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Physical characterization of the liquid adhesive from orb-weaving spiders
Materials Science and Engineering C 34 (2014) 341–344
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Short communication
Physical characterization of the liquid adhesive from orb-weaving spiders Fernando G. Torres ⁎, Omar P. Troncoso, Fernando Cavalie Department of Mechanical Engineering, Catholic University of Peru, Lima 32, Peru
a r t i c l e
i n f o
Article history: Received 22 March 2013 Received in revised form 27 August 2013 Accepted 21 September 2013 Available online 29 September 2013 Keywords: Spider adhesive Spider silk Nanoindentation
a b s t r a c t Orb-weaving spiders produce bioadhesives that are used to capture their prey. In this paper, the physical properties of these adhesives are characterised. The liquid adhesive from Argiope argentata spiders has been studied and the morphological properties of the droplets, including size, shape and volume were determined. An estimation of viscosity and Young's modulus using atomic force microscopy has also been carried out. Morphological characterization confirmed that the liquid adhesive displayed a typical beads-on-a-string (BOAS) morphology on the silk fibres. The experimental data confirmed that the elastic modulus of the liquid adhesive from A. argentata was in the range 20–100 kPa which is in agreement with the Dahlquist criterion for adhesives. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Orb-weaving spiders produce several types of silks, each of which has a specific functional role. For instance, drag line silk produced by the major ampullate glands makes the radii of the orb-web. The orbweb must be able to cope with the kinetic energy of flying insects that arrive on it. The mechanical properties of spider silk such as tensile strength and extensibility determine the behaviour of orb-webs. Orb-weaving spiders use a special thread coated with an aqueous adhesive to capture their prey. This kind of thread is known as viscid thread and it is produced together with the aqueous adhesive by three spigots. The thread is spun in the flagelliform gland after which it is coated with the adhesive produced in the aggregate glands [1]. Volrath et al. [2] have investigated the chemical composition of the water soluble fraction of this aqueous adhesive. They found hygroscopic components related to neurotransmitters such as GAB-amide, choline, lysine, serine, etc. Tillinghast [3] and Sahni et al. [4] were able to analyse the non-soluble fraction of the adhesive. The presence of galactosamina, galactose, mannose, glucosamine, fucose and glucose would indicate that glycoproteins are responsible for the stickiness of this adhesive as they are the only components with long branches. Foelix [5] and Edmonds et al. [6] have reported that when the viscid thread is coated with the adhesive, the glue covers it evenly but it tends to form droplets due to Rayleigh instability, displaying a beads-on-astring (BOAS) morphology [7–13]. Opell and Hendricks [14] have performed drop adhesion measurements. They stretched single adhesive drops until separation from a glass probe. The maximum adhesion forces they found were in the order 0.1–0.4 mN. Sahni et al. [4] carried out load–relaxation tests. The forces they measured were around
0.2 mN. They also discussed the rheology of adhesive drops. They claimed that such drops behave neither as a viscous liquid nor as a viscoelastic liquid. Instead, the adhesive drops exhibit the characteristics of a viscoelastic solid. The studies previously published in the literature assessed the chemical and mechanical properties of the spider adhesive. However, in order to fully understand the physics of the adhesion of spider glue, other properties must be studied. One of the basic rules of adhesion is that one of the surfaces to be adhered should be relatively soft. It has been shown that the elastic modulus of the components strongly influences adhesion in biological systems [15]. In this paper, we give an estimation of other physical properties of the spider adhesive glue such as surface area, contact angle, viscosity and Young's modulus. 2. Experimental section 2.1. Materials Argiope argentata (Fabricius, 1775) spiders, an American species of the Araneidae family, were used in this study. Female adult spiders of 3.5–5 mm in prosoma width were collected in the outskirts of Lima, Peru. They were housed in 30 × 20 × 10 cm cages and were fed larval stage mealworms (Tenebrio molitor) three times per week. Cardboard frames with a rectangular opening were used to collect viscid silk samples. The ends of the silk samples were glued to the frames so that a part of the sample remained untouched. Silk samples were collected from the webs of three different spiders. At least three webs from each spider were used. 2.2. Characterization techniques
⁎ Corresponding author. E-mail address: [email protected] (F.G. Torres). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.030
Micrographs of the viscid silk were taken in a FEI-QUANTA 200 scanning electron microscope (SEM) in low vacuum with a voltage in the
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F.G. Torres et al. / Materials Science and Engineering C 34 (2014) 341–344
range of 20–30 kV with a working distance of 10 mm. Three different silk samples were viewed directly without drying and without conductive coating. At least four SEM micrographs were taken from each specimen. This allowed for the measurement of sixty different drops. Nanoindentation tests were performed in a Nanosurf Easy Scan atomic force microscope (AFM) at 25 °C and 80% RH. A monolithic silicon NanoWorld Pointprobe® probe with a spring constant of 0.17 N/m was used. The tip radius of the probe is around 8 nm and its half cone angle is 50°. Mica TO-220 was used as substrate. Samples were tested immediately after being collected. Three samples were used. Each indentation test was repeated 5 times. The duration of each test was 1 s. 2.3. Young's modulus estimation The indentation tests were repeated 5 times for each point. The average values were used to plot the curve of the cantilever deflection against the height of the sample. If we consider an infinitely stiff tip and a soft flat sample, the Hertz model can be used to predict the relation between indentation and loading force [16–18]. The Young's modulus can be estimated by following Eq. (1): vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiqffiffiffiffiffiffiffiffiffiffiffiffiffi u k u d−d0 : z−z0 ¼ d−d0 þ u . . t π E tan ð α Þ 2 ð1−υ2 Þ
ð1Þ
Where E is the Young's modulus, ν is the Poisson ratio (taken as 0.5 corresponding to an incompressible material), k is the spring constant of the cantilever (0.28 N/m), α is the opening angle of the cone (50°), z is the height of the sample and d is the deflection of the cantilever. The 0 subscript accounts for the offset values. 3. Results and discussion Fig. 1 shows a SEM image of the spider adhesive glue forming drops on the spider silk thread. This image resembles other examples of the well described beads-on-a-string (BOAS) morphology. The BOAS phenomenon can be observed while pulling apart a thin film of a viscoelastic fluid such as saliva, fish slime, cellular protoplasm, etc. [19]. It can also be observed when blending incompatible polymers in the melt
Fig. 1. SEM micrograph of viscid silk showing the typical beads-on-a-string (BOAS) morphology of the adhesive droplets on the silk fibres.
state. During shear flow, these polymers are extended into long threads, but when the flow stops, the external thread breaks up into small droplets [20]. Fig. 2 shows a schematic representation of the BOAS morphology. According to Sahni et al. [21], when the threads come out of the spider spigot, the axial silk thread is coated by a regular cylindrical layer of glue. The glue layer then breaks down into an equally spaced array of droplets due to Rayleigh instability. For the adhesive droplets studied here, we have used some of the equations that describe the BOAS phenomenon to assess the physical characteristics of the spider glue from A. argentata. Sahni et al. [21] have related the thickness of the glue cylinder (e), the wavelength of the array of drops (λ) and the radius of the uncoated fibre (d) by using Eq. (2): h i1=3 2 2 : R ¼ ð3λ=4Þ ðd þ eÞ −d
ð2Þ
Image analysis was used to measure the radius of the drops (R), the wavelength and the ‘d + e’ length. The average values obtained for these parameters were 6.5 μm, 111.2 μm and 1.3 μm, respectively. Then, according to Eq. (2) the thickness of the glue coating is around 0.37 μm and the radius of the uncoated fibre is 0.93 μm. The equation proposed by Ryck and Quéré [22] for the capillary number when withdrawing a fibre from a reservoir (3) is: e ¼ 1:34 d Ca
2=3
; CaT1:
ð3Þ
The Capillary number being: Ca ¼ ηV=γ:
ð4Þ
Where η, V, and γ are the viscosity, velocity of the coating and surface tension. From Eq. (3), the value of Ca is 0.228. Eq. (4) relates the surface tension of the droplets with the coating velocity and the fluid viscosity. Volrath et al. [23] have estimated the reeling velocity of spider silk at around 20 mm·s−1. However, as far as the authors are concerned, there are no published data for the viscosity of the spider adhesives. Common values for the surface tension of other polymer solutions range 30 × 10−3–60 × 10−3 Nm−1 [24–26]. Considering these surface tension values, the viscosity of the spider glue should be in the range 340–680 Pa·s. For the sake of simplicity, Fig. 2 shows spherical drops. However, in order to estimate the contact angle, a different approach must be used. When a liquid is placed on a flat solid surface it will form a drop with a specific contact angle between the liquid and solid phases. By contrast, when the solid phase is a fibre the liquid forms a surface of revolution as shown in Fig. 3. Within the framework of Laplace's formula of surface tension and Young's equation of wetting, Carroll [27] has studied the wetting properties of fluids on fibre surfaces. Fig. 3 depicts the analysis of Carroll [27] for the static shape of a droplet on a fibre neglecting the effect of gravity. Carroll proposed a method for estimating the contact angle of a drop-on-fibre system. This method uses two dimensionless
Fig. 2. Typical representation of the beads on a string (BOAS) morphology for the droplets of adhesive on a thread.
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4. Conclusions
Fig. 3. Representation of Carroll's scheme for a fluid droplet on a solid fibre.
combinations of the three parameters x1, x2, and L (Fig. 3) to obtain the contact angle θ = F(x2/x1, L/x1) = F(n, L), using theoretically derived tabulations of the function F (see Supplementary information). The contact angle found in this study was around 45°. AFM nanoindentation tests were carried out on the silk droplets. E was estimated by using the procedure described in the experimental section. The estimated values of E are in the range 20–110 kPa. The average value is 70.02 ± 47.19 MPa (average of 300 points). The values obtained for E are in agreement with the Dahlquist criterion, which states that good adhesives should have an elastic modulus lower than 100 kPa [28,29]. The relatively high standard deviation might be due to the intrinsic variation of mechanical properties in biological materials. Ashby et al. [30] have reported the mechanical properties of a variety of natural materials such as wood, muscle, bone, etc. They concluded that biological materials show a large variability in their mechanical properties and claimed that the value of a specific property varies within one organism, between organisms and between species. Experimental factors of AFM nanoindentation tests may also be another source of error. Tan and Lim [31] have reviewed some of the issues concerning AFM nanoindentation tests. These include the effect of the underlying substrate, ambiguous tip shape, surface roughness, curvature of surface and nonperpendicular loading. Some authors, including Clifford and Seah [32] suggest that the experimental errors observed in AFM indentation tests depend heavily on the uncertainty in the determination of certain constants such as the AFM cantilever stiffness or spring constant and the tip radius. On the other hand, Schäfer et al. [33] propose that the main source of experimental errors in AFM indentation tests of spider silk might be due to the lack of Hertzian contact during the test. We believe that the variation found among our data might be more due to the intrinsic variation of the adhesive properties rather than due to experimental errors, although further studies would be needed to confirm this. The data obtained for the elastic modulus E of the silk adhesive droplets given here is the first estimation of the elastic properties for this kind of adhesive to be reported in the literature. Further studies of the elastic modulus of these adhesives including other AFM nanoindentation tests will allow for more accurate average values and value ranges of E. As far as the authors are concerned, there is no information regarding the Young's modulus of the adhesive of other spider species. Studies of other biological systems have reported the values of Young's modulus of the adhesive pads of animals and insects. Barnes et al. [34] used micro-indentation to measure the Young's modulus of whole toe pads of the tree frog, Litoria caerulea and found Young's modulus values in the range 4–25 kPa. The adhesive pads of the tarsi of the cricket Tettigonia viridissima were studied by Jiao et al. [35] and Young's modulus values in the range 21.9–64.1 kPa were reported. Perez et al. [36] have reported that the Young's modulus of Locusta migratoria was in the range 250–750 kPa.
The viscoelastic adhesive obtained from the silk of A. Argentata spiders displayed the typical beads-on-a-string (BEADS) morphology observed among other species. It was found that the adhesive drops had an average radius of 6.5 ± 1 μm and their periodicity was characterised by a wavelength of 111 μm. The contact angle of the droplets was around 10°. The apparent viscosity of the adhesive was found to be in the range 340–680 Pa·s. AFM indentation measurements provided an average Young's modulus of 70 ± 47 kPa. This value is in agreement with the Dahlquist criterion for adhesives [27,28], which states that an adhesive with a Young's modulus lower than 100 kPa can perform as a good adhesive. As usual, data obtained from AFM indentation tests have to be considered cautiously due to a wide variety of potential error sources related to the intrinsic variation of properties in biological materials, as well as to errors associated with the experimental technique. Further studies need to be performed in order to fully characterise the viscoelastic properties of this fascinating adhesive. Acknowledgements The authors would like to thank the Vice-Rectorate for Research of the Catholic University of Peru (VRI-PUCP) for their financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.09.030. References [1] T.A. Blackledge, N. Scharff, J.A. Coddington, T. Szüts, J.W. Wenzel, C.Y. Hayashi, I. Agnarsson, Reconstructing web evolution and spider diversification in the molecular era, PNAS 106 (2009) 5229–5234. [2] F. Vollrath, W.J. Fairbrother, R.J.P. Williams, E.K. Tillinghast, D.T. Bernstein, K.S. Gallagher, M.A. Townley, Compounds in the droplets of the orb spider's viscid spiral, Nature 345 (1990) 526–528. [3] E.K. Tillinghast, Selective removal of glycoproteins from the adhesive spiral of the spiders orb web, Naturwissenschaften 68 (1981) 526–527. [4] V. Sahni, T.A. Blackledge, A. Dhinojwala, Viscoelastic solids explain spider web stickiness, Nat. Commun. 1 (2010) 19. [5] R. Foelix, Biology of Spiders, Second ed. Oxford University Press, USA, 1996. [6] D.T. Edmonds, F. Vollrath, The contribution of atmospheric water vapour to the formation and efficiency of a spider's capture web, Proc. R. Soc. Lond. B 248 (1992) 145–148. [7] P.P. Bhat, S. Appathurai, M.T. Harris, M. Pasquali, G.H. McKinley, O.A. Basaran, Formation of beads-on-a-string structures during break-up of viscoelastic filaments, Nat. Phys. 6 (2010) 625–631. [8] J. Eggers, E. Villermaux, Physics of liquid jets, Rep. Prog. Phys. 71 (2008) 1–79. [9] S.L. Goren, The instability of an annular thread of fluid, J. Fluid Mech. 12 (2) (1962) 309–319. [10] S.L. Goren, The shape of a thread of liquid undergoing break-up, J. Colloid Sci. 19 (1) (1964) 81–86. [11] S. Kalliadasis, H. Chang, Drop formation during coating of vertical fibres, J. Fluid Mech. 261 (1994) 135–168. [12] T. Hesselberg, F. Vollrath, The mechanical properties of the non-sticky spiral in Nephila orb webs (Araneae, Nephilidae), J. Exp. Biol. 215 (2012) 3362–3369. [13] G.H. Mckinley, Viscol-Elasto-Capillary Thinning and Break-Up of Complex Fluids, in: D.M. Binding, K. Walters (Eds.), Rheology Reviews 2005, The British Society of Rheology, London, 2005. [14] B.D. Opell, M.L. Hendricks, The role of granules within viscous capture threads of orb-weaving spiders, J. Exp. Biol. 213 (2010) 339–346. [15] J.H. Dirks, W. Federle, Fluid-based adhesion in insects — principles and challenges, Soft Matter 7 (2011) 11047–11053. [16] A.L. Weisenhorn, M. Khorsandi, S. Kasas, V. Gotzos, H.J. Butt, Deformation and height anomaly of soft surfaces studied with an AFM, Nanotechnology 4 (1993) 106–113. [17] C. Rotsch, F. Braet, E. Wisse, M. Radmacher, AFM imaging and elasticity measurements on living rat liver macrophages, Cell Biol. Int. 21 (1997) 685–696. [18] M. Radmacher, Measuring the elastic properties of biological samples with the AFM, Eng. Med. Biol. Mag. 16 (1999) 47–57. [19] P.P. Bhat, S. Appathurai, M.T. Harris, M. Pasquali, G.H. McKinley, O.A. Basaran, Formation of beads-on-a-string structures during break-up of viscoelastic filaments, Nat. Phys. 6 (2010) 625–631. [20] P.H.M. Elemans, J.M.H. Janssen, H.E.H. Meijer, The measurement of interfacial tension in polymer/polymer systems: the breaking thread method, J. Rheol. 34 (1990) 1311–1326.
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[21] V. Sahni, D.V. Labhasetwar, A. Dhinojwala, Spider silk inspired functional microthreads, Langmuir 28 (2012) 2206–2210. [22] A. Ryck, D. Quere, Inertial coating of a fibre, J. Fluid Mech. 311 (1996) 219–237. [23] F. Vollrath, D.P. Knight, Liquid crystalline spinning of spider silk, Nature 410 (2001) 541–548. [24] A.W. Adamson, A.P. Gast, Physical Chemistry of Surfaces, Sixth ed. John Wiley & Sons Inc., New Jersey, 1997. [25] Y. Christanti, L.M. Walker, Surface tension driven jet break up of strain-hardening polymer solutions, J. Non-Newtonian Fluid Mech. 100 (2001) 9–26. [26] J. Cooper-White, J.E. Fagan, V. Tirtaatmadja, D.R. Lester, D.V. Boger, Drop formation dynamics of constant low-viscosity, elastic fluids, J. Non-Newtonian Fluid Mech. 106 (2002) 29–59. [27] B. Carroll, The accurate measurement of contact angle, phase contact areas, drop volume, and Laplace excess pressure in drop-on-fiber-systems, J. Colloid Interface Sci. 57 (1976) 488–495. [28] C.A. Dahlquist, An investigation into the nature of tack, Adhes. Age 2 (1959) 25–29. [29] C. Creton, Pressure-sensitive adhesives: an introductory course, MRS Bull. 28 (2003) 434–439.
[30] M.F. Ashby, L.J. Gibson, U. Wegst, R. Olive, The mechanical properties of natural materials. I. Material property charts, Proc. R. Soc. Lond. A 450 (1995) 123–140. [31] E.P.S. Tan, C.T. Lim, Nanoindentation study of nanofibers, Appl. Phys. Lett. 87 (2005) 123106. [32] C. Clifford, M.P. Seah, Quantification issues in the identification of nanoscale regions of homopolymers using modulus measurement via AFM nanoindentation, Appl. Surf. Sci. 252 (2005) 1915–1933. [33] A. Schäfer, T. Vehoff, A. Glisović, T. Salditt, Spider silk softening by water uptake: an AFM study, Eur. Biophys. J. 37 (2008) 197–204. [34] W.J.P. Barnes, P.J. Perez-Goodwyn, M. Nokhbatolfoghahai, S.N. Gorb, Elastic modulus of tree frog adhesive toe pads, J. Comp. Physiol. A. Neuroethol. Sens. Neural Behav. Physiol. 197 (2011) 969–978. [35] Y. Jiao, S. Gorb, M. Scherge, Adhesion measured on the attachment pads of Tettigonia viridissima (Orthoptera, Insecta), J. Exp. Biol. 203 (2000) 1887–1895. [36] P. Perez-Goodwyn, A. Peressadko, H. Schwarz, V. Kastner, S. Gorb, Material structure, stiffness, and adhesion: why attachment pads of the grasshopper (Tettigonia viridissima) adhere more strongly than those of the locust (Locusta migratoria) (Insecta: Orthoptera), J. Comp. Physiol. A. 192 (2006) 1233–1243.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Short communication
Physical characterization of the liquid adhesive from orb-weaving spiders Fernando G. Torres ⁎, Omar P. Troncoso, Fernando Cavalie Department of Mechanical Engineering, Catholic University of Peru, Lima 32, Peru
a r t i c l e
i n f o
Article history: Received 22 March 2013 Received in revised form 27 August 2013 Accepted 21 September 2013 Available online 29 September 2013 Keywords: Spider adhesive Spider silk Nanoindentation
a b s t r a c t Orb-weaving spiders produce bioadhesives that are used to capture their prey. In this paper, the physical properties of these adhesives are characterised. The liquid adhesive from Argiope argentata spiders has been studied and the morphological properties of the droplets, including size, shape and volume were determined. An estimation of viscosity and Young's modulus using atomic force microscopy has also been carried out. Morphological characterization confirmed that the liquid adhesive displayed a typical beads-on-a-string (BOAS) morphology on the silk fibres. The experimental data confirmed that the elastic modulus of the liquid adhesive from A. argentata was in the range 20–100 kPa which is in agreement with the Dahlquist criterion for adhesives. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Orb-weaving spiders produce several types of silks, each of which has a specific functional role. For instance, drag line silk produced by the major ampullate glands makes the radii of the orb-web. The orbweb must be able to cope with the kinetic energy of flying insects that arrive on it. The mechanical properties of spider silk such as tensile strength and extensibility determine the behaviour of orb-webs. Orb-weaving spiders use a special thread coated with an aqueous adhesive to capture their prey. This kind of thread is known as viscid thread and it is produced together with the aqueous adhesive by three spigots. The thread is spun in the flagelliform gland after which it is coated with the adhesive produced in the aggregate glands [1]. Volrath et al. [2] have investigated the chemical composition of the water soluble fraction of this aqueous adhesive. They found hygroscopic components related to neurotransmitters such as GAB-amide, choline, lysine, serine, etc. Tillinghast [3] and Sahni et al. [4] were able to analyse the non-soluble fraction of the adhesive. The presence of galactosamina, galactose, mannose, glucosamine, fucose and glucose would indicate that glycoproteins are responsible for the stickiness of this adhesive as they are the only components with long branches. Foelix [5] and Edmonds et al. [6] have reported that when the viscid thread is coated with the adhesive, the glue covers it evenly but it tends to form droplets due to Rayleigh instability, displaying a beads-on-astring (BOAS) morphology [7–13]. Opell and Hendricks [14] have performed drop adhesion measurements. They stretched single adhesive drops until separation from a glass probe. The maximum adhesion forces they found were in the order 0.1–0.4 mN. Sahni et al. [4] carried out load–relaxation tests. The forces they measured were around
0.2 mN. They also discussed the rheology of adhesive drops. They claimed that such drops behave neither as a viscous liquid nor as a viscoelastic liquid. Instead, the adhesive drops exhibit the characteristics of a viscoelastic solid. The studies previously published in the literature assessed the chemical and mechanical properties of the spider adhesive. However, in order to fully understand the physics of the adhesion of spider glue, other properties must be studied. One of the basic rules of adhesion is that one of the surfaces to be adhered should be relatively soft. It has been shown that the elastic modulus of the components strongly influences adhesion in biological systems [15]. In this paper, we give an estimation of other physical properties of the spider adhesive glue such as surface area, contact angle, viscosity and Young's modulus. 2. Experimental section 2.1. Materials Argiope argentata (Fabricius, 1775) spiders, an American species of the Araneidae family, were used in this study. Female adult spiders of 3.5–5 mm in prosoma width were collected in the outskirts of Lima, Peru. They were housed in 30 × 20 × 10 cm cages and were fed larval stage mealworms (Tenebrio molitor) three times per week. Cardboard frames with a rectangular opening were used to collect viscid silk samples. The ends of the silk samples were glued to the frames so that a part of the sample remained untouched. Silk samples were collected from the webs of three different spiders. At least three webs from each spider were used. 2.2. Characterization techniques
⁎ Corresponding author. E-mail address: [email protected] (F.G. Torres). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.030
Micrographs of the viscid silk were taken in a FEI-QUANTA 200 scanning electron microscope (SEM) in low vacuum with a voltage in the
342
F.G. Torres et al. / Materials Science and Engineering C 34 (2014) 341–344
range of 20–30 kV with a working distance of 10 mm. Three different silk samples were viewed directly without drying and without conductive coating. At least four SEM micrographs were taken from each specimen. This allowed for the measurement of sixty different drops. Nanoindentation tests were performed in a Nanosurf Easy Scan atomic force microscope (AFM) at 25 °C and 80% RH. A monolithic silicon NanoWorld Pointprobe® probe with a spring constant of 0.17 N/m was used. The tip radius of the probe is around 8 nm and its half cone angle is 50°. Mica TO-220 was used as substrate. Samples were tested immediately after being collected. Three samples were used. Each indentation test was repeated 5 times. The duration of each test was 1 s. 2.3. Young's modulus estimation The indentation tests were repeated 5 times for each point. The average values were used to plot the curve of the cantilever deflection against the height of the sample. If we consider an infinitely stiff tip and a soft flat sample, the Hertz model can be used to predict the relation between indentation and loading force [16–18]. The Young's modulus can be estimated by following Eq. (1): vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiqffiffiffiffiffiffiffiffiffiffiffiffiffi u k u d−d0 : z−z0 ¼ d−d0 þ u . . t π E tan ð α Þ 2 ð1−υ2 Þ
ð1Þ
Where E is the Young's modulus, ν is the Poisson ratio (taken as 0.5 corresponding to an incompressible material), k is the spring constant of the cantilever (0.28 N/m), α is the opening angle of the cone (50°), z is the height of the sample and d is the deflection of the cantilever. The 0 subscript accounts for the offset values. 3. Results and discussion Fig. 1 shows a SEM image of the spider adhesive glue forming drops on the spider silk thread. This image resembles other examples of the well described beads-on-a-string (BOAS) morphology. The BOAS phenomenon can be observed while pulling apart a thin film of a viscoelastic fluid such as saliva, fish slime, cellular protoplasm, etc. [19]. It can also be observed when blending incompatible polymers in the melt
Fig. 1. SEM micrograph of viscid silk showing the typical beads-on-a-string (BOAS) morphology of the adhesive droplets on the silk fibres.
state. During shear flow, these polymers are extended into long threads, but when the flow stops, the external thread breaks up into small droplets [20]. Fig. 2 shows a schematic representation of the BOAS morphology. According to Sahni et al. [21], when the threads come out of the spider spigot, the axial silk thread is coated by a regular cylindrical layer of glue. The glue layer then breaks down into an equally spaced array of droplets due to Rayleigh instability. For the adhesive droplets studied here, we have used some of the equations that describe the BOAS phenomenon to assess the physical characteristics of the spider glue from A. argentata. Sahni et al. [21] have related the thickness of the glue cylinder (e), the wavelength of the array of drops (λ) and the radius of the uncoated fibre (d) by using Eq. (2): h i1=3 2 2 : R ¼ ð3λ=4Þ ðd þ eÞ −d
ð2Þ
Image analysis was used to measure the radius of the drops (R), the wavelength and the ‘d + e’ length. The average values obtained for these parameters were 6.5 μm, 111.2 μm and 1.3 μm, respectively. Then, according to Eq. (2) the thickness of the glue coating is around 0.37 μm and the radius of the uncoated fibre is 0.93 μm. The equation proposed by Ryck and Quéré [22] for the capillary number when withdrawing a fibre from a reservoir (3) is: e ¼ 1:34 d Ca
2=3
; CaT1:
ð3Þ
The Capillary number being: Ca ¼ ηV=γ:
ð4Þ
Where η, V, and γ are the viscosity, velocity of the coating and surface tension. From Eq. (3), the value of Ca is 0.228. Eq. (4) relates the surface tension of the droplets with the coating velocity and the fluid viscosity. Volrath et al. [23] have estimated the reeling velocity of spider silk at around 20 mm·s−1. However, as far as the authors are concerned, there are no published data for the viscosity of the spider adhesives. Common values for the surface tension of other polymer solutions range 30 × 10−3–60 × 10−3 Nm−1 [24–26]. Considering these surface tension values, the viscosity of the spider glue should be in the range 340–680 Pa·s. For the sake of simplicity, Fig. 2 shows spherical drops. However, in order to estimate the contact angle, a different approach must be used. When a liquid is placed on a flat solid surface it will form a drop with a specific contact angle between the liquid and solid phases. By contrast, when the solid phase is a fibre the liquid forms a surface of revolution as shown in Fig. 3. Within the framework of Laplace's formula of surface tension and Young's equation of wetting, Carroll [27] has studied the wetting properties of fluids on fibre surfaces. Fig. 3 depicts the analysis of Carroll [27] for the static shape of a droplet on a fibre neglecting the effect of gravity. Carroll proposed a method for estimating the contact angle of a drop-on-fibre system. This method uses two dimensionless
Fig. 2. Typical representation of the beads on a string (BOAS) morphology for the droplets of adhesive on a thread.
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343
4. Conclusions
Fig. 3. Representation of Carroll's scheme for a fluid droplet on a solid fibre.
combinations of the three parameters x1, x2, and L (Fig. 3) to obtain the contact angle θ = F(x2/x1, L/x1) = F(n, L), using theoretically derived tabulations of the function F (see Supplementary information). The contact angle found in this study was around 45°. AFM nanoindentation tests were carried out on the silk droplets. E was estimated by using the procedure described in the experimental section. The estimated values of E are in the range 20–110 kPa. The average value is 70.02 ± 47.19 MPa (average of 300 points). The values obtained for E are in agreement with the Dahlquist criterion, which states that good adhesives should have an elastic modulus lower than 100 kPa [28,29]. The relatively high standard deviation might be due to the intrinsic variation of mechanical properties in biological materials. Ashby et al. [30] have reported the mechanical properties of a variety of natural materials such as wood, muscle, bone, etc. They concluded that biological materials show a large variability in their mechanical properties and claimed that the value of a specific property varies within one organism, between organisms and between species. Experimental factors of AFM nanoindentation tests may also be another source of error. Tan and Lim [31] have reviewed some of the issues concerning AFM nanoindentation tests. These include the effect of the underlying substrate, ambiguous tip shape, surface roughness, curvature of surface and nonperpendicular loading. Some authors, including Clifford and Seah [32] suggest that the experimental errors observed in AFM indentation tests depend heavily on the uncertainty in the determination of certain constants such as the AFM cantilever stiffness or spring constant and the tip radius. On the other hand, Schäfer et al. [33] propose that the main source of experimental errors in AFM indentation tests of spider silk might be due to the lack of Hertzian contact during the test. We believe that the variation found among our data might be more due to the intrinsic variation of the adhesive properties rather than due to experimental errors, although further studies would be needed to confirm this. The data obtained for the elastic modulus E of the silk adhesive droplets given here is the first estimation of the elastic properties for this kind of adhesive to be reported in the literature. Further studies of the elastic modulus of these adhesives including other AFM nanoindentation tests will allow for more accurate average values and value ranges of E. As far as the authors are concerned, there is no information regarding the Young's modulus of the adhesive of other spider species. Studies of other biological systems have reported the values of Young's modulus of the adhesive pads of animals and insects. Barnes et al. [34] used micro-indentation to measure the Young's modulus of whole toe pads of the tree frog, Litoria caerulea and found Young's modulus values in the range 4–25 kPa. The adhesive pads of the tarsi of the cricket Tettigonia viridissima were studied by Jiao et al. [35] and Young's modulus values in the range 21.9–64.1 kPa were reported. Perez et al. [36] have reported that the Young's modulus of Locusta migratoria was in the range 250–750 kPa.
The viscoelastic adhesive obtained from the silk of A. Argentata spiders displayed the typical beads-on-a-string (BEADS) morphology observed among other species. It was found that the adhesive drops had an average radius of 6.5 ± 1 μm and their periodicity was characterised by a wavelength of 111 μm. The contact angle of the droplets was around 10°. The apparent viscosity of the adhesive was found to be in the range 340–680 Pa·s. AFM indentation measurements provided an average Young's modulus of 70 ± 47 kPa. This value is in agreement with the Dahlquist criterion for adhesives [27,28], which states that an adhesive with a Young's modulus lower than 100 kPa can perform as a good adhesive. As usual, data obtained from AFM indentation tests have to be considered cautiously due to a wide variety of potential error sources related to the intrinsic variation of properties in biological materials, as well as to errors associated with the experimental technique. Further studies need to be performed in order to fully characterise the viscoelastic properties of this fascinating adhesive. Acknowledgements The authors would like to thank the Vice-Rectorate for Research of the Catholic University of Peru (VRI-PUCP) for their financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.09.030. References [1] T.A. Blackledge, N. Scharff, J.A. Coddington, T. Szüts, J.W. Wenzel, C.Y. Hayashi, I. Agnarsson, Reconstructing web evolution and spider diversification in the molecular era, PNAS 106 (2009) 5229–5234. [2] F. Vollrath, W.J. Fairbrother, R.J.P. Williams, E.K. Tillinghast, D.T. Bernstein, K.S. Gallagher, M.A. Townley, Compounds in the droplets of the orb spider's viscid spiral, Nature 345 (1990) 526–528. [3] E.K. Tillinghast, Selective removal of glycoproteins from the adhesive spiral of the spiders orb web, Naturwissenschaften 68 (1981) 526–527. [4] V. Sahni, T.A. Blackledge, A. Dhinojwala, Viscoelastic solids explain spider web stickiness, Nat. Commun. 1 (2010) 19. [5] R. Foelix, Biology of Spiders, Second ed. Oxford University Press, USA, 1996. [6] D.T. Edmonds, F. Vollrath, The contribution of atmospheric water vapour to the formation and efficiency of a spider's capture web, Proc. R. Soc. Lond. B 248 (1992) 145–148. [7] P.P. Bhat, S. Appathurai, M.T. Harris, M. Pasquali, G.H. McKinley, O.A. Basaran, Formation of beads-on-a-string structures during break-up of viscoelastic filaments, Nat. Phys. 6 (2010) 625–631. [8] J. Eggers, E. Villermaux, Physics of liquid jets, Rep. Prog. Phys. 71 (2008) 1–79. [9] S.L. Goren, The instability of an annular thread of fluid, J. Fluid Mech. 12 (2) (1962) 309–319. [10] S.L. Goren, The shape of a thread of liquid undergoing break-up, J. Colloid Sci. 19 (1) (1964) 81–86. [11] S. Kalliadasis, H. Chang, Drop formation during coating of vertical fibres, J. Fluid Mech. 261 (1994) 135–168. [12] T. Hesselberg, F. Vollrath, The mechanical properties of the non-sticky spiral in Nephila orb webs (Araneae, Nephilidae), J. Exp. Biol. 215 (2012) 3362–3369. [13] G.H. Mckinley, Viscol-Elasto-Capillary Thinning and Break-Up of Complex Fluids, in: D.M. Binding, K. Walters (Eds.), Rheology Reviews 2005, The British Society of Rheology, London, 2005. [14] B.D. Opell, M.L. Hendricks, The role of granules within viscous capture threads of orb-weaving spiders, J. Exp. Biol. 213 (2010) 339–346. [15] J.H. Dirks, W. Federle, Fluid-based adhesion in insects — principles and challenges, Soft Matter 7 (2011) 11047–11053. [16] A.L. Weisenhorn, M. Khorsandi, S. Kasas, V. Gotzos, H.J. Butt, Deformation and height anomaly of soft surfaces studied with an AFM, Nanotechnology 4 (1993) 106–113. [17] C. Rotsch, F. Braet, E. Wisse, M. Radmacher, AFM imaging and elasticity measurements on living rat liver macrophages, Cell Biol. Int. 21 (1997) 685–696. [18] M. Radmacher, Measuring the elastic properties of biological samples with the AFM, Eng. Med. Biol. Mag. 16 (1999) 47–57. [19] P.P. Bhat, S. Appathurai, M.T. Harris, M. Pasquali, G.H. McKinley, O.A. Basaran, Formation of beads-on-a-string structures during break-up of viscoelastic filaments, Nat. Phys. 6 (2010) 625–631. [20] P.H.M. Elemans, J.M.H. Janssen, H.E.H. Meijer, The measurement of interfacial tension in polymer/polymer systems: the breaking thread method, J. Rheol. 34 (1990) 1311–1326.
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