**2.5 Amino acid analysis**

Protein extracts were separated by SDS-PAGE, followed by transfer of the protein bands to PVDF membrane using Invitrogen NuPAGE Transfer buffer (NP0006-1). Amino acid analysis of PVDF membrane pieces used vapour-phase hydrolysis (5.8 M HCl at 108 °C for 18 h), followed by precolumn derivatisation with 6-aminoquinolyl-*N*-hydroxysuccinimidyl carbamate (Cohen & DeAntonis, 1994). Derivatives were separated and quantified by reversed phase (C18 Waters AccQTag) HPLC at 37 C (Cohen, 2001) (Australian Proteome Analysis Facility), using a Waters Alliance 2695 Separation Module, a Waters 474 Fluorescence Detector and a Waters 2487 Dual Absorbance Detector in series.

Biomimetic Materials as Potential Medical Adhesives – Composition and Adhesive

**3. Results and discussion**

normally around 8-12.

mm, Bars (B, C, D) = 1 mm.

**3.1 Tubule structure and microscopy** 

Properties of the Material Coating the Cuvierian Tubules Expelled by *Holothuria dofleinii* 249

Dissection of euthanised *H. dofleinii* showed that the body cavity contained a large number, several dozen, Cuvierian tubules in their compressed form (Figure 1A). In their compressed form the tubules were not sticky, but they rapidly became sticky on mechanical extension, even from the dead animals. The compressed, individual tubules showed a corrugated and folded surface (Figure 1B), which would allow extension when required, like a concertina bellows. In cross-section (Figure 1C) a three-lobed channel could be seen that would allow fluid insertion for expansion of the tubules. When expelled and fully extended *in vivo*, these tubules became instantly sticky and changed from the 25-35 mm compressed length up to around 350-400 mm. The fully extended tubules (Figure 1D) were typically about 4mm flat width when fully inflated, and still showed some patterning from the folding that was present in the compressed state. Individual animals contained several dozen non-inflated tubules, but when the animals were stimulated only a small number were expelled,

Fig. 1. Cuvierian tubules for *H. dofleinii*. (A, B, C) After dissection of a euthanised animal, showing, (A) the total mass of tubules, (B) the tips of compressed tubules, and (C) the crosssection of compressed tubules. (D) The surface of an *in vivo* expelled tubule. Bar (A) = 10

#### **2.6 Adhesion properties**

Adhesion properties were measured using a 90 Degree Adhesive Peel Strength Test (Dimas et al., 2000), adapted to the rapid evaluation of tubules from a single animal under different experimental conditions. Individual expelled tubules, as above, were transferred to a wash solution in a plastic trough containing a wash solution determined by the particular test (see below). The numbers of samples tested in a given experiment are given in the results Tables; in each case, tubules from a minimum of 3 separate animals were used. After 60 ± 2 sec, the tubule was removed from the wash solution and allowed to drain for 5 sec. It was then laid across the width of a 25 mm wide strip of substratum, selected for the particular test. The tubule was allowed to adhere to the test substratum under its own weight for 60 ± 2 sec. This differs from previous studies where a load was applied during adhesion (Flammang et al., 2002). During the adhesion period the tubule was trimmed to leave <10 mm overhanging one side of the substratum and about 50 mm on the other side. The flat width of the tubule was measured and also recorded photographically, with a ruler placed adjacent, for subsequent verification. At the end of the 60 sec adhesion period, each tubulesubstratum assembly was then transferred to a frame that allowed the substratum to be held horizontally with the adhered tubule on the underside, i.e. with the free *c.* 50 mm length of tubule hanging below. The load was then increased stepwise (2.5 g/5 sec) to the overhang of the tubule until the tubule-substratum adhesion failed by peeling. The total load at failure was recorded. The maximum force tested was 0.2 N (approximately 20 g load) which equated to about 0.05 N/mm for an average tubule, because higher loads typically took too long to add and the tubule could have begun to desiccate at that stage, potentially changing the adhesive strength. A minimum of six determinations was made for each test condition. Data are presented as the total force at failure (N) divided by tubule width (mm). Although we did not test values above 0.05 N/mm, our conservative approach did not hinder examination of conditions that led to reduction of adhesive strength.

Experiments to test adhesion to different substrata used a wash solution of simulated seawater comprising 3.5% NaCl, 10 mM sodium phosphate buffer, pH 7.6. Various substrata were tested, including clean glass (microscope slide), aluminium, polyvinyl chloride, chitin (from crab), polycarbonate, poly(methyl methacrylate) (PMMA) and polytetrafluoroethylene (PTFE), all cut to a similar size. As the chitin substratum lacked stiffness, the samples were first glued with cyanoacrylate onto a glass microscope slide. The chitin sample also had an irregular surface and was not uniform like the other materials.

The effects on tubule-glass adhesion of various chloride or sodium salts (50 mM) were examined by supplementing the 3.5% NaCl, 10 mM sodium phosphate buffer, pH 7.6, before washing the tubules. The effect of NaCl concentration on tubule-glass adhesion was examined using different NaCl concentrations in 10 mM sodium phosphate buffer, at final pH 7.6. The effect of pH on tubule-glass adhesion was examined using solutions prepared using three salts: Tris/chloride, sodium citrate and sodium acetate, each at 50 mM in 3.5% NaCl, 10 mM sodium phosphate. Similarly, the effect of urea on tubule-glass adhesion was examined using different urea concentrations in 3.5% NaCl, 10 mM sodium phosphate at a final pH of 7.6. Glass was used as the standard substratum as it was readily available in uniform quality, and had previously been shown to be an excellent material for adhesion of *H. forskåli* tubules (Flammang et al., 2002).
