Bio-derived three-dimensional hierarchical nanostructures as efficient

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Biological and Medical Applications of Materials and Interfaces

Bio-derived three-dimensional hierarchical nanostructures as efficient SERS substrates for cell membrane probing Stefano Managò, Gianluigi Zito, Alessandra Rogato, Maurizio Casalino, Emanuela Esposito, Anna Chiara De Luca, and Edoardo De Tommasi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19285 • Publication Date (Web): 23 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

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ACS Applied Materials & Interfaces

Bio-derived three-dimensional hierarchical nanostructures as efficient SERS substrates for cell membrane probing Stefano Managò,†,k Gianluigi Zito,†,k Alessandra Rogato,‡,¶ Maurizio Casalino,§ Emanuela Esposito,§ Anna Chiara De Luca,∗,† and Edoardo De Tommasi∗,§ †Institute of Protein Biochemistry, Italian National Research Council, Naples, Italy ‡Institute of Biosciences and Bioresources, Italian National Research Council, Naples, Italy ¶Stazione Zoologica Anton Dohrn, Department of Integrative Marine Ecology, Naples, Italy §Institute for Microelectronics and Microsystems, Italian National Research Council, Naples, Italy kauthors contributed equally E-mail: [email protected]; [email protected]

Abstract In this work, we propose the use of complex, bio-derived nanostructures as efficient surface-enhanced Raman scattering (SERS) substrates for chemical analysis of cellular membranes. These structures were directly obtained from a suitable gold metalization of Pseudonitzchia multistriata diatom silica shell (the so called frustule), whose grating-like geometry provides large light coupling with external radiation, whereas its extruded, sub-wavelength lateral edge provides excellent interaction with cells without steric hindrance. We carried out numerical simulations and experimental characterizations of the supported plasmonic resonances and optical near-field amplification. We

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thoroughly evaluated the SERS substrate enhancement factor (EF) as a function of the metalization parameters, and finally applied the nanostrucures for discriminating cell membrane Raman signals. In particular, we considered two cases where membrane composition plays a fundamental role in assessment of several pathologies, i.e. red blood cells and B-leukemia REH cells.

Keywords SERS, plasmonics, bio-derived nanomaterials, biophotonics, sensing, diatoms, cell membrane.

Introduction Over the last forty years, surface-enhanced Raman scattering (SERS) spectroscopy has taken benefit of progressive advances both in chemical synthesis of nanomaterials and nanofabrication techniques for engineering metallic substrates. 1,2 Raman scattering is a formidable tool for non-invasive investigations in the biological field. However, the weak Raman scattering often requires to be enhanced by means of near-field optical amplification mediated by localized surface plasmon resonances (LSPRs) in plasmonic nanostructures. This is at the foundation of SERS spectroscopy technique which allows to achieve even single-molecule sensitivity. 3 Therefore, large efforts have been dedicated to increase SERS performances towards engineering biomedical applications where extreme Raman sensitivity is required. Bottom-up chemical synthesis processes have allowed to obtain a large variety of nanostructures supporting LSPRs, such as colloidal silver and gold nanoparticles (NPs) of many shapes (nanoprisms, nanocubes, nanostars and nanosheets), 4–8 silica-coated metal NPs, 9,10 monolayers of metal nanowires, 11 two-dimensional self-assembling nanostructures, 12,13 etc. On the other hand, top-down technologies based on electron beam lithography have allowed fabricating SERS substrates with designer geometric properties, such as plasmonic nano2


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antennas arrays, 14–16 subwavelength nano-porous gold gratings, 17,18 nanostructured metallic fishnets 19,20 and hybrid substrates based on metallic nanoparticles coupled to dielectric photonic crystals. 21,22 Furthermore, a recent technology based on secondary electron lithography generated by ion milling allowed to obtain a variety of three-dimensional (3D) hollow nanostructures with tunable geometries. 23 Beyond analytic applications of SERS, extruded 3D nanostructures are highly promising for proper SERS interrogation of complex organisms with non-colloidal substrates. Indeed, they can overcome the typical steric hindrance of two-dimensional architectures. However, costly complex 3D geometries are prohibitive to nanofabrication and routine practical applications. An alternative way to obtain efficient SERS substrates relies on biomimetics. 24 Indeed, replicas of natural periodic micro and nanostructures allow to achieve efficient plasmonic substrates more feasible for mass production. Insects, birds and flowers have developed during evolution sub-micron periodic and quasi-periodic architectures able to manipulate light in a similar way as in artificial photonic crystals. 25–33 However, one of the most complex 3D dielectric nanostructure present in nature belongs to diatom shells. 34,35 Diatoms are ubiquitous monocellular micro-algae living in all aquatic environments. 34 In all the species, the cell is contained in a hydrated porous silica armour called frustule, whose functionalities comprise mechanical protection, sorting of nutrients from harmful agents and optimization of photosynthesis by light confinement. 36–40 The frustule is made of two valves showing complex multilayered or hierarchical multiscale nanostructure of pseudo-periodic patterns of pores and ridges. Payne et al. 41 obtained silver replicas of Synedra sp. valves. They were used as SERS substrates for analytical measurements showing SERS Enhancement Factor (EF) up to 106 . Similarly, Ren et al. 42 incorporated silver NPs in Pinnularia sp. with amine-functionalized diatom frustules. The same technique was also used for a SERSbased immunoassay. 43 An interesting aspect is that a diatom culture is a nearly zero-cost large-scale bio-factory in which a suitable 3D nanostructure can be produced by natural self-replication with high reproducibility. This might be advantageous in specific problems

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where 3D nano-architectures play a fundamental role. In this work, we study one of the most convenient, naturally-developed diatom frustule aiming at SERS probing of cells. Elongated, bilaterally symmetric needle-like frustules belonging to the Pseudonitzchia genus posses unique geometries provided by subwavelength extruded nanostructures at the edges that can be highly convenient for SERS probing. Being formed by a complex, intricate but regular dielectric 3D nanostructure, the properly metalized frustule triggers broadband LSPR in the red and infrared spectral range. In particular, we show that a suitable 3D plasmonic silica/metal heterostructure based on Pseudonitzchia multistriata valve is capable of robust SERS amplification (EF ∼ 106 ) with good reproducibility. A thorough morphological investigation is followed by a rigorous SERS EF characterization. 2,44,45 Finally, our diatom-derived SERS substrates were applied, for the first time to the best of our knowledge, for probing of both red blood and leukemic cells. In this context, we prove that the SERS spectra are mainly related to the cell membranes whose impairment is responsable of several pathologies. This allows overcoming the limitations of conventional spontaneous Raman spectroscopy where the scattered light comes from the cell as a whole. The nanostructure is naturally self-standing and can be easily integrated in microfluidic devices. In principle, the structure can be manipulated externally with the use of dielectric or magnetic beads.

Results and discussion Frustule morphology Pseudonitzchia multistriata, whose genome has been recently sequenced, 46 is one of the 48 known species of Pseudonitzchia diatom genus. Figure 1 shows the morphological details of a single valve after removal of the organic content at different magnifications. P. multistriata is a pennate diatom, and as such its frustule presents bilateral symmetry. The frustule is linear with margins tapering towards rounded tips (Fig.1a-c; in Fig.1f a detail of a tip is 4


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Figure 1: Bright field (a), dark field (b) and SEM (c) images of a single valve of Pseudonitzchia multistriata diatom after removal of the organic content. Progressive zooming of SEM (d, e, f ) and TEM (g, h, i) images allows to visualize nanometric pores and ridges of the frustule. In (f ) a detail of a valve tip is shown. Scale bars: 10 µm (a, b, c); 2 µm (d); 1 µm (e, h); 500 nm (f ); 5 µm (g); 400 nm (i).

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shown). In our strain, the apical axis has an average length of about 35 µm, whereas the transapical axis is of about 3 µm. The frustule valve has the shape of a double tip needle with a folded and vertically extruded side edge. The thickness of the silica is ∼ 30-50 nm. At the edges, the thickness progressively becomes smaller reaching the 10-nm range with a blade profile. Throughout its length the valve presents a series of interstitiae, about 100 nm wide, orthogonal to the apical axis. In between the interstitiae, in the so called striae (about 150 nm width), two (rarely three) rows of pores can be observed, whose average diameter is about 70 nm in length and whose mean center-to-center distance is of about 110 nm (see Fig.2a). On one side of the valve (see Fig.1d-e and Fig.2a) a band constituted by a periodic sequence of narrow fibulae (about 100 nm in width) and raphe interspaces (up to 350 nm wide) takes place. This last subwavelength structure at the edge of the diatom is vertically extruded of about 200 - 400 nm (the edge progressively becomes shorter toward the tip of the frustule) (see Fig.2b) and is characterized by extremely sharp edges (with a radius of curvature of about 13 nm). In plasmonic nanostructures, the regions of highest amplification correspond to those where a strong near-field localization occurs. Large SERS enhancements thus occur in two typical geometries: i ) nano gaps and interstices between coupled NPs 44 and ii ) sharp nanoscale tips and edges. 47,48 In planar SERS substrates, the former rarely provide easy and controllable access to complex bio-environments like cell membranes in fluid because of steric hindrance and Brownian motions. On the contrary, tips and vertically extruded edges are exquisitely prone for sensing biological membranes basically because they provide a sort of pointwise-probing, 23 a well-known mechanism also exploited in tip-enhanced Raman scattering. 48,49 However, extruded sharp tips and edges are challenging to fabricate and costly. The diatom frustule, instead, provides unique nanostructures with very sharp edges in which a strong SERS enhancement factor is expected to occur and that can be used to probe cells, as we demonstrate in this work. From an optical point of view, the single valve of P. multistriata provides a natural three-dimensional hierarchical diffraction grating comprising several interrelated periodic-

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Figure 2: (a) TEM image showing the details of a P. multistriata single valve (scale bar = 1 µm). (b-d) AFM scans revealing the actual 3D morphology of the structure consisting of relief modulations (interstitiae) (1) and inner gratings of nanopores (striae) (2); an outer side-bridge grating (fibulae) connecting the body to the side wall (3), and finally an extruded edge wall with a blade profile on top of which a further tooth-like modulation in relief is visible (4).

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ities, which can also be termed super-grating 50,51 as the frustule can be seen as the superposition of several structures. In particular, the complex pattern of the frustule can be schematized with four main sub-patterns, as indicated in Fig. 2 in which several atomic force microscopy (AFM) topographic scans reveal more insight into the morphology. Indeed, we can see: (1) a relief grating with protruded modulations (interstitiae) of about 20 nm in the body of the diatom (Fig. 2b); (2) a density grating produced by alternating couples of rows of nanopores (striae) in the body of the diatom (Fig. 2d); (3) a vertically extruded grating made of side bridges (fibulae) connecting the inner body to the side wall (Fig. 2d - please, consider that the AFM topography cannot render the voids inside the fibulae but they can be clearly seen in the TEM image in Fig. 2a); (4) a relief grating in the vertically-extruded side wall consisting in a periodic modulation of the height of the blade with a tooth-like shape of approximately 40 - 60 nm (Fig. 2c) (probably providing a better sealing of the two valves of the diatom). Such ordered structures provide an efficient coupling of the incident radiation with the uniformly-metalized valve. Indeed, a key challenge of plasmonic-based sensors is coupling the surface plasmons of uniform metal layers with the free-space optical excitation. The surface-plasmon wavevector (evanenscent field) is larger than the free-space wavevector, as such the momentum mismatch must be compensated with prisms or gratings for a good coupling. 52 In particular, the grating made of fibulae/raphe provides a strong scattering efficiency, as indeed revealed by the dark-field image in Fig. 1b where - as clearly visible - the side walls of the diatom appear as intense bright spots because of the larger light coupling. The scattering efficiency of NPs is usually small, and large spatial density of NPs is required to achieve good efficiency and hence high SERS sensitivity. 53 In our case, the metalized diatom geometry provides an excellent coupling with radiation. On the other hand, the poroids in the striae (Fig. 2a) and the blade-like wall on the side (Fig. 2d) are expected to provide a tight nanoscale confinement of the optical fields after metalization, as discussed in the next section of numerical simulations.

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In this work, we obtained SERS active substrates by means of gold thermal evaporation of P. multistriata frustules (see Methods). In particular, we studied the conditions for which a uniform gold layer is formed onto the superstructure of the frustules without altering the morphology of the architecture. Figure 3 shows the effect of different thicknesses of gold evaporated on the diatom. The inspection of the nanopores after gold deposition by means of AFM revealed that interstitiae, striae and pores were still visible and not occluded after metalization, except that for 50 nm of thickness. Statistics on pore dimensions reveal a mean pore diameter reduction of about 6-7% for a maximum thickness level of 40 nm (Fig. S1 in the Supporting Information). Further insights into the gold layer characteristics were inferred from scanning electron microscopy. A comparison of SEM micrographs as a function of the gold layer thickness is reported in Fig. S2 of the Supporting Information. In particular, we observed that for a thickness of 20 nm, the gold film presented cracks and clear discontinuities (specific ones showing fractal-like branching are also shown in Fig. 3a). The resulting nanogaps between metal islands are typically sites of large SERS enhancement, but also accompanied by random and poorly useful amplification. The uniformity of the layer increased for 30, 40 and 50 nm (Fig. 3 and Fig. S2). The particulate in the films was ascribed to the surface roughness, which was in the range of 10 nm as resulting from AFM analysis (Fig. S3 in Supporting Information).

Numerical Simulations Before optical characterization of single, metalized valves, we performed 3D numerical FEMbased simulations in order to estimate the electromagnetic field enhancement and the related plasmonic resonances over gold, nano-patterned slabs reproducing the main morphologies of the frustule (Fig. 4a). We simulated two approximated geometries consisting of a periodic arrangement of fibulae and raphe interspaces (Fig. 4b) and interstitia-stria sequence (with relative double rows of pores) (Fig. 4c) in order to qualitatively reveal the effect of near-field 4 0 amplification. The relative electromagnetic near-field amplification G = ElocE+E (with 0 9



Figure 3: The uniformity of the gold layer was evaluated by AFM. The AFM scans of a 1 µm2 region of a single valve of P. multistriata (in the central area of the diatom) for different thicknesses of the gold layer deposited on the frustule, reveal a non-uniform layer with fractal like gold islands for a thickness of 20 nm. The homogeneity of the gold layer increased for 30 and 40 nm. For a thickness of 50 nm, the nanofeatures of the valve appeared mostly occluded. Scale bar = 250 nm.

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E0 and Eloc incident and local field amplitudes, respectively), mapping distinct cut planes of the complex structure, are reported in Fig.4b,c. Edges were rounded to avoid sharpedge effects in the CAD design. The mesh grid had a resolution < 1 nm. Full retarded electrodynamic simulations were carried out with a 512 GB - RAM PC operating Comsol Multiphysics 5a in the frequency domain. The absorption and scattering efficiency spectra were calculated with Sommerfeld propagator as described in refs. 44,45 The overall extinction spectrum was peaked at around 705 nm (Fig. 4d). Finally, in order to have a more realistic view of the actual near-field amplification expected, the panels (e)-(f) in Fig. 4 show the CAD design and the related enhancement factor map generated from the actual experimental AFM topography of a region near the side edge of a diatom. In Fig. 4f,g we can observe propagating surface plasmons characterized by a lightening-rod effect at the vertically extruded edges. In summary, the main contribution in terms of enhancement of the optical field derives from the fibulae and edges, where values of the electromagnetic amplification G up to 106 and more are possible for normal plane wave incidence.

SERS Enhancement Factor characterization Irradiation by a supercontinuum white source in cross-polarization configuration (see Methods for details) allowed measuring the forward scattering spectrum of the single metalized frustule valve. In Fig.5, the dipole LSPR spectrum resulted centered around 695 nm, in reasonable agreement with numerical simulations based on a simplified version of the structure. In the inset, the visible portion of the scattered radiation coming from the single metalized valve is shown. The criteria to estimate the performance of the final SERS nanostructures is the enhancement of the Raman intensity. Therefore, we measured the experimental SERS substrate EF on the metalized diatoms as a function of the gold layer (20, 30, 40 and 50 nm), using the following equation: 13 EF =

ISERS /Nsurf , IRS /Nvol 11


(1)


Figure 4: (a) Approximated geometry reproducing the main subpatterns of the diatom valve. 4 0 Enhancement factor maps G = ElocE+E (log10-scale) related to a gold slab (thickness = 0 40 nm) with the geometry of a periodic sequence of fibulae (b), and a stria (c), respectively (evaluated in representative cut planes). (d) Relative overall extinction spectrum. (e) Computer-aided design (CAD) of the side wall of a diatom directly obtained from the actual AFM scan of the nanostructure (thickness 40 nm). (f ) Corresponding enhancement factor map over the surface of the CAD in (e). (g) The inset shows a particular transverse plane revealing the near-field amplification at the edge (same colormap as in (b)-(c)).

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Figure 5: Plasmonic resonance detected by spectral analysis of the radiation scattered by a single metalized valve. Inset: a metalized single valve scattering radiation. where ISERS and IRS are SERS and Raman intensities, Nsurf is the average number of molecules adsorbed to the frustule in the scattering area for the SERS measurements, whereas Nvol represents the number of molecules present in bulk in the scattering volume for spontaneous Raman scattering measurements. The SERS and Raman spectra were acquired with 785 nm laser excitation, 0.5 mW power and 1 s integration time. Scattering area and volume were thoroughly characterized by means of a knife-edge technique with a Si crystal wafer blade in backscattering configuration. 44 The beam waist of the incident laser was found of ∼ 1.4 µm. The height of the scattering volume was chosen as twice as the Rayleigh length, which was approximately of 13 µm in our configuration with a pinhole of 500 µm. In particular, the volume was calculated by integrating the Gaussian intensity of the beam over 26 µm of axial range. In our experiments, we made use of self-assembling monolayers of biphenyl4-thiol (BPT) as test analytes in order to quantitatively estimate Nsurf = 1.23 × 107 , known the packing density of BPT (conservatively 4 molecules per nm2 ) and the scattering area of the laser. IRS was estimated by measuring under same exposure conditions a sample of bulk powder of BPT (186.27 g/mol, ρ = 1.08 g/cm3 ) leading to a number of molecules in the scattering volume Nvol = 1.24 × 1011 . For SERS measurements, a comparative analysis of the flat gold-coated region outside the

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Figure 6: (a) Optical images and relative SERS intensity maps of the biphenyl-4-thiol (BPT) monolayer on SERS substrates for different gold thicknesses: 20 nm, 30 nm, 40 nm, and 50 nm. Scale bar: 10 µm. (b) SERS spectra of the diatom side (grey), with the relative average spectra (orange) and average spectra outside the diatom (red) for the BPT. The Raman spectrum of the pure BPT is additionally shown for comparison (blue).

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diatom and the patterned region of the gold-coated diatom was carried out in order to elicit both light coupling and efficiency gain introduced by the frustule architecture. To evaluate the spatial SERS reproducibility, we acquired confocal maps raster-scanning the area of interest around a diatom. The SERS intensity signal were plotted as a function of the coordinates of the sample. Several scans of 35 × 10 µm2 around the metalized diatom at different gold thickness are reported in Fig. 6a (pixel size of 0.7 µm). For all the considered thickness values, the highest SERS EF (bright yellow spots in Fig. 6a) were detected on the side of the frustule valve. The frustule side band had a width of approximately 400 nm (our beam radius was instead 1.4 µm), therefore producing an effective SERS scattering area reduced of a factor 5.5 with respect to the whole beam area. This leads to a more accurate effective value Nsurf = 2.2 × 106 . Figure 6b shows the different SERS spectra acquired from the diatom extruded side edge (gray lines) together with the average SERS spectra on (orange line) and outside (red line) the metalized valve as a function of the thickness of the gold layer. These mean spectra are the result of averaging 50 spectra acquired on the edge of the valve and outside the valve. With a nominal thickness of 20 nm deposited on the diatoms, the gold film resulted actually not uniform (Fig. 3) and presented discontinuities in the film (Fig. S2). The corresponding SERS reproducibility was quite low, not allowing a precise assessment of the SERS EF, with a value of (4.4 ± 3.0)×106 . The nano-granularity and crack-nanogaps between the nanoislands gave rise to BPT SERS signals also out of the valve. The SERS amplification and its spatial reproducibility was comparable with that of random gold NP aggregates (Fig. S4). The SERS signal on the pseudo-flat region out of the frustule was 3-fold less intense than that coming from the metalized valve edge. Therefore, the enhancement of the signal was ascribed mainly to the gold nano-granularity and cracks in the film, also in agreement with the results reported in Khlebtsov et al. 54 Nonetheless, the diatom morphology provides a significant boost to the SERS signal on the diatom side edge because of the much larger light coupling offered by the edge subwavelength structure. The homogeneity of metallic films was

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largely improved for 30 and 40 nm gold evaporations (see Fig. 3 and corresponding SERS analysis in Fig.6). In these cases, the BPT signals coming from the valve (especially from the valve side edge) were several orders of magnitude larger than the surrounding flat film of gold. The estimated EF for 30 and 40 nm gold layers along the valve side edge was of about (8.8 ± 3.0)×105 and (4.6 ± 0.9) × 106 , respectively. Additionally, the 80% of SERS spectra of BPT measured in different regions of the valve edge with 40 nm-tick gold layer appear well overlapped (gray lines displayed in Fig.6b, 40 nm), thus the spatial reproducibility of the SERS signals is very high. With a thickness of 50 nm, the corresponding SERS signal on the diatom was not uniform and scarcely reproducible, with some hot spots where a local 3-fold increase in SERS amplitude can be observed compared to the 40 nm-thick gold layer. In this case, we speculate that the gold occluded most of the pores also smoothing the nanofeatures of the frustule (Fig. 3). Concluding, a precise individuation of the plasmonic resonances and a quantitative evaluation of the SERS EF was carried out as a function of the gold layer thickness. The highest SERS EF achievable is estimated to be 4.6 × 106 , generally speaking consistent with theoretical predictions of amplification achievable in the approximated structure according to the FEM simulations. Specifically, the experimental data pointed out that the metalized diatoms show a huge amplification in terms of SERS EF and reproducibility with respect to the surrounding flat gold as a result of the strong coupling with far field light provided by the complex grating-like architecture of the diatom. In particular, the best performances were obtained for a uniform gold layer of 40 nm.

SERS probing of model cellular membranes Our diatom-derived substrates were applied to SERS probing of cellular systems. In particular, the metalized valves were exploited in the chemical analysis of red blood cells (RBCs) and leukemic cell (LC) membranes. The study of chemical and physical properties of cellular membranes plays a crucial role in the diagnosis of several pathologies and in the design of 16


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targeted molecular therapies. However, non-colloidal and non-targeted SERS nanostructures typically fail to provide a significant signal from cells because of poor amplification, low reproducibility and steric hindrance. Although AuNPs may show a good enhancement, planar substrates based on random colloidal aggregation have a low spatial reproducibility (see Fig. S4), which does not allow one probing complex biological samples such as cell membranes. Crucially, the hot spots are mainly limited to the gaps between the NPs (of a few tens of nanometers) and therefore, not really useful to enhance the signal of the cell membrane, as previously reported. 23,55 More complicate 3D plasmonic architectures are useful to promote the binding of the cell membrane to the SERS hot spots. 23,55 We used our most enhancing and reproducible diatom frustule valves with a gold layer of 40 nm as SERS substrates for probing RBC and LC membranes, according to the reproducibility results shown in Fig.6. The RBC membrane properties are fundamental, since several blood diseases are related to their chemical and physical alteration. 56,57 Usually, the acquisition of Raman spectra from RBC membranes is prevented by the strong contribution of hemoglobin (Hb). 13 Preliminarily, we studied the spontaneous Raman spectra (RS) of single RBCs for comparison with SERS measurements. RBCs were obtained from the peripheral blood of healthy donors. RBCs were washed in PBS and transferred on a quartz slide free of SERS structures. The RBCs were suspended in osmomolar PBS solution infiltrated in a post-sealed quartz chamber. The spectra were acquired at 785 nm, off-resonance with respect to the electronic transition of porphyrin in Hb, with a laser power of 10 mW and an integration time of 10 s. The RBC Raman spectrum, shown in Fig.7b, is an average signal on 30 different cells acquired after the cells had reached their stability in shape and position. The spectrum corresponds to the bond vibrations of the heme group. 13,58 In particular, it is possible to identify the Raman peaks at 676 cm−1 symmetric pyrrole deformation mode; 754 cm−1 ν15 pyrrole breathing mode; 1003 cm−1 : phenylanalyne; 1124 cm−1 asymmetric pyrrole half-ring stretching vibration; 1224 cm−1 combination band relative to C-H methine deformation;

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1342 cm−1 mode of the pyrrole ring stretching; 1564 cm−1 band of the spin state marker; 1621 cm−1 ν(C=C) mode of the porphyrin macrocycle (see Ref. 58 for band assignments). SERS measurements were conducted instead in a sealed chamber where SERS diatom frustules had been previously transferred to. Since the Raman signals of the molecules adsorbed to the SERS diatom substrate are highly enhanced because of near field amplification, minimal laser power of 0.5 mW with integration times of 1 s were sufficient to investigate the cell membrane, allowing not to damage the sample and not to lose spectral information caused by interference with Hb content, which did not mask, in this case, the membrane contribution. Figure 7c and d show the average RBC SERS spectra acquired from the contact region between a single RBC and a metalized diatom and repeated on 30 cells. By comparing the RS and SERS spectra of the RBCs, we can observe that the SERS spectra together with the main Hb bands show additional features with higher intensities which are almost unpronounced in the RS spectrum. Erythrocyte membranes contains 40% lipid, 52% protein and 8% carbohydrate, thus the RBC SERS spectra rise from these major membrane components more than Hb (see Fig.7e for the detailed Raman assignments). Basically, the bands at 690, 1108, 1240-1244, 1408-1417 cm−1 are assigned to the vibrational modes of lipids; bands at 909, 954, 1003, 1206, 1604 cm−1 represent vibrational modes of membrane proteins; and bands at 820, 1408-1417 and 1529 cm−1 are tentatively assigned to the glycocalyx and carotenoids. 13,59 Indeed, in case of RBCs interacting with the diatom-derived substrates, SERS signal came, as expected, only from the extruded region of the diatom nanostructure (the side edge) providing the most favorable interaction with cell molecules. In particular, as shown in the hyperspectral image in Fig.7a, the localization of the enhanced Raman signals produce a position dependent average contribution revealing the heterogeneity of the local membrane environment explored. Two main SERS spectra can be identified depending on the local membrane chemicals analyzed and shown in Fig.7a. The study of chemical composition of cell membranes plays a fundamental role also in the diagnosis of several oncological pathologies, including leukemia, as well as in most of the

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Figure 7: (a) Optical image of RBCs over a single metalized diatom valve. In the inlet, a detail of the Raman map superimposed to the optical image and acquired around a single cell. The signal comes from the region of contact between the cell and the valve. The map is relative to the 1300-1500 cm−1 spectral range. (b) Conventional Raman spectrum of an oxygenated single RBC, with typical spectral features of hemoglobin: 13,58 (c) and (d) SERS spectra acquired at the point of contact between a single RBC and the metalized diatom valve. The main contributions come from membrane constituent: lipids (L), proteins (P) and carbohydrates (C), the latter mostly belonging to glycoproteins. Integration time: 1 s. (e) Main Raman and SERS spectral assignments obtained from RBC based on ref. 13,59 The Raman shift in the SERS spectra can vary slightly, depending on the local membrane molecules analysed. Abbreviations: Pyr, pyrrole; def, deformation; sym, symmetric,; asym, asymmetric; Cys, Cysteine; Lys, lysine; Glu, glutamic acid; Ile, isoleucine; Phe, Phenylalanine; Asp, Aspartic acid; Asn, Asparagine; Gln, glutamine; Ala, Alanine; Leu, leucine; Val, valine; Trp, tryptophan; Ser, serine; br, breathing; sciss, scissor; str, stretching; wag; wag19 ging. Vibrations: ν, valence; δ, ACS deformation; deformation out of plane. Scale bar: 10 Paragon Plusγ, Environment µm.


Figure 8: (a) Optical image of REH leukemic cells over a single metalized diatom valve. In the inlet, a detail of the Raman map superimposed to the optical image and acquired around a single cell. The two hotspots correspond to the regions of contact between the cell and the metalized valve. The map is relative to the 1100-1300 cm−1 spectral range. (b) Conventional Raman spectrum of a single REH cell. (c) and (d) SERS spectra acquired at different points of contact between a single REH cell and the metalized diatom valve. The main contributions come from membrane constituents (lipids, proteins ans carbohydrates, the latter mostly belonging to glycoproteins). See text for the relative peak assignments. Integration time: 1 s. Scale bar: 10 µm. associated molecular target therapies. The assessment of different stages of differentiation of leukemia cells collected from the patient peripheral blood, indeed, is related to the detection of specific antigens localized at the plasma membrane. This kind of investigation is traditionally based on the use of fluorescent tags and, thus, is limited by photobleaching and affected by the difficulty to detect simultaneously multiple dyes. In this sense, a label-free technique based on Raman spectroscopy can represent an efficient alternative. 60 Nevertheless, in conventional spontaneous Raman spectroscopy, the collected, scattered light comes from the cell as a whole, thus carrying information from all its main components (nucleus, cytoplasm, membrane). As can be seen, for example, in Fig.8b, the spontaneous Raman spectrum of a single B-leukemia REH cell is strongly characterized by vibrational bands related to DNA, RNA, DNA-protein complexes, and DNA sugar-phosphate backbone, in addition to lipid contributions arising from the stretching of CH and CH2 groups and protein contribution due mainly to amide groups. In particular, we can distinguish the following 20


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spectral regions: 60 748 cm−1 : T, C (NA); 783 cm−1 : T, C, DNA bk (NA); 930 cm−1 : BK, deoxyrib., C-C (NA/P); 1000 cm−1 : Phe, C-C (P); 1126 cm−1 : O-P-O sym. str., BK (NA); 1308 cm−1 : G, Amide III, CH2 (NA/P/L); 1336 cm−1 : A, G, C-H def (NA/P); 1446 cm−1 : CH def (P/L); 1577 cm−1 : A, G (NA); 1655 cm−1 : Amide I, C=C (P/L) (abbreviations: def, deformation; str, stretching; bk, vibration of backbone; sym, symmetric; A, adenine; C, cytosine; G, guanine; T, thymidine; Phe, phenylalanine; NA, nucleic acids; P, proteins; L, lipids). On the other hand, when a REH cell interacts with a metalized, nanostructured diatom valve, the detected SERS signal comes only from the points of contact between the cell and the substrate - see the superposition of optical image and Raman map in Fig.8a. The most intense peaks of the SERS spectra are related mainly to membrane components, with no presence of vibrational bands characteristic of nucleic acids. Always referring to Fig.7e for band assignments, we can identify the following main peaks, with relative intensities strongly dependent on the investigated membrane region. 680 cm−1 : C-S strain (P+L); 764 cm−1 : choline (L); 820-847 cm−1 : C-C, C-O, C-C-H, Tyr (C+P); 954 cm−1 : Amide III (P); 980 cm−1 : -N+ (CH3 )3 (P); 1120-1139 cm−1 : =C-C= (L+P); 1170-1195 cm−1 : Tyr, Phe, Trp (P); 1225-1270 cm−1 : Amide III (P); 1344 cm−1 : Trp, CH2 sciss (P+L); 1467 cm−1 : CH2 (L); 1517 cm−1 : Amide II (P); 1585 cm−1 : Amide II, COO− (P+C) (please notice that in this case C stands for carbohydrates).

Conclusions SERS substrates obtained after uniform thermal deposition of gold onto frustule valves of P. multistriata diatom present several advantages. A hierarchical nanostructure as complex as that of the valve is hardly reproducible by lithographic approaches, even by means of the most recent nano-fabrication techniques. Instead, the bio-mimetic metalization takes advantage of nanostructures naturally self-replicated at each duplication cycle of the alga. The grating-like geometry of the valve provides an efficient coupling of the plasmonic field with

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the external optical radiation. The 3D vertically extruded side edges with sharp contours of the diatoms favor a high near-field localization providing strong SERS signals and optimal interaction with a complex biological system like a cell. We demonstrated the capability to detect the SERS signal coming from membrane constituents avoiding the contribution of the cell as a whole, which is crucial in the diagnosis of most blood and oncological diseases. Plasma membrane proteome analysis can be used to define biomarkers for diagnosis, classification, prognosis and progression monitoring in leukemia patients, as well as to predict therapeutic response or resistance. 64 Therefore, a method allowing the specific analysis of the cell membranes without interference with other cell components may have important diagnostic and prognostic applications. In perspective, the achievement of externally driven free-standing metalized valves from pennate diatoms may allow us obtaining SERS micro-sensors that can be optically trapped and conducted to the sensing site through, e.g., a microfluidic system. This will reverse the traditional scheme of Raman spectroscopy combined with optical tweezers usually applied in biochemical analysis of cells, where the cell itself is spectroscopically interrogated after its trapping by a tightly focused laser beam. 57 In our envisaged approach, indeed, it will be the sensing element to be trapped and dragged towards the target cell providing subwavelength resolution triggered by the intrinsic nanoscale morphology of the valve. Finally, the peculiar shape of P. multistriata or even sharper pennate diatoms may be used to take advantage of the intense tip effects coming from the sub-wavelength apex of the frustule.

Methods Sample preparation. Diatom frustules preparation. Pseudonitzschia multistriata strains were isolated at the Long Term Ecological Research Station MareChiara in the Gulf of Naples (40 ◦ 48.5 N, 14 ◦ 15 E). The strains have been successively grown at 18 ◦ C, under 100 µmol photons m−2 s−1 irra-

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diance with 12 L : 12 D h (light:dark) photoperiod. Cultures were grown in Guillard F/2 medium 61 made with autoclaved filtered natural sea water collected from the Gulf of Naples and Guillards F/2 solution (Sigma-Aldrich). Organic matter has been removed from the frustules of cultured cells according to von Stoch’s method: 62 concentrated sample pellets have been led to near dryness; an equal volume of HNO3 has been added plus a 3 × sample amount of H2 SO4 ; the mixture has been boiled for about 3 minutes and then cooled; the obtained samples have been rinsed with distilled water until free of acid and then resuspended in ethanol. Every rinsing cycle consisted in a centrifugation at 3500 rpm for 20 minutes. 20 µl of the valve suspension was pipetted in a clean quartz slide (150 µm thickness) and allowed to dry at room temperature for 10 minutes. Frustule metalizations. 20-50 nm-thick gold (Au) layers were deposited by thermal evaporation at a pressure of 3 × 10−6 mbar at room temperature. Deposition rate was 2 ˚ A/s and the sample, given by sparse diatom valves deposited onto a quartz slide, was put in rotation during the deposition process in order to promote film uniformity. BPT sample preparation. Biphenyl-4-thiol (Sigma-Aldrich, 99%) was dissolved in ethanol to achieve a final concentration of 1mM. By incubating the SERS slide in the BPT solution for 24h, an highly uniform self-assembled monolayer, with a resulting surface density of about 4 molecules over nm2 , is obtained. Cells. Red blood cells have been extracted from blood samples provided by a healthy donor. 100 µl of blood has been diluted in 900 µl of PBS solution. 10 µl of the diluted sample have been used for the SERS/ Raman experiments. The REH B-leukemia cell line was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Germany). The cell line was maintained in RPMI supplemented with 20% (v/v) fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin and 50 µg/mL streptomycin. To be suitable for the SERS (or Raman) analysis, the cells were removed from the culture medium, washed, suspended in a buffer solution (PBS) and transferred on the SERS

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substrate (or quartz slide) and the spectra measured within 30 minutes, after stability in shape and position of the cells has been reached. The SERS (quartz) chamber was sealed to keep unvaried the liquid content and avoid bacterial infection during measurements.

Morphological characterization. Scanning Electron Microscope (SEM) images of P. multistriata valves have been recorded after deposition of the samples on flat doped silicon wafers by drop casting. Imaging was performed at 5 kV accelerating voltage and 30 µm wide aperture by a Field Emission Scanning Electron Microscope (Raith 150). Transmission Electron Microscopy (TEM) images have been performed on valves dispersed in ethanol by a Tecna G2 Spirit BioTWIN microscope by FEI Company. Atomic Force Microscopy (AFM) measurements have been performed by means of a XEI-70 microscope from Park Systems using high-aspect-ratio probes of 5 nm of radius of curvature. The instrument is provided with two independent, closed-loop XY and Z flexure scanners for sample and tip, respectively. Flat and linear XY scan up to 15×15 µm2 with low residual bow is provided. Out of plane motion is less than 2 nm over entire scan range. Topographic resolution is below 1 nm.

Optical characterization. Radiation from a high power, fiber-based supercontinuum source (model SuperK Extreme, NKT Photonics) emitting in 400-2400 nm spectral range, was focused on single, metalized diatom valves deposited onto a quartz slide by a microscope objective (100×, NA = 0.75). A xyz micrometric translation stage was used to select single valves and transmitted radiation was collected by a second microscope objective (10×, NA = 0.2). A cross-polarization configuration allowed the detection of the scattered radiation by a spectrometer (model HR 4000, Ocean optics) with a resolution of 0.25 nm and response in 200-1100 nm spectral range.

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Raman spectra acquisition and processing. Raman spectra were recorded using an inverted confocal Raman microscope (XploRA INV, Horiba-Jobin Yvonne), 63 equipped with a 785 nm wavelength diode laser. A 60× water immersion objective (Nikon, Ti-2000 Eclipse, NA = 1.2) was used to focus the laser beam onto the sample and collect the scattered light. The beam waist of the laser was 1.4 µm. The scattered light from the sample was spectrally filtered by a holographic notch filter, spatially filtered by a pinhole (500 µm) and finally directed to the spectrometer, equipped with an 1800 lines/mm holographic grating. The Raman scattered light was focused onto the spectrometer entrance slit (set at 100 µm) and detected by a thermoelectrically cooled CCD camera. Each Raman spectrum was background corrected with polynomial fit to the fourth degree. The SERS intensity image was recorded by raster scanning the region of interest through the laser focus, with a step size of 0.7 µm, and acquiring a 2D array of SERS spectra on a selected area around the diatom valves.

Associated content Supporting information The Supporting Information includes Figures S1-S4: AFM images of a diatom valve before and after metalization process; SEM images of the diatoms after metalization and data about gold film morphology; AFM analysis of the film roughness; comparative SERS analysis of chemically synthesized AuNPs.

Author contributions G.Z., A.C.D.L., and E.D.T. conceived the experiments; S.M., G.Z., and E.D.T. performed measurements; S.M., G.Z., A.C.D.L., and E.D.T. analyzed data; G.Z. performed numerical simulations; A.R. grew diatom cells and designed the protocol for frustule cleaning; E.E. 25



performed electron microscopy morphological characterization of the frustules; M.C. and G.Z. carried out AFM characterization of the frustules; M. C. developed, optimized, and performed the metalization process. All the authors discussed the results.

Acknowledgments The authors are grateful to Dr. Maria I. Ferrante from Stazione Zoologica Anton Dohrn, Department of Integrative Marine Ecology, Naples, for providing P. multistriata cultures and to the Euro-BioImaging facility staff at the Institute of Protein Biochemistry (CNR), Naples, for support with TEM microscopy experiments. This work was partially supported by grants from the Italian Association for Cancer Research-AIRC (Start-up Grant 11454).

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Bio-derived three-dimensional hierarchical nanostructures as efficient

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