Supplementary Information

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1 SUPPLEMENTARY INFORMATION Breaking the diffusion limit with super hydrophobic delivery of few molecules to plasmonic nanofocusing structures F. De Angelis 1,2, F. Gentile 1,2, F. Mecarini 1, G. Das 1, M. Moretti 1, P. Candeloro 2, M.L. Coluccio 1,2, G. Cojoc 2, A. Accardo 1,2, C. Liberale 1, R.P. Zaccaria 1, G. Perozziello 1, L. Tirinato 2, A. Toma 1, G. Cuda 2, R. Cingolani 1, and E. Di Fabrizio 1,2 1 Department of Nanostructrures, Istituto Italiano di Tecnologia (IIT), via Morego 30, I16163 Genova, Italy. 2 BIONEM Lab, University of Magna Graecia, Campus S. Venuta, Germaneto, viale Europa, I88100 Catanzaro, Italy, Supplementary Information SI1. DESIGN OF SUPERHYDROPHOBIC SURFACES SI2. FABRICATION OF SUPERHYDROPHOBIC SURFACES SI3. SAMPLE PREPARATION ANDCHARACTERIZATION TECHNIQUES SI4. -DNA DEPOSITION AT DIFFERENT DILUTION SI5. FDTD simulation nature photonics 1

2 supplementary information SUPPLEMENTARY INFORMATION #1: DESIGN OF SUPERHYDROPHOBIC SURFACES #1.A Introduction It is well known that a drop post upon a solid surface makes a contact with the solid described by the sole parameter θ e (Fig.S1A,B), that is, the equilibrium contact angle at the interface between the liquid and the solid. θ e obeys the Young equation [1]: cos θ e γ = SV γ γ LV SL where γ ij is the surface tension between the phase i and j, and the letters S, L, V stand for the solid, liquid and vapour. The equation (1) can be regarded as a simple balance of forces. For sufficiently small droplets the dominant force becomes the liquid-vapor surface tension and gravitation may be neglected [2]. The dimensionless Bond number can be consequently introduced as Bo=ρ g R 2 / γ LV, where ρ is the density of the liquid, R is the radius of the spherical drop prior the deposition upon the surface, and g is the acceleration due to gravity. When Bo<<1 gravitational effects vanish and the shape of the droplet may be assumed spherical everywhere [3-5]. For a drop of water with γ LV =72.9 mj/m 2, ρ=1000 kg/m 3, and diameter d=2 R=1 mm, it follows that Bo~0.035, and thus the physics of micrometric or submillimetric drops is correctly governed by surface tension solely 1. (1) Fig.S1.1 Flat surfaces may have, via chemical modifications, a contact angle that is 120 at most [6]. Artificial super hydrophobic states are possible for surfaces comprising an underlying pattern or texture, in fact it is widely observed and very well understood that roughness increases hydrophobicity [7]. Two distinct mechanisms exist that yield superhydrophobic states, and these are well described by the models of Wenzel [8] and Cassie Baxter [9]. The first mechanism (Wenzel model) is based on the increased contact area between the surface and the liquid as due to roughness, and thus relies upon geometric effects solely. The modified angle θ e * is related to the unmodified contact angle θ e as: cos θ e = r cos θ e (2) 1 Another way of looking at the problem is considering the capillary length k -1 =(γ LV/ρg) 1/2 that for water assumes the value k -1 (water)=2.73 mm. Drops smaller than k -1 (water) are then small to the extent that tension dominates over gravitation. 2 nature photonics

3 supplementary information r is the solid roughness defined as the ratio between the real surface and the projected one [10], and thus the effect of surface roughness is to amplify the wetting. Noticeably, when θ e <π/2, r increases the hydrophilicity of the surface, whereas for θ e >π/2 roughness promotes hydrophobicity. In the second mechanism (Cassie model), air may remain trapped below the drop thus enhancing hydrophobicity. The increased contact angle θ e c >θ e can theoretically be written as ( cos θ 1) c cos θ 1 + φ + e = s e where φ s is the fraction of solid in contact with the drop. The less φ s the larger the apparent contact angle. The Cassie model is intuitive and predicts that a drop upon a patterned surface 'sees' a contact angle that is proportional to the fraction of air in contact with the drop. A flat surface would have φ s =1 and accordingly θ e c =θ e. In spite of the fact that they do both induce hydrophobicity, these situations are very different when considering their adhesive properties. In Wenzel state drops are found to be highly pinned (Fig.S1C). On the contrary, in the Cassie state the drop sits mainly upon air, and this increases the contact angle (Fig.1SD). Rigorously, Wenzel and the Cassie states are distinct, meaning that they cannot coexist. In other terms, a drop upon a micro textured surface would undergo the Wenzel or the Cassie state, not both simultaneously. A drop would assume either one configuration or the other depending on the geometry of the substrate (thus on φ s and r) and on the surface chemistry (thus on θ e ). A transition from Cassie to Wenzel occurs when cosθ e =cosθ e T = (φ s 1)/(r φ s ) or, in terms of the aspect ratio τ (the ratio between the height and the base of a structure): θ T e = arccos φ s φ s 1 ( 4τ 1) + 1 This means that a Cassie state holds for surfaces whose chemical induced hydrophobicity is sufficiently large, and for a fixed τ, the smaller the value of φ s, the larger θ T e [7, 11]. Since small values of θ T e are preferable, one should design surfaces having a texture with the largest ratio of the solid to the total projected area (large φ s ), and this would guarantee broad working areas where Cassie dominates over Wenzel [12, 13]. Notice though that large φ s would induce small contact angles, and thus the choice for the best parameters in (4) is not trivial, and it is indeed a matter of optimization, as exposed in paragraph 1C. In the Cassie state the drop would roll upon the surface and, most importantly for biological applications, it would progressively reduce its contact area during a process of evaporation. The drop would then maintain the Cassie state over time, thus avoiding collapse and any irreversible transition to Wenzel until a critical radius of impalement is achieved. The mechanisms inducing collapse in a slowly evaporating droplet are two and, namely, (i) the drop could either touch the surface below the posts, or (ii) the surface free energy gained as the drop collapses wins over the surface free energy lost by increased contact with the hydrophobic posts [14]. While the first mechanism regards surfaces decorated with short posts or pillars, the second relies upon an energetic argument and is independent on the pillars' height. In many practical situations one should consider the latter criterion solely. In particular, both analytical calculations and numerical simulations show that the critical radius of impalement depends upon the distance between the pillars δ and θ e as r min = δ cos θ e and thus the closer the pillars, or the larger θ e, the smaller the final area of contact [14-17]. Experimental evidences confirm this theoretical finding [18]. (3) (4) (5) nature photonics 3

4 supplementary information #1.B Adhesive Properties of Superhydrophobic Surfaces In this paragraph we show that super hydrophobic surfaces can exhibit vanishingly small friction coefficients. A drop sitting upon a surface experiences an equilibrium contact angle θ e that follows equation (1). Considering a drop sufficiently small, as to neglect gravitational effects (Bo<1), the radius r of the solid liquid contact can be written as r = R sinθe, β = ( 1 cos θe )( 2 + cos θe ) (6) β R is radius of the spherical drop prior the deposition on the surface. For symmetry solely half a drop is considered as in Fig.S1.2A. Fig.S1.2 The net adhesive force F a acting along x may be calculated [19, 20] as F a = γ Γ lv ( cosθ cosθ ) i dl r a (7) where θ r and θ a denote the receding and advancing contact angles, respectively, i denotes the unit vector along the x axis and dl a differential length of boundary Γ. From the geometry of the problem, Eq.(7) may be expressed as F a = π 2 π 2 γ lv ( cosθ cosθ ) r cosϕdϕ = 2γ ( cosθ cosθ ) r. r a lv r a (8) Thus the adhesion force F a would depend upon the length of the contact line (2r), the liquidvapor surface tension (γ lv ) and the term (cosθ r cosθ a ) that is directly related to the contact angle hysteresis (CAH): θ a θ r 2. We consider a drop upon a micro textured surface with a periodic hexagonal lattice of cylindrical pillars (Fig.S1.2B), where d is the diameter and δ the distance (gap) between the 2 The larger the hysteresis, the more the drop is stuck to the substrate. The CAH is due to heterogeneities (in topography and chemical composition) which are always present at a solid surface, inducing fluctuations of the surface tensions between the solid-vapor and solid-liquid phases [21, 22]. Notice that the equilibrium contact angle that may be experimentally observed is not unique in that it can range between the lower and upper limits θ r and θ a. An ideally smooth surface has no hysteresis effects (θ a=θ r=θ e) and a drop would roll or slip upon the application of any infinitesimal external force. 4 nature photonics

5 supplementary information pillars. The fraction φ s of solid remaining in contact with the drop is φ s =πd 2 /4(d+δ) 2, and the fraction of air is φ a =1 φ s. We calculate the adhesive force considering the textured substrate. Equation (8) still holds true, but now (i) the radius r of the solid liquid contact is reduced due to the increased contact angle (Eq.(6)) and (ii) the CAH the drop sees is not unique due to the heterogeneous contact surface (the CAH is maximum at the solid fraction, zero otherwise). Relation (8) can be thus rewritten as F c a c ( cosθr cosθa ) = 2r γ lv ( cosθ r cosθ a ) φ s = (9) c 2r γ lv where <Ω> is the spatial average taken on the surface of contact of the drop (it is here assumed that the radius of contact is large compared to the pillars dimension). From this, it follows that tiling the surface (φ s <1) the hydrophobicity increases and drastically reduces adhesion. Using relations (6) and (3) it is possible to recast equation (9) in terms of θ r, θ a, θ e, R and φ s : F c a 1 / s e 2 = 2R γ lv ( φ ( ) ) ( ) ( ( )) ( ( )) s cosθ e cosθ r cosθ a φ s (10) 2 φs cos 1+ θe 1+ φ cos 1+ θ When the drop and the material are given, the quantities θ r, θ a, θ e, R are constant and the sole variable in (10) is φ s. The considerations above help to draw a comparison between ideally flat and textured surfaces. The adhesion force is: 4 3 F a = ν π R 3 ρ g cosα Whereby F a is written in terms of the critical angle α that is the tilting angle above which the drop would begin to roll off the plane of contact. The friction coefficient ν may be then conveniently determined using the well known equation ν = tanα For the configurations at study (say pillars 10 µm wide with a gap of 20 µm) tilting angles as low as 1 were measured, and correspondingly Eq.(12) reads ν~ Evaporation of a drop upon the surface. Initially the drop is pinned and the contact line is fixed. The solvent evaporates over time thus decreasing the volume of the drop. The spherical shape of the liquid has to be preserved and consequently also the contact angle decreases. This unbalances the forces acting upon the drop and would generate a radial force that tends to recall the contact line towards the centre of the drop, thus recovering the initial equilibrium contact angle θ e c (Fig.S1.3). (11) (12) nature photonics 5

6 supplementary information Fig.S1.3 Following the reasoning above, the total force in the x direction at a generic angle θ e <θ e c can be evaluated as F p = c ( cosθ cosθ ) c 2r γ lv e and accordingly the condition for depinning is F p >F a c or, equivalently, F p /2r c γ lv >F p c /2r c γ lv. When F p F a c, the drop is stuck on the surface. (This condition is valid, strictly speaking, in close proximity of θ e ). This model is effective in that allows predicting with sufficient accuracy the tendency of a drop to reduce its contact area while evaporating, thus concentrating the solute therein initially dispersed. Consider for instance a sessile water droplet post upon a continuous, smooth Teflon surface, having the parameters φ s =1, θ e =114, θ r =100, θ a =125. Conveniently using these values within equations (13) and (9) it is found that F c a ~0.8Rγ lv and F p ~2Rγ lv ( cosθ), and thus F p would be smaller than F c a for a large range of θ. In other words, during the evaporation process, the drop will predominantly be pinned on the surface. Consider instead the same Teflon surface decorated with a regular array of pillars as to have φ s ~ It then follows that F a c ~0.022Rγ lv and F p ~2Rγ lv (0.94+cosθ). These values suggest that a small deviation from the equilibrium position (in other words, a small decrease θ e Øθ) would induce F p >F a c, as a consequence the contact line would jump inward from a thread of pillars to another while the drop evaporates. Let`s now define a new parameter Υ as the ratio Υ=F p /F a c, useful for evaluating the tendency of a drop to slide upon a surface: when Υ>1 the depinning dominates over the friction force, and thus the drop slides toward its centre upon evaporation. For the configuration above (φ s ~0.087, that is 10 µm wide pillars with a gap of 20 µm) Υ may be plotted against the angle θ=θ e θ, that is the deviation from equilibrium. Initially, at t=0, Υ<1, meaning that the drop would be sticky. As evaporation starts, the contact angle decreases, and accordingly θ increases. The depinning force rises with θ, and so does Υ. When Υ is sufficiently large, the contact line of the drop would recede, the system would assume a state that is energetically favourable and the contact angle would match again the expected equilibrium contact angle θ e. Under these conditions, θ=υ=0. The process starts over again and the same mechanism is repeated. In Fig.S1.4 the calculations are accomplished for 10 cycles, and, at each cycle, the contact radius is reduced by a quantity that is the distance between two lines of pillars. When r<r min, the drop gets pinned, and the process ends. (13) 6 nature photonics

7 supplementary information Fig.S1.4 For sufficiently high pillars the minimum contact radius achievable is limited by energetic considerations and depends upon the distance of the pillars δ and θ e as described by Eq.(5). When r c r min the drop is impaled and an irreversible transition to the Wenzel state occurs. In this situation the drop is definitely pinned and no further contact area shrinkage is possible. Fig 1.5 illustrates the same process following the reduction of the contact radius of the drop with time. The diagram is a discontinuous step function, whereby the radius is constant while the ratio Υ is lower or equal to one. When Υ>1, (when the angle of contact becomes sufficiently low to yield a depinning force significantly larger than the adhesion force), a transition occurs, and the line of contact would jump inward from a line of pillars to another. Beyond the lower bound given by Eq.(5) the contact radius is hold constant. nature photonics 7

8 supplementary information Fig.S1.5 #1.C Considerations for an optimal design c A closer glance to equations (1) and (5) evidences how the apparent contact angle θ e decreases monotonically with increasing φ s and, similarly, for a given pillars' diameter d the smallest contact radius r min would be attained for large values of φ s. In short, φ s has coincidental effects on both θ c e and r min. Let the microtextured surface be described by the parameters d and δ (see above for the significance of these terms). If d is fixed, then the system would be described by the sole δ and thus φ s. The aim here is to determine the optimal subset (d 0, δ 0 ), or equivalently φ 0 s, that would induce the higher θ c e still retaining the advantages of a small r min. To do this, consider the radius r of the solid liquid contact as in Eq.(6), where it is intended that the contact angle θ e is actually the apparent, Cassie's θ e c : r would depend upon the sole independent variable θ e c and thus φ s (Eq.(3)). As φ s gets smaller even r does it. Let's then divide r by R, and call the output function Ψ 1 (Ψ 1 =r/r). Ψ 1 is normalized to one and accounts for the effects of the micro structure of the surface on the contact line. Consider now the minimum contact radius r min before the drop collapse as in equation (5). r min is a function of δ r min ( ) ( ( ) ) 1 φs = r = r f d = r d min δ min 1 φs min φs Notice that r min is not a function of the sole φ s, and may be instead cast into a form that is consistent with Ψ 1 if divided by d: r min *= r min /d. r min * is consequently normalized to one upon dividing by the value of r min * that is obtained at φ s : θ e c (φ s )=ξ, where ξ is an angle of contact very high and still physically achievable (170 <ξ<180 ). Let's term the latter quantity Ψ 2 (14) Ψ 2 = rmin / rmin c θ e =ξ (15) Ψ 2 accounts for the effects of the micro structure of the surface upon the smallest radius of 8 nature photonics

9 supplementary information contact prior the drop collapse. Let's then introduce Ψ as the sum Ψ = Ψ1 + Ψ 2 For a given d, Ψ varies with δ. Most importantly, a value of δ exists where Ψ attains a minimum: this, for a given hexagonal lattice, would be the best value of δ, δ=δ 0, providing a sufficiently small contact line (or equivalently, high contact angle) still assuring a small impalement radius. δ 0 was computed for different d s (0 d 30 µm) and for ξ ranging between 162 and 170. The results are shown in Fig.S1.6. (16) Fig.S1.6 Fig.S1.6 is a contour plot of the contact angle θ e c as a function of the pillars' diameter (d) and distance (δ). Each point of the diagram would thus represent a lattice configuration. The radial straight lines stemming from the origin of the axes represent the sites at constant contact angle or constant φ s. The region of the diagram in light turquoise (region of interest) comprises all the possible couples (d 0, δ 0 ) of optimal design according to the reasoning above. The points in the diagram are the actual parameters used for nano-patterning the surface and mostly fall in the region of interest. Generally, d should be as small to consent a sufficiently large number of pillars to interact with the drop. The lower boundary for d depends upon limitations intrinsic to the process of fabrication. #1.D Symmetry Breaking and Spontaneous Impalement Here we consider the geometry of the device as in Fig.2E of the main text, where a cone is enclosed in a regular pattern of cylindrical pillars. A rationale is given that would explicate nature photonics 9

10 supplementary information how a slowly evaporating droplet would preferentially collapse in close proximity of the cone. An argument is introduced that relies on the mean field theory approach proposed by Moulinet and colleagues in [15, 16]. On the application of a drop of water upon a regular hexagonal array of pillars, the portion of the water exposed to the air experiences a force due to the internal Laplace pressure, which may be written as F p ( ) = P A 1 φ L where A is the projected area of the surface of interest, φ=4/3π(d/δ2) 2 has the meaning given above, and P L P L = 2γ R() t with R(t) the radius of the drop that is, in general, function of time. F P would push the drop towards the substrate and thus the larger F P the more the transition to the Wenzel state is likely to take place. F p would be balanced by the capillary force N p f where N p = φa/π(d/2) 2 is the number of pillars in the area of interest, and f is the elementary force per pillar given by f = 2πγ d cos θ. 2 The drop would penetrate inside the pillars and collapse if F p >N p f or, equivalently, combining all the quantities in Eq.s from (17) to (19), if 2γ R 2 2π d 4π d 1 > γ cos θ (20) 2 () t 3 2δ 3 δ That can be rearranged as B P L δ d π 2 B Ξ < 1, = B = d PL 4π γ cosθ 3 And thus the criterion for the stability of the Fakir state would be the left hand part of Eq.(21) to be larger than one. Eq.(21) is true on average (mean field theory approach), and here we make the assumption that the above results may be extended locally to a lattice where the distribution of pillars in x (horizontal coordinate) and y (vertical coordinate) is described by the sole function d(x,y). Fig.S1.7 reproduces Ξ(d) for a network of micro pillars with a central defect (that is, a pillar is substituted by a cone, with radius of curvature at the apex of few nm, thus severely smaller than the pillars` diameter) where the parameters d=10 µm and d=30 µm were used. Notice that at `defect` Ξ(d) attains a minimum, meaning that, over the area of interest, this is the position where the probability of impalement is the highest. This result is valid for a generic drop`s radius, that is, when the drop is sufficiently small and energetically favourable for impalement, the collapse would occur, most likely, at the cone. (17) (18) (19) (21) 10 nature photonics

11 supplementary information Fig.S1.7 nature photonics 11

12 supplementary information 1. Young, Phil. Trans., 1805; (84):1; edit by Peacock. 2. McHale G., Shirtcliffe N. J. and Newton M. I., Analyst, 2004; (129): Mahadevan L., Pomeau Y., Phys. Fluids, 1999; (11): Aussillous P. and Quéré D., Nature, 2001 ; (411): Blossey R., Nature Materials, (2): , Shafrin, E. G. & Zisman, W. A. in Contact Angle, Wettability and Adhesion Advances in Chemistry series, Vol. 43, ed. Fowkes, F. M., , American Chemical Society, Washington D.C., Lafuma A. and Quéré D., Nature Materials (2): , Wenzel model - Wenzel, R. N., Ind. Eng. Chem. 28, , Cassie model - Cassie, A. B. D. & Baxter, S., Trans. Faraday Soc. 40, , J. Bico et al, Pearl Drops, Europhys. Lett., 47 (2), pp , Bico J., Thiele U. & Quéré D., Colloids Surf.A 206, 41-46, Neelesh A. Patankar, Langmuir 2004, 20, Michael Nosonovsky and Bharat Bhushan, J. Phys.: Condens. Matter 20, 2008, pp. 14. H. Kusumaatmaja, M. L. Blow, A. Dupuis and J. M. Yeomans, EPL, 81, 2008, S. Moulinet and D. Bartolo, Eur. Phys. J. E 24, , D. Bartolo, F. Bouamrirene, E. Verneuil, A. Buguin, P. Silberzan and S. Moulinet, Europhys. Lett., 74 (2), pp , M. Reyssat, A. Pepin, F. Marty, Y. Chen and D Quéré, Europhys. Lett., 74 (2), pp , M. Reyssat, J. M. Yeomans and D. Quéré, EPL, 81, 2008, Dussan E. B. and Chow R. Tao-Ping, J. Fluid Mech. (1983), 137, Quéré D., Lafuma A. and Bico J., Nanotechnology 14 (2003), De Gennes P G, 1985, Rev. Mod. Phys. (57): De Gennes P G, 1985, Rev. Mod. Phys. (57): G. McHale, S. Aqil, N. J. Shirtcliffe, M. I. Newton and H. Y. Erbil, Langmuir 2005, 21, nature photonics

13 supplementary information SUPPLEMENTARY INFORMATION #2: FABRICATION OF SUPERHYDROPHOBIC SURFACES #2.A Fabrication process o micropillars covered with electroless silver nanograin (100) silicon wafers (from Jocam, Milan, Italy) were cleaned with acetone and isopropanol to remove possible contaminant and then etched with a 4% wet HF solution. The wafers were then rinsed with DI water and dried with N2. Standard optical lithography technique was employed to realize regular patterns of disks within a layer of positive resist (S1813, from Rohm and Haas) that was spin-coated onto clean silicon wafers. Electroless deposition techniques were employed to grow silver in form of grains within the holes. The electroless deposition is based on an autocatalytic or a chemical reduction of aqueous metal ions. This process consists of an electron exchange between metal ions and a reducing agent. In this work, Si substrate was used itself as reducing agent. A fluoridric acid (HF) solution containing silver nitrate (AgNO 3 ) was used, where Ag was reduced to metal form by the Si substrate oxidation. In particular, the patterned silicon wafer was dipped in a 0.15 M HF solution containing 1 mm silver nitrate for 60 s at a constant temperature T=313 K. After the growth process the silicon wafer was rinsed with water and dried under nitrogen flux. The driving force in this process is the difference between redox potentials of the two half-reactions, which depends on solution temperature, concentration and ph. Consequently, these parameters influence the particles size and density. Reactive Ion etching. After residual resist removal with acetone, a Bosch Reactive Ion Etching (MESC Multiplex ICP, STS, Imperial Park, Newport, UK) process was utilized to obtain the final structures that are cylindrical pillars having an aspect ratio greater than 5, diameters ranging from 5 to 15 µm and a heights larger than 20 µm, in accordance with the model introduced in Suppl. Info #1. The electroless grown Ag layer served as mask during the RIE process. The substrates, as a whole, were then covered with a thin (few nm) film of a Teflon like (C 4 F 8 ) polymer to assure hydrophobicity. The masks necessary for optical lithography were fabricated using standard Electron Beam Lithography (CRESTEC CABL- 9000C Electron beam lithography system) methods. Fig.S2.1 shows SEM images of microstructured surfaces. The cartoon in Fig.S2.2 briefly recapitulates the fabrication process. Fig.S2.1 Micropillars covered with silver nanograin assemblies nature photonics 13

14 supplementary information Fig.S2.2 Electroless and RIE Fabrication process of micropillars #2B fabrication of micropillars decorated with regular arrays of silver nanodots Optical lithography, Electron beam lithography, RIE and electroless techniques were used for the fabrication of the devices as in Fig.2c in the main text. (100) silicon wafers were cleaned with acetone and isopropanol to remove possible contaminant and then etched with a 4% wet HF solution. The wafers were then rinsed with DI water and dried with N 2 ; thereafter they were baked on a covered hot plate for 30 min at 170 C to remove residual humidity. A high resolution positive electron resist (PMMA 950k A2) was spin coated for 60 s at 4000rpm to obtain a ~50 nm thick layer of resist. Prior to the EBL exposure, the sample was pre-baked at 170 C for 2 min to evaporate the solvent from the resist. A regular pattern of dots spanning above an area of µm 2 was written on the sample using an EBL system, employing a 50 kev acceleration voltage. Disc-shaped structures were obtained using the spot-scan modality, with a dose of 150 C/cm 2. The sample was then immersed for 30 s in PMMA 950k developer (MIBK:IPA=1:4 v/v) to selectively remove the exposed resist. Si was then processed using reactive ion etching (RIE) in oxygen for 40 s to remove any residual resist from the discs. Arrays of empty discs with 80nm diameter and an interspatial gap ranging from 10 to 30nm were finally obtained. The successive Ag growth was accomplished using an electroless growth as described above. Due to resist patterning, silver grains could grow solely inside the holes. The remaining PMMA resist was removed with solvent. Grains assemblies have a 100nm diameter and 80nm thickness with interspatial gap of nm. AZ 5214 image reversal photoresist (MicroChemicals GmbH, Germany) was spin-coated onto the nano structured samples. Standard optical lithography techniques were employed to realize regular micro-patterns of AZ 5214 squares. A Bosch RIE process was utilized to etch the samples thus yielding, upon removal of the residual AZ 5214, prismatic Si micropillars decorated with regular arrays of Ag nanodots. Please notice that these regular patterns or nano structures induce, to different extents, a giant, local electromagnetic field and a consequent enhancement of the Raman signal. In Fig.S2.3 is reported the flow chart description of the process. Several sample were produced and SEM micrographs were taken to assess uniformity and reproducibility (Fig.S2.4). 14 nature photonics

15 supplementary information Fig.S2.3 Fig.S2.4 #2.C Fabrication micropillars combined with plasmonic nanocones Silicon micropillars were obtained as described in supplementary information #2.A. Ag nanograins were removed using binary mixture of HF:H20=1:4 v/v. The circular grating was nature photonics 15

16 supplementary information milled on the surface of the silicon micropillar by focused ion beam milling (DUAL BEAM (SEM-FIB) - FEI Nova 600 NanoLab, acceleration voltage 30 kv, ion beam current 500pA, polar coordinates, grating pitch 360 nm, depth 200 nm). Afterwards, a nanocone was growth on the top of the silicon tapered pillar by employing electron beam induced deposition (EBID) from a Platinum-based gas precursor. Finally, a thin layer of silver (40 nm) was deposited of the device by means of thermal evaporation. The apex of the silver tip has a radius of curvature of less than 10 nm. Fig.S2.5 #2.D FABRICATION OF Micropillars surrounding a plasmonic nanocone The body of the nanocone was obtained by tapering a silicon micro-pillar with Focused Ion Beam (acceleration voltage 30 kv, ion beam current 1nA, polar coordinates). A grating was milled on the side of the cone to allow an efficient laser coupling, and surface plasmon generation through the grating order -1: grating pitch 490 nm, depth 200 nm, number of lines 12. Afterwards, a nanocone was growth on the top of the silicon tapered pillar by employing electron beam induced deposition (EBID) from a Platinum-based gas precursor. The body of the cone is about 10 µm height, whereas the base is 2 µm large. Finally, a thin layer of silver (40 nm) was deposited of the device by means of thermal evaporation. The apex of the silver tip has a radius of curvature of less than 10 nm, and the vertex angle (about 7 ) was adjusted to be compatible with adiabatic compression of plasmons. 16 nature photonics

17 supplementary information Fig.S2.6 nature photonics 17

18 supplementary information SUPPLEMENTARY INFORMATION #3: SAMPLE PREPARATION AND CHARACTERIZATION TECHNIQUES #3.A Materials Rhodamine R6G, myoglobin, ribonuclease B, lysozyme and sodium chloride were purchased from Sigma; Lambda DNA was purchased from Fermentas (Genbank/EMBL accession number J02459). De-ionized (D.I.) water (Milli-Q Direct 3, Millipore) was used for all experiments. All chemicals, unless mentioned otherwise, were of analytical grade and were used as received. Rhodamine6G is an organic compound and is used extensively in biotechnology applications. It is a dye which can be observed very clearly by fluorescence microscopy. Its absorption and emission wavelength are 530 and 556 nm, respectively. Lysozyme is an enzyme able to damage bacterial cell walls by hydrolysis of the glycosidic bond in peptidoglycans and chitodextrins. In humans, it is part of the immune system and is present in a number of secretions such as tears, milk, saliva and mucus. A lack of the enzyme is related to bronchopulmonary displasia in newborns [1] and disease in infant fed with enzyme lacking formula [2]. Lambda DNA is the double stranded DNA of the Enterobacteria Phage Lambda. It is a virus bacteriophage that infects Escherichia coli. It is a base pair long flanked by 12 base single stranded sequence that make up the cos site matching 5 with 3 end. It is usually used as a model organism and as a useful tool in molecular biology laboratory. In particular it is used as a cloning vector, for the shuffling of the cloned DNAs in the Gateway method and in the method called "recombeenering". #3.B Methods Small drops (V<10 µl, R<1.35 mm) of D.I. water containing infinitesimal amounts of analytes were gently posted upon the surfaces and the entire process of evaporation followed over time. An automatic contact angle meter (KSV CAM 101, KSV INSTRUMENTS LTD, Helsinki, Finland) was used at room temperature. Notice that the energy of adhesion γ per unit area at the gas/water interface is ~72.8 mj/m 2 at 20 C. The process enabled to concentrate very tiny amounts of agents over micrometric areas. The evaporation processes were performed in a clean room to reduce the presence of external contaminants and lasted approximately 20 min. The residual solute was observed using scanning electron microscope (SEM), fluorescent microscopy and Raman spectroscopy techniques. Protocol for Lambda DNA Lambda DNA (Fermentas. Genbank/EMBL accession number J02459) comes with a concentration of 0.3 ug/ul (10 nm). A 1:100 dilution of the stock solution is necessary to make DNA combing on microscopy glass to clearly distinguish one molecule from the other. More concentrated solutions yield a green carpet of DNA covering the entire surface. Stained Lambda. For the single binding event experiments, 10 ul of Lambda (dilution at M) were stained with 10 ul of a 0.1 um freshly prepared YOYO solution (in PBS) (see protocol Invitrogen MP03600) for 1 hour at room T in the dark. (For the multiple binding 18 nature photonics

19 supplementary information event experiments, as in supplementary information #4, starting concentration of M was used). The solution was taken with the suitable syringe and put on the superhydrophobic pillars. The drop volume was about 2-3 ul. The drop volume is variable depending on the pillars structure. The drop was let de-wet in the dark for 20 min. Unstained Lambda. Following the same protocol as before, a drop of Lambda at same dilution was put on the pillars without staining. Background control. PBS solution. PBS YOYO solution. The sample was gold sputtered at 40 ma for 20 before SEM imaging. Fluorescence microscopy Characterization Fluorescence microscopy measurements were performed using an inverted microscope, with infinity-corrected optics (Nikon-ECLIPSE TE 2000-U). The microscope objectives used were a Plan Fluor 40x and a Plan Apo 60x, with 0.75 and 0.95 numerical apertures. For probe excitation a violet diode laser source emitting at 408 nm, an Argon source emitting at 488nm, Helium Neon laser source emitting at 543 nm were used. A Nikon D - Eclipse C1 scanning head with three channels was utilized for the measurements. Raman Characterization of Rhodamine deposits Micro-probed Raman spectra were obtained using Renishaw invia Raman microscope at room temperature through 20x objective of a Leica microscope. The Raman spectra were excited by the nm line of an Ar + laser in backscattering geometry. The laser power was 0.18 mw with an integration time of 50 s. Mapping Raman measurements were carried out with the step size µm and µm in x and y-axis direction, respectively. Raman Characterization of Lysozyme on cone Lysozyme M solution was deposited through a glass micropipette (World Precision Instruments) on top of the superhydrophobic device. The micropipette has a tip with an outer diameter of 1µm and it is driven by means of micrometer screws (microinjector from Eppendorf GmbH), thus allowing an accurate positioning over the plasmonic antenna. The whole operation was carried out under an optical microscope in order to have visual inspection of the deposition process. A microinjector with resolution in the 10 femtoliter regime was used to release the drops of solution on the super hydrophobic chip. Evaporation of the drop occurred in reduced time lapse (that is, few seconds) because of the small liquid volume. After evaporation, Raman measurements were performed over the plasmonic antenna, and also in the surrounding areas (both on top and at the bottom of the pillars) as control measurements. For all Raman spectra the laser light had wavelength of 514nm with 100 X objective, the power at the sample level was about 0.1 mw, and the accumulation time was 50 s. Measurements on the plasmonic antenna were recorded by focusing the laser light on the tip of the cone. Several measurements were also recorded along the cone axis at different heights below the tip. 1. Revenis ME, Kaliner MA, August "Lactoferrin and lysozyme deficiency in airway secretions: association with the development of bronchopulmonary dysplasia". J. Pediatr. 121 (2): Lönnerdal B, June "Nutritional and physiologic significance of human milk proteins". Am. J. Clin. Nutr. 77 (6): 1537S 1543S. 3. JC Kendrew, G Bodo, HM Dintzis, RG Parrish, H Wyckoff, and DC Phillips, "A Three-Dimensional Model of the Myoglobin Molecule Obtained by X-Ray Analysis". Nature 181 (4610): nature photonics 19

20 supplementary information SUPPLEMENTARY INFORMATION #4: λ-dna DEPOSITION AT DIFFERENT DILUTION #4.A single binding event detection The experiments heretofore described would prove that superhydrophobic devices deliver the ability to concentrate solutions with extremely high efficiency. Nevertheless, the recognition of one single binding event would ultimately establish the novelty and adequacy/efficiency of these devices. In this context, lambda DNA solutions were analysed. When the concentration is sufficiently low (10-18 M), single molecules may be detected: single double stranded Lambda DNA molecule (48502 bp) keeps stretched on top of two pillars. Fig.4 in the main article, and a collection of SEM images reported in Fig.S4.1 show this. Fig.S4.1 #4.B multiple binding event detection When the initial concentration of the lambda DNA solution gets higher, more filaments are likely to be detected upon evaporation. Fig. S4.2 shows two DNA filaments connecting 3 pillars as to form a row. For the present configuration the initial concentration is M. Increasing the concentration (10-16 M) yields an arrangement as in Fig.S5.3. Here, multiple filaments thread the pillars over a large area as to form a dense λ-dna network (Fig.45.3). Fig.S nature photonics

21 supplementary information Fig.S4.3 In some cases, intricate network of DNA complexes are created: DNA filaments combine and overlap yielding multifaceted complexes where different diameters are observed. This is evidenced in Fig.S4.4, where three or more filaments cross link simultaneously in the same point (concentration 1fM). Fig.S4.4 nature photonics 21

22 supplementary information Supplementary information #5: FDTD simulation Three-dimensional Finite difference time domain (FDTD) calculations were performed to optimize surface plasmons generation, propagation and accumulation at the tip end. Simulations were carried out employing commercial software specifically developed to FDTD calculation of photonic structure ( A sketch of devices cross section is reported in the figure below, where the main geometrical parameters are also indicated. As showed, the overall simulation volume was divided in two regions in order to save simulation time, required memory, and to improve accuracy. The bottom region comprehends the body of the cone and the metallic grating, and it takes the most part of the simulation volume (3x3x8 µm 3, minimum mesh size 4 nm). The top region comprehends the final part of the nanocone, and being much smaller allows a fine mesh refinement (volume 0.5x0.5x0.2 µm 3, minimum mesh size 0.5 nm). First, a simulation was run in the bottom region to calculate the generation of SPPs through the coupling between the incoming laser and the metallic grating. SPPs travelling toward the tip were stored in a monitor placed at the boundary of the two regions. The stored electric field was then used as a source to run a second simulation involving the top region. When the radius of curvature of the tip is 10 nm, the calculated electric field enhancement is in the order of 30 with the respect of the incident laser amplitude. Simulation details. Source: 532 nm laser, continuous pulse, focused on the metallic grating through a 0.9 Numerical Aperture objective. Simulation time 100 fs (time step 0.01 fs). Simulation volume: bottom region 3x3x8 µm 3, minimum mesh size 4 nm; top region 0.5x0.5x0.2 µm 3, minimum mesh size 0.5 nm Index of refraction of the materials: Palik database. Grating details: pitch=480 nm; depth= 200 nm; order=-1; Ag= 40 nm thick. 22 nature photonics

23 supplementary information nature photonics 23