the order of 100 nm range. Figures 7 D-F show globules with 450 ± 180 nm size bounded between
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them by a fiber like structure; similar in size to the imaged by optical microscopy and imaged by
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AFM. Figure 7 F shows a globules interconnected by fibers. On top of the globules smaller particles
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are imaged. The results suggest that the globules, crystals, fibres and fractal structures are not an
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artefact from the vacuum drying. The adhesive is partially composed of lipids and/or proteins that
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can be aggregated and form these macromolecular structures [17,22,32].
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Figure 7. TEM images of the solid slime secretion A)300 B) 8000 X C) 6000 X D)1200X E) 2500X
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10000X
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Similar images were obtained after preparing the samples by A and B procedures. However, for EDX,
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preparation B was preferring not to have any contamination from additional reagents. EDX analyses
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of different areas, that were considered representative of the solid adhesive, were conducted to give
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a rough overview of the elemental composition of the solidified adhesive [34-36]. The elements
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carbon (C), oxygen (O) and nitrogen (N) had the highest counts in the spectrum, followed by sulphur
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(S) and silicium (Si),magnesium (Mg). Calcium (Ca), phosphorus (P), (Al) and potassium (K). The
Cu, Si and Al could be related to the TEM grid in concentrations lower than 0.1 at %, according to
the values obtained when the grid without the sample was analyzed. The Si concentrations of 2 at. %
and Al at 0.2 at. % could be related to the impurities from the substrate or soil, but all the samples
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and areas analyzed presented Si; suggesting this element as part of the chemical composition of the
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slime. The globules presented mainly high concentration of C, N, O and S and Si. Graham et al.
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suggested that disulphide bonding is a feature common to salamander, frog and onychophoran
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secretions [7]. The crystal like structures, figure 7 B, presented higher concentration of C, N, O, Cl
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and K. Table 1 summarizes the results. Figure S 8 shows an example of the spectra obtained by EDX.
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Further biochemical characterization is necessary to determine the composition of the globular
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structures, the crystals, the nanoparticles and the fibers; and to correlated them with the role in
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adhesion and mechanical properties, but the scope of this research is focus on the general morphology
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presented in the dried state.
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Table 1. TEM/EDX analysis of the velvet worm slime. Concentrations given in at%. Traces of
[N]
[O]
[Al]
[Si]
[S]
General
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41.8
0.2
2.0
ND
Globules
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21.4
9.1
0.1˂
0.1˂
0.1˂
Crystal like
66.3
24.9
8.3
ND
ND
0.1˂
[K]
[Ca]
[Cu]
[Mg]
[P]
0.1˂
0.1˂
0.1˂
0.1˂
0.1˂
0.1˂
0.1˂
0.1˂
0.1˂
0.1˂
ND
ND
0.3
0.2
ND
ND
ND
ND
elements ˂0.1 were labelled as ˂0.1 at% due to detection limit of the technique.
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*ND: not detected
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AFM imaging under laboratory conditions revealed nanoparticles with a spherical morphology that
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were separated into two groups according to their diameter. Those in the first group, called “globules”,
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have a circular shape with a diameter of 660±109 nm, as it can be seen in figure 8 A and B. Those in
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the second group have a diameter of 83 ±18 nm and are homogeneously distributed inside the matrix
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and assemble in a fibre-like structure, figure 8 D-F. Figure 8 C shows μm length fibers that could be
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correlated with the fibers showed in figure 7 A. The nanometric particles arrangement observed in
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figure 8 D could suggest an organization in a 2D crystal like structure (XY); forming a high density
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coverage film. Some proteins as hydrophobin can form crystalline structure and organize
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preferentially in 2D arrangements [37]. Figure S 9 shows an AFM cross-section of the globular
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particles that are more exposed to the surface; the nano-roughness produced by the nanoparticles and
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the amorphous matrix is 2.9±0.8 nm.
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Figure 8. AFM height images of the dried adhesive at A) 20 µm X 20 µm B) 10 µm X 10 µm C)
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4.1 µm X 4.1 µm D) 2.6 µm X 2.6 µm.
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The AFM and TEM results suggest an adhesive that is formed by roundish globules and smaller
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particles that self-assemble in fibre-like structures. The sizes cannot be directly compared due to the
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different conditions used by the TEM and AFM analyses. Figure 9 and S D shows the top surface of
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the called “globules “. In one of the cases the surface is smooth, figure 9 A, and heights of 25 ±8 nm
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and a second globule with smaller particles on top with sizes of 62 ± 18 nm between the base and
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the upper part. A fiber like structure is connect to this globule, showing similar to the images showed
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in figure 7 F. Figure S 10 A shows similar nanoparticles forming particles in the material before
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adding water. The phase image shows a contrast where the particles are located, figure S 10 B.
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Therefore, it shows the nanoparticles forming globules and fibres. Phase changes could be associated
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with differences in composition, adhesion, friction, and viscoelasticity properties, with respect to the
surrounding matrix [38]. Figure S 10 C and D shows agglomeration of this globules after adding
water. Globular organic nanoparticles have been observed in the adhesives of several marine animals,
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including polychaetes, mussels, barnacles and sea stars as well as in terrestrial plants such as ivy. The
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reported sizes are 50–100 nm and it is suggested that these form building blocks, similar to the AFM
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results obtained in this study. The functions of the nanoparticles in other natural adhesives have been
Page 13 of 17
attributed to increased strength, stiffness and toughness by preventing cracking formation and
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propagation and increasing the surface contact between the adhesive material and the substratum [12].
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Bauer et al. suggested that this nanoparticle could form fibers and agglomerate in contact with water
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[22]. This self-assemble mechanism could be the responsible for the stretching and contracting of the
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material in a macroscale due to the changes at nanoscale. The material mechanically mixture with
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the tweezers before hardening showed fibers and more random agglomerates of the particles, figure
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Figure 9. AFM height images of the dried adhesive after contact with water A) 2,5 µm X 2,5 µm C)
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3,2 µm X 3,2 µm. B and D phase images of A and C respectively.
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In other natural animal adhesives, lipids have been associated with the prevention of slime adhesion
to the secretory glandules and excretory pathway [13] and also with the function of displacing water
from the surfaces to improve the adhesion of proteins [33]. Bauer et al. suggested the role of the
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lipids to prevent premature protein self- assembly before the mechanical stimulus [18].
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To determinate the possible changes of the surface due to drying without mechanical stimulus, the
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sample was heated at 95°C for 1 hour. Figure S 12 A and B show the thread before and after drying.
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The thread remained with similar sizes and no crystalline phases were formed, figure S 4 C. The
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nanoparticles and globules were observed by AFM in the surface and some changes related to a more
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elongated morphology and agglomeration, figure S 12 B. However, it suggested the stability of the
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material.
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Conclusions
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In summary, we described the morphology of the solidified velvet worm secretion at environmental
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conditions of relative humidity 85 % humidity, 22 °C and after contact with water. The solidified
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secretion contracts under water and it can be stretched in semidried state. Extracellular nanostructures
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such as nanoparticles of 70 nm nanoparticles that can organize in fiber like structures and 2D low
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roughness films, particles with approximately 0.7 μm diameter, micro meter length fibres and
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amorphous matrix forming the solidified adhesive secretion of the onychophoran Epiperipatus hilkae
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were observed on the surface. The material presented a birefringent phase with different crystal and
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fiber like structures. After being in contact with water the materials suffer a change; showing higher
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density of globular particles connected by a fiber like material. The chemical composition is based
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on proteins and carbohydrates and contains traces of metals such as Na, K, Mg, Ca, Al, and Si.
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In addition, physicochemical studies are necessary to understand o the correlation between the
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structures and the mechanical and rheological properties to synthesize a high strength new bioinspired
adhesive that could be hardened in humid environments and applied in biomedical fields. This
mechanical material could be used in the future not only as a universal adhesive because it adheres to
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plastic, metal and glass and biological material but also as material for biological actuators due to
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mechanical response to the dry and humid state.
Page 15 of 17
Acknowledgments
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The authors would like to thank Dr Orlando Argüello-Miranda for his contributions to the
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development of the topic of “nanobiodiversity” in Costa Rica and to Prof. Dr. Pedro León for sharing
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his knowledge related to the velvet worms.
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