Insulin Amyloid Superstructures as Templates for Surface Enhanced

pubs.acs.org/Langmuir © 2010 American Chemical Society

Insulin Amyloid Superstructures as Templates for Surface Enhanced Raman Scattering Szawomir W
ojcik,†,‡ Viktoria Babenko,†,‡ and Wojciech Dzwolak*,† †

Institute of High Pressure Physics, Polish Academy of Sciences, Sokolowska 29/37, 01-142 Warsaw, Poland, and ‡Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland Received August 28, 2010. Revised Manuscript Received October 10, 2010

Nanostructuring of noble metal surfaces with biomorphic and biological templates facilitates a variety of applications of surface enhanced Raman scattering (SERS). Here we show that the newly reported insulin amyloid superstructures may be employed as stable nanoscaffolds for metallic Au films providing an effective substrate for SERS on covalently bound molecules of 4-mercaptobenzoic acid (4-MBA). The vortex-aligned insulin fibrils are capable of templating nanopatterns in sputtered Au layers without overlapping the SERS spectra of 4-MBA with vibrational bands stemming from the protein. This holds true regardless of whether the incident laser beam is directly backscattered from the 4-MBA layer, or after passage through the insulin amyloid layer.

Introduction New hybrid materials and building blocks of molecular devices may be obtained through a direct coupling of biological components to metal surfaces. The significant interest in these entities often stems from the unique optical properties of metal surfaces affecting molecules in their nearest vicinity, as is the case of the phenomenon of surface enhanced Raman scattering (SERS).1 That, in turn, has opened fascinating perspectives for applications of such systems in areas ranging from plasmonics and microelectronics to clinical diagnostics. Depending on the synthetic goal, there are two basic practical approaches to the problem of merging biocomponents (such as protein molecules, nucleic acids, virus capsids or even cell fragments) with metals: (i) a biomaterial serves as a topological guide for the structuring in statu nascendi metal phase (either bulk or in the form of nanoparticles2-4), or (ii) preexisting and usually chemically functionalized metal surfaces recruit and immobilize biomolecules for their subsequent use as molecular sensors.5-8 The complexity and variability of peptide conformations, and the fact that external stimuli may switch between them, have made proteins and polypeptides very promising scaffolds for metal nanoparticles4,9 and have led to some spectacular results such as synthesis of conducting nanowires from functionalized yeast Sup35 protein reported by Lindquist et al.10 In fact, most of the *Corresponding author. Phone: þ48 22 8880237. Fax: þ48 22 632 4218. E-mail: [email protected]. (1) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1974, 26, 163. (2) Fan, T. X.; Chow, S. K.; Zhang, D. Prog. Mater. Sci. 2009, 54, 542. (3) Sotiropoulou, S.; Sierra-Sastre, Y.; Mark, S. S.; Batt, C. A. Chem. Mater. 2008, 20, 821. (4) Guo, Y.; Ma, Y.; Xu, L.; Li, J.; Yang, W. J. Phys. Chem. C 2007, 111, 9172. (5) Murphy, C. J.; Gole, A. M.; Hunyadi, S. E.; Stone, J. W.; Sisco, P. N.; Alkilany, A.; Kinard, B. E.; Hankins, P. Chem. Commun. 2008, 8, 544. (6) Xu, H.; Bjerneld, E. J.; K€all, M.; B€orjesson, L. Phys. Rev. Lett. 1999, 83, 4357. (7) Kneipp, J.; Kneipp, H.; McLaughlin, M.; Brown, D.; Kneipp, K. Nano Lett. 2006, 6, 2225. (8) Sun, L.; Sung, K.-B.; Dentinger, C.; Lutz, B.; Nguyen, L.; Zhang, J.; Qin, H.; Yamakawa, M.; Cao, M.; Lu, Y.; Chmura, A. J.; Zhu, J.; Su, X.; Berlin, A. A.; Chan, S.; Knudsen, B. Nano Lett. 2007, 7, 351. (9) Guan, J.; Li, J.; Guo, Y.; Yang, W. Langmuir 2009, 25, 2679. (10) Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin, X. M.; Jaeger, H.; Lindquist, S. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4527.

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thus far developed hybrid biopolymer/metal materials employ natively fibrillar proteins such as collagen,11 microtubule filaments,12 bacterial flagella13 or filamentous temperature-sensitive protein Z14 as scaffolds for Au or Ag nanoparticles. On the other hand, there is a growing interest in accomplishing this task using more durable material: amyloid fibrils, which are nanometric β-sheetrich aggregates composed of orderly stacked misfolded protein molecules. Although their presence in vivo is often associated with fatal degenerative maladies such as Alzheimer disease,15 amyloidlike fibrils may also be induced in vitro from benign proteins or even synthetic peptides and polypeptides. Because of their unique (among other structural forms of proteins) thermodynamic stability,16 the advantage of using amyloid fibrils in nanotechnology became immediately obvious.10,17,18 Recently, it has been shown that such fibrils may form superstructures, which, through the lateral alignment and hierarchical intertwining of filaments, extend topological order and periodicity into second and third dimensions and into meso- and microscales inaccessible to single fibrils.19,20 Because of their geometry and stability, amyloid superstructures could become appropriate sacrificial templates for SERS-active metallic surfaces withstanding harsh conditions of metal vapor deposition or sputtering.21 Our previous studies have shown that hydrodynamic forces, such as shear flow, align early insulin fibrils into highly organized superstructures with powerful chiroptical properties termed -ICD/ þICD after the observed strong extrinsic Cotton effect detected (11) Sun, Y.; Wang, L.; Sun, L.; Guo, C.; Yang, T.; Liu, Z.; Xu, F.; Li, Z. J. Chem. Phys. 2008, 128, 074704. (12) Zhou, J. C.; Wang, X.; Xue, M.; Xu, Z.; Hamasaki, T.; Yang, Y.; Wang, K.; Dunn, B. Mater. Sci. Eng., C 2010, 30, 20. (13) Kumara, M. T.; Tripp, B. C.; Muralidharan, S. Chem. Mater. 2007, 19, 2056. (14) Ostrov, N.; Gazit, E. Angew. Chem., Int. Ed. 2010, 49, 3018. (15) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329. (16) Gazit, E. Angew. Chem., Int. Ed. 2002, 41, 257. (17) Gazit, E. Nanobiotechnology 2005, 1, 286. (18) Reches, M.; Gazit, E. Science 2003, 300, 625. (19) Krebs, M. R. H.; MacPhee, C. E.; Miller, A. F.; Dunlop, I. E.; Dobson, C. M.; Donald, A. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14420. (20) Corrigan, A. M.; M€uller, C.; Krebs, M. R. H. J. Am. Chem. Soc. 2006, 128, 14740. (21) Payne, E. K.; Rosi, N. L.; Xue, C.; Mirkin, C. A. Angew. Chem., Int. Ed. 2005, 44, 5064.

Published on Web 11/01/2010

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through induced circular dichroism (ICD) in amyloid-bound achiral dye thioflavin T (ThT).22-25 These structures, once assembled, remain stable after transferring to quiescent solutions and do not change properties upon prolonged storage at room temperature. Also, in a manner typical for other amyloids, -ICD fibrils are resistant to proteases and chemical denaturants. The fine microarchitecture of the -ICD insulin aggregate has triggered our interest in its application as a nanotemplate for metallic SERS-active surfaces. Because formation and maintaining of -ICD insulin superstructures requires the presence of NaCl,24 which could per se lead to roughening of a Au layer (and enhancement of SERS signal), we have employed a routine protocol based on glutaraldehyde (GA) in order to cross-link amyloid superstructures and provide an additional stabilizing factor upon elution and removal of salts. Properties of the Au/-ICD sandwich system as a scaffold for SERS were assessed using 4-mercaptobenzoic acid (4-MBA) as a model inelastic light scatterer often used in Raman studies.26-30 As it becomes increasingly clear that the formation of nonnative protein assemblies opens another synthetic route to rigid nanostructures that are inaccessible to conventional biomaterials; merging of these entities with unbiological components, such as noble metals, may create new and fascinating opportunities for photonics, plasmonics, and material chemistry.

Materials and Methods Preparation of Cross-Linked -ICD Insulin Amyloid Superstructures. -ICD fibrils were prepared by vortexing a 1 wt % solution of bovine insulin (from Sigma, USA) in 0.1 M NaCl, pH 1.9, for 24 h at 60 °C/1400 rpm using an Eppendorf Thermomixer Comfort accessory.25 Once fibrillation of insulin was complete, a small sample was collected for Fourier transform infrared (FT-IR)/Raman measurements, while the remaining suspension of -ICD fibrils was mixed with an aqueous solution of GA from Sigma to give the final concentration of GA 2.5 v/v %. Meanwhile, a 2.5 v/v % solution of GA in saturated sodium phosphate (Na2HPO4) buffer in 0.1 M NaCl, pH 9 was prepared. The buffered GA solution was added slowly to a vigorously stirred suspension of -ICD fibrils in 2.5% GA, until the pH reached 7.2. This was accompanied by the liquid becoming orange-yellow, reflecting the ongoing formation of the Schiff base between GA and insulin amine groups. The reacting mixture was agitated for 1 h at room temperature. This experimental routine was designed to maintain constant (0.1 M) concentration of chloride ions during the reaction, and to allow GA molecules to penetrate pores of -ICD fibrils at low pH before the Schiff reaction could take place and restrict accessibility of the cross-linker to voids of amyloid superstructures. After the completion of the cross-linking step, orange-yellow -ICD fibrils were centrifuged at 13600 rpm and washed repeatedly with a total of 105-fold volume excess of deionized water in order to remove traces of the cross-linker and salts. Finally, the chemically annealed fibrils were either deposited directly onto glass slides and dried up (to serve as SERS scaffold material) or suspended in an aqueous solution of ThT for ICD measurements. Wet suspensions of GA-cross-linked -ICD fibrils were stable for at least several weeks when stored at 4 °C. (22) Dzwolak, W.; Pecul, M. FEBS Lett. 2005, 579, 6601. (23) Dzwolak, W.; Loksztejn, A.; Galinska-Rakoczy, A.; Adachi, R.; Goto, Y.; Rupnicki, L. J. Am. Chem. Soc. 2007, 129, 7517. (24) Loksztejn, A.; Dzwolak, W. J. Mol. Biol. 2008, 379, 9. (25) Loksztejn, A.; Dzwolak, W. J. Mol. Biol. 2010, 395, 643. (26) Michota, A.; Bukowska, J. J. Raman Spectrosc. 2003, 34, 21. (27) Orendorff, C. J.; Gole, A.; Sau, T. K.; Murphy, C. J. Anal. Chem. 2005, 77, 3261. (28) Hunyadi, S. E.; Murphy, C. J. J. Mater. Chem. 2006, 16, 3929. (29) Zhang, S.; Ni, W.; Kou, X.; Yeung, M. H.; Sun, L.; Wang, J.; Yan, C. Adv. Funct. Mater. 2007, 17, 3258. (30) He, D.; Hu, B.; Yao, Q. F.; Wang, K.; Yu, S. H. ACS Nano 2009, 3, 3993.

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FT-IR, ICD, AFM, and SEM measurements. For FT-IR measurements, a small droplet of insulin fibrils suspension was cast onto a CaF2 window and dried up in vacuum (0.05 mbar) at room temperature. Infrared spectra of the protein film were collected on a Nicolet NEXUS FT-IR spectrometer equipped with a liquid nitrogen-cooled MCT detector. For a single spectrum, 256 interferograms of 2 cm-1 resolution were coadded. During measurements, the sample chamber was continuously purged with CO2-free dry air. Data processing was performed with GRAMS software (Thermo Nicolet, USA). All further experimental details were the same as specified earlier.31 Prior to the acquisition of ICD spectra, suspensions of fibrils in water (0.1 wt %) were stained with an aqueous solution of ThT (from Sigma, USA) to a final concentration of 200 μM. After mixing insulin fibrils with the dye and brief incubation, ICD measurements at 25 °C followed using a Jasco J-815 S spectropolarimeter, as specified in our previous works.23-25 For atomic force microscopy (AFM) measurements, freshly collected 2 μL samples of insulin fibrils were swiftly diluted with 500 μL portions of deionized water. Subsequently, tiny amounts (5-8 μL) of diluted samples were placed onto freshly cleaved mica and left to dry overnight. After 24 h, AFM tapping-mode measurements followed using a Nanoscope III atomic force microscope from Veeco, U.S.A., and TAP300-Al sensors (res. frequency 300 kHz) from BudgetSensors, Bulgaria. Other details of AFM imaging were the same as described earlier.25 For scanning electron microscopy (SEM), droplets of aqueous suspensions of cross-linked -ICD fibrils were deposited on silicon wafers and dried under vacuum at room temperature. Films of fibrils were covered with thin (approximately 5 nm) layers of Au (condensation of vapor) or Au/Pd alloy (sputtering). The images were collected on a Zeiss Leo 1530 microscope. SERS and Raman Measurements. In order to prepare amyloid scaffolds for SERS, aqueous suspensions of cross-linked -ICD fibrils were deposited onto microscopic glass slides (from MenzelGl€aser, Germany) and allowed to dry in vacuum (0.05 mbar, room temperature, 2 h). Protein films were then covered with a 100 nm layer of Au through sputtering. A fresh 2 wt % solution of 4-MBA in tetrahydrofuran (THF) (both from Sigma-Aldrich, USA) was dropped onto an Au-sputtered surface. After a few minutes, the excess of unbound 4-MBA was removed through a prolonged washing with pure THF. The samples were subjected to SERS measurements in a 180° backscattering mode, as is indicated in Figure 2. All Raman measurements were carried out using a Thermo Scientific Nicolet 6700 spectrometer equipped with a Raman NXR 9650 appliance and a liquid nitrogen-cooled NXR Genie germanium detector and a Nd:YVO4 laser operating at 1064 nm and 0.5 W power. Typically, 512 interferograms of 6 cm-1 resolution were coadded for a single SERS spectrum. Estimation of the Enhancement Factor in SERS Spectra. The enhancement factor (EF) is defined as: EF ¼ ðISERS =Ibulk Þ  ðNbulk =Nads Þ where Nbulk is the number of molecules sampled in the bulk, Nads is the number of molecules adsorbed and sampled on the SERSactive substrate, ISERS is the intensity of a vibrational mode in the surface-enhanced spectrum, and Ibulk is the intensity of the same mode in the Raman spectrum. All the intensities were obtained directly from the experiment. We chose to calculate the EF value according to intensity of the v(C-C)ring ring-breathing mode at 1073 cm-1 (SERS)/1093 cm-1 (bulk). When determining Nads in the laser spot, it was assumed that 4-MBA molecules are adsorbed as a monolayer on the Au surface with a density of 0.5  l0-9 mol/cm2.32 Under the experimental conditions of our study, the penetration depth of the laser beam was assumed to be ca. 25 μm, which, given the density of 4-MBA (1.35 g/cm3), (31) Dzwolak, W.; Loksztejn, A.; Smirnovas, V. Biochemistry 2006, 45, 8143. (32) Taylor, C. E.; Pemberton, J. E.; Goodman, G. G.; Schoenfisch, M. H. Appl. Spectrosc. 1999, 53, 1212.

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Figure 1. (a) FT-IR spectrum of a film cast from suspension of -ICD insulin amyloid superstructures. The spectral component of the amide I band marked in blue corresponds to the parallel β-sheet conformation. (b) The effect of cross-linking of -ICD amyloid superstructures on stability against elution with water probed by ICD spectra after staining with ThT. (c) An AFM amplitude image of GA-cross-linked -ICD superstructures. (d,e) The corresponding SEM images obtained after covering amyloid fibrils with conducting metal films deposited through Au vapor condensation (d) and Au/Pd sputtering (e). yielded Nbulk equal to 2  l0-5 mol/cm2. On the basis of these assumptions, we estimate the EF to be l04.

Results and Discussion The vortex-induced formation of chiral insulin amyloid superstructures belongs to a class of strongly irreversible dissipative processes wherein hydrodynamic forces cause emergence of a new structural order in the dispersed solid phase.33 The assembly of -ICD fibrils reduces conformational fluctuations of the polypeptide chains, resulting in the narrowing of bandwidth of protein vibrational bands.31 Figure 1a shows the amide I/II band region of a dry film of -ICD insulin fibrils prior to further modifications. The fibrils were obtained under typical conditions, i.e., at low pH, in the presence of NaCl and under strong agitation. The narrow amide I band is the hallmark of the parallel β-sheet conformation, the main secondary component of insulin amyloid. The net charge of insulin at pH 1.9 is positive, and this, through electrostatic repulsion, is a factor that decelerates aggregation and prevents self-assembly of higher superstructures. However, in the presence of chloride counterions (from NaCl), Debye screening of Coulombic forces is provided, and precipitation of -ICD fibrils is facilitated. As was indicated earlier, after completion of the selfassembly process, it is necessary to remove NaCl, which when coprecipitating with the fibrils, could become a competing artifact scaffold for a metallic layer. Because elution of the salt from -ICD fibrils promotes their disassembly,24 this was prevented by crosslinking with GA, as described in the Materials and Methods section. The degree of net stability of -ICD fibrils can be naturally probed by ICD of amyloid-bound ThT molecules.22 Tightly twisted ordered superstructures show profoundly negative extrinsic Cotton effect which quickly diminished upon their disintegration. (33) Vermant, J; Solomon, M. J. J. Phys.: Condens. Matter 2005, 17, R187.

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Figure 2. Preparation of glass slides with sandwiched 4-MBA/Au/ cross-linked -ICD amyloid layers for SERS measurements. The red arrows indicate the two directions of the backscattered incident laser beam examined in this study: direct and reverse.

The ICD spectra in Figure 1b reflect the fact that the cross-linking step does stabilize the insulin fibrils against elution of NaCl with water. The AFM and SEM images in Figures 1(c-e) depict surface alignment of individual insulin fibrils into larger domains of the size of single micrometers. Importantly, these domains come about in the process of a multistage hierarchical assembly of single amyloid protofilaments ca. 2 nm in diameter. This renders the surface of -ICD insulin fibrils covered with a variety of spikes and combs of different sizes, and roughens Au film deposited directly on the protein film, as is indicated in Figure 2. DOI: 10.1021/la103433g

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Figure 4. Comparison of SERS spectra of 4-MBA bound to Au/-ICD superstructures obtained through direct (bottom), and reverse through-the-glass (top) backscattering. The shadowed area corresponds to overlapping Rayleigh scattering.

Figure 3. (a) SERS spectra (direct scattering) of 4-MBA bound to Au/cross-linked -ICD amyloid superstructures (top), and of 4-MBA bound to smooth Au film on a clean glass slide (bottom). (b,c) Raman spectra of bulk 4-MBA and insulin fibrils, respectively. The shadowed area corresponds to overlapping Rayleigh scattering.

In Figure 3, a Raman spectrum of 4-MBA deposited on the Au/-ICD sandwich system (a) is juxtaposed with spectra of bulk 4-MBA (b), and insulin fibrils (c). The spectrum of the Au/-ICDbound 4-MBA film features two prominent peaks at 1584 and 1073 cm-1 assigned to v(C-C)ring ring-stretching and v(C-C)ring ring-breathing modes, respectively,28 but lacks other bands showing up in the Raman spectrum of 4-MBA (Figures 3a and 3b). The absence of the 2564 cm-1 band corresponding to S-H stretching vibrations is understandable for thiol molecules bound directly to the substrate surface via covalent Au-S bonds. However, the disappearance of the C-H stretching band at 3065 cm-1 from the Raman spectra of Au/-ICD-immobilized 4-MBA film must be explained by a slight alteration of the selection rules when the inelastic Raman scattering is enhanced through the SERS phenomenon. In fact, the spectrum shown in Figure 3a is very similar to SERS spectra of 4-MBA presented in earlier works.26-28 The red-shifting of the v(C-C)ring ring-stretching and ring-breathing peaks compared to the bulk Raman spectra has also been described therein. All in all, the spectrum of 4-MBA on the Au/-ICD sandwich shows all the characteristics of SERS rather than of 18306 DOI: 10.1021/la103433g

bulk Raman scattering. We have estimated the EF to be around 104 (Materials and Methods) by comparing intensities of the v(C-C)ring ring-breathing modes for bulk and surface-bound 4-MBA samples (Figure 3a,b). Importantly, the SERS spectrum of 4-MBA does not seem to be “contaminated” by contributions from bulk 4-MBA or the protein scaffold, as a comparison with the ensuing Figure 3c proves. None of the conspicuous Raman bands of insulin fibrils (e.g., C-H stretches above 3000 cm-1, amide I around 1650 cm-1, amide III below 1300 cm-1) can be detected. This observation is quite similar to data shown in earlier SERS studies where protein molecules, despite being in the direct vicinity of the metallic surface, remained absent from the SERS spectrum.11,34 In the latter work by Beier et al, whose objective was to detect Alzheimer amyloid protein using SERS, accomplishing this goal was only possible after using an auxiliary amyloid-binding molecule (Congo Red) with strong scattering properties.34 Although the lack of protein bands in SERS spectra is beneficial, as scaffolds are selected on the basis of being biocompatible but otherwise preferentially inert, it nevertheless remains puzzling. Three well-recognized factors leading to quenching of SERS signals are (i) spatial separation of scattering molecules from the metallic surface, (ii) incorrect orientation of transition dipole moments versus the surface, and (iii) insufficient roughening of the metal layer. Because of the preparation method of the metal/protein interface used in this study and the expected broad distribution of local topological metal/fibril arrangements (most of which should include SERS-permissible orientations), we doubt that either of the two former factors would play a significant role. On the other hand, the exact nature of surface defects induced in a sputtered Au layer upon direct contact with -ICD insulin amyloid superstructure is, at the present, the least understood. It should be stressed that SERS signals of proteins (and of insulin in particular, albeit in the native state conformation35) are easily obtainable, e.g., from electrochemically roughened surfaces.35 The transparent glass slide on which the 4-MBA/Au/-ICD system is deposited (Figure 2) enables collection of Raman spectra in the reverse “through-the-glass” mode. This was of interest, as we expected that the SERS signal from 4-MBA would be weakened or altogether absent. Surprisingly, the scattering intensity turned out to be approximately an order of magnitude higher than when spectra were collected in the direct backscattering mode (Figure 4). (34) Beier, H. T.; Cowan, C. B.; Chou, I. H.; Pallikal, J.; Henry, J. E.; Benford, M. E.; Jackson, J. B.; Good, T. A.; Cot
e, G. L. Plasmonics 2007, 2, 55. (35) Stewart, S.; Fredericks, P. M. Spectrochim. Acta A 1999, 55, 1615.

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The minor asymmetric peak protruding above 500 cm-1 could be attributed to insulin’s S-S bridges. However, it is likely that this peak is an overlap from Raman scattering on bulk fibrils rather than surface-enhanced scattering. Namely, disulfides in a direct contact with Au surfaces are prone to breakage at the S-S sites and formation of covalent Au-S bonds. Studying multilayer sandwich systems, such as the one described in this study, has one important advantage for research on the relationship between surface defects and SERS intensity on metallic substrates, as it provides a controllable spatial separation between the site where the defect is physically caused, and the site where its optical consequences are probed. Certainly, this can be attempted using unbiological or even inorganic templates as long as these provide proper nanopatterns. Such experiments should first look into the dependence between the thickness of the Au layer and EF magnitude. In this study, deliberately thick (about 100 nm) Au layers have been sputtered onto insulin fibrils in order to withstand the subsequent steps of 4-MBA deposition and elution with THF. We suggest that the resulting modest EF value

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could be explained in terms of a “cushioning effect” that the thick metal layer has on the roughening fibrous scaffold. In conclusion, we have shown that newly synthesized insulin amyloid superstructures may be adopted as nanoscaffolds for Au films becoming an effective substrate for SERS of 4-MBA. The protein layer is capable of roughening sputtered Au layers without contributing spectral artifacts to the proper SERS data. One of the most important objectives for studies in the field of metal/ protein composite materials is to obtain more effective and biocompatible substrates for clinically oriented SERS applications. The non-native protein assemblies, such as the -ICD insulin amyloid superstructures are novel robust structurally tunable and easily functionalizable entities that may prove advantageous for such applications. Acknowledgment. We are grateful to Mr. Adam Presz for his kind help with SEM measurements. This work was supported by the Polish Ministry of Education and Science - SPUB COST 518/ N-COST/2009/0.

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