Near-Field Lithography by Two-Photon Induced Photocleavage of Organic Monolayers

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Near-Field Lithography by Two-Photon-Induced Photocleavage of Organic Monolayers
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By Marta Álvarez , Andreas Best , Andreas Unger , José M. Alonso , Aránzazu del Campo ,
Marcus Schmelzeisen , Kaloian Koynov , and Max Kreiter *

We prove that the enhanced electromagnetic near-fi eld around metallic nanostructures can be used for localized two-photon-induced activation of surfaces, obtaining a defi ned chemical pattern with nanometric resolution. Gold nanoparticles (Au-NP) are deposited on glass slides that were modifi ed with a polysiloxane layer containing a nitroveratrylcarbonyl (NVoc) photore-movable group. Upon illumination with a femtosecond laser, the NVoc entity is removed. Due to the electromagnetic fi eld enhancement of the nano-particles, the threshold of this process is lowered in the nm-scale vicinity of the metal structures. Upon cleavage, an amine functional group is released, which can be used to site-selectively bind species with complementary chemical functionality on the surface. This method can be utilized for sub-wavelength chemical structuring.

1. Introduction

The interaction of electromagnetic radiation with noble metal particles much smaller than the incident wavelength induces an oscillation of the conduction electrons of the metal, which is called localized surface plasmon (LSP). [ 1–4 ] As a result, an optical resonance (LSPR) appears at specifi c wavelengths that can be detected using UV–vis/NIR extinction spectroscopy. The spectral position and magnitude of the LSPR depends on the size, shape, composition, and local dielectric environment of the particles. These oscillations of the conductive electrons can induce very high local charge accumulations and thus strong enhancement of the electromagnetic fi eld in the prox-imity of the particle. These enhanced near-fi elds are spatially confi ned close to the particle, typically within 10 to 100 nm

© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheiAdv. Funct. Mater. 2010, 20, 4265–4272

DOI: 10.1002/adfm.201000939

[∗] Dr. M. Álvarez , Dr. M. Kreiter Max Planck Institute for Polymer Research Ackermannweg 10, 55128 Mainz (Germany) E-mail: [email protected] A. Best , Dr. A. Unger , Dr. A. del Campo , M. Schmelzeisen , Dr. K. Koynov Max Planck Institute for Polymer Research Ackermannweg 10, 55128 Mainz (Germany) Dr. J. M. Alonso [+]

Max Planck Institute for Metal Research Heisenbergstrasse 3, 70569 Stuttgart (Germany)

[+] CIC nanoGUNE Consolider, Tolosa Hiribidea, 76, 20018 San Sebastián (Spain)

from the metal surface depending on the structure. Because of the enhancement of the radiation in the near fi eld, the metallic nanoparticles have been inten-sively studied in the last years for their application in different fi elds such as chemo- and biosensing [ 5 , 6 ] and near fi eld lithography. [ 7 ]

In near fi eld induced photolithography light can be applied for sub-wavelength structuring because feature sizes in the near fi eld are not diffraction-limited. Such processes require very small dis-tances (below half of the applied wave-length) between the light source (or light-forming element such as mask or structure) and the light-sensitive entity.

Structure generation can be based on photochemistry or on ablative processes. Photochemically, the near-fi eld distribution has been imaged by using a photoresist or an azobenzene-dye polymer. In the former case, the exposure of the resist to the radiation induces a change in the polymer structure which leads to changes in the solubility and therefore in the topog-raphy of the polymer after development. The irradiation can be performed through a mask [ 8–11 ] or from nanoparticles plas-mons’. [ 7 , 12 ] For azo-dye functionalized polymers, irradiation induces a conformational change between the trans and the cis isomers of the azo-group. As a consequence mass trans-port takes place which induces a pushing or pulling of the polymer, creating topographic modifi cations of the polymer fi lm surface. [ 13–15 ] These topographic features were recorded directly by atomic force microscopy without the need of an extra chemical treatment. In typical ablative experiments nanoparticles are placed on substrates such as silicon or glass, and then the system is irradiated with a high-power short-pulse laser. In the positions with high fi eld enhancement, substrate material is removed. [ 16 , 17 ]

Nitroveratryloxycarbonyl (NVoc) is a photolabile pro-tecting group [ 18 ] that has been used to cage organic func-tional groups (such as carboxy, hydroxy or amino) at surface layers. [ 19 ] Upon irradiation the NVoc group under-goes an intramolecular redox reaction and is cleaved, leaving the reactive group on the surface which allows for further selective chemical functionalization [ 19–21 ] offering in this way a new and useful advantage in comparison to the other methods of near-field structuring described in the literature. Photocleavage of the NVoc at the surface

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Figure 1 . a) Strategy for optical structuring on the 10-nm scale: 1) irradiation of noble metal nanoparticles with femtosecond laser pulses inducing the removal of the photosensitive NVoc group from the silane layer; 2) attachment of nanometric objects to the photoactivated areas; 3) deposition of colloids on non-activated areas. b) Scheme of the photocleavage of the photoreactive surface modifi cation agent, R-TEG-NH-NVoc, bearing an NVoc caging group and decoration of the irradiated areas (with free NH 2 groups) with 1) biotin-NHS and 2) Streptavidin-Cy5 dye. c) UV–Vis spectra of a quartz substrate after surface modifi cation with R-TEG-NH-NVoc. d) Fluorescence image of a masked-illuminated R-TEG-NH-NVoc modifi ed substrate after reaction with a fl uorophore.

has been achieved using single [ 18 , 19 ] ( λ = 365 nm) and multiphoton excitation [ 22 ] ( λ = 780 nm). Here we present a simple method for both near-fi eld diagnostics and nanolithography, which is performed by combining gold nanoparticles with a polysiloxane layer containing NVoc. Structures written in monolayers with photosensitive moieties are not blurred by diffusion of radicals or movement of polymer chains, which are the typical problems which arise by working with polymers. The NVoc group shows an absorption maximum in the UV region ( λ max = 345 nm), whereas the optical resonances of small metal objects are typically observed in the red part of the visible spectrum and in the near infrared. [ 1 ] Thus, in order to match the spectral ranges, a two-photon excitation process is needed for the cleavage of the photosensitive moiety from the silane protected amines. Two-photon cleavage of the NVoc was achieved at localized regions close to the nanoparticles where the near-fi eld was enhanced and proteins were bound to these positions. Our results provide a direct proof of prin-ciple for sub-wavelength chemical structuring of surfaces.

2. Results and Discussion

The central idea of this work is to show that light triggered processes at the surface require lower energy thresholds when they occur close to metallic nanoparticles (NP) due to near-fi eld enhancement effects. The area of infl uence of these metallic NP is limited to some tens of nanometers, allowing therefore for sub-wavelength patterning. These effects will

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be proven using a decoration method that enables contrast between affected and non-affected areas.

Figure 1a represents the general sub-wavelength pat-terning strategy: upon illumination at 780 nm the elec-tromagnetic near-fi eld of the noble metal nanoparticles is expected to induce, by a two-photon process, photoac-tivation of a surface layer by cleavage of the photolabile NVoc (see Figure 1a , step (1)). Since the near fi elds decay on typical length scales of 10–100 nm, [ 1 ] deprotection is expected to happen on this length scale far below the diffraction limit. After selective decoration of the depro-tected areas by reacting the activated groups with a com-plementary molecule (Figure 1a , step (2)), or of the non-deprotected areas with colloidal particles (Figure 1a , step (3)), true sub-wavelength structuring is revealed. In the following, we provide experimental proof for these processes.

2.1. Photocleavage of Silane Layers Without Metal Nanoparticles

2.1.1. Properties of Photocleavable Silane Layers

As a fi rst step the conditions in our system for the photo-cleavage of NVoc by one and two photons – for systems in the absence of metallic nanoparticles – will be presented.

Quartz slides were modifi ed with a tetraethylene glycol (TEG) triethoxysilane with a terminal amino group protected by nitroveratryl (NVoc). Its structure is shown in Figure 1 b [ 23 ]

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Figure 2 . Fluorescence microscopy image of a biotin-NHS + SAv-Cy5 decorated R-TEG-NH-NVoc silane sample after Ti:Sa femtosecond-laser patterning at 780 nm. Each square corresponds to a specifi c laser inten-sity and irradiation time. Square size: 45 μ m × 45 μ m. The values of D 2p−t indicated are D 2p−t = (6%) 2 · 62 s = 2232% 2 s, D 2p−t = (12%) 2 · 31 s = 4464% 2 s and D 2p−t = (25%) 2 · 8 s = (50%) 2 · 2 s = 5000% 2 s.

and it will be abbreviated in the following as R-TEG-NH-NVoc. The UV-Vis absorption spectrum of quartz substrates modifi ed with R-TEG-NH-NVoc is shown in Figure 1c . From the absorb-ance value at λ max = 345 nm it is possible to calculate the den-sity of the chromophore at the surface assuming that the molar absorption coeffi cient in solution ( ε 345 = 3028 M − 1 cm − 1 ) is the same as at the surface. [ 24 ] Under this assumption, a surface density of ∼ 3 × 10 12 molecules/mm 2 was obtained. This value is comparable to the typical surface density of a self-assembled monolayer (SAM) of thiols on gold with maximum coverage (4.5 × 10 12 molecules/mm 2 ). [ 25 ]

2.1.2. One-Photon Photocleavage

In the fi rst place the conditions for the NVoc removal from the surface in a 1-photon process will be tested. Also, the condi-tions for a selective decoration in the areas where cleavage took place will be established.

Photocleavage of the chromophore was performed at λ = 365 nm through a mask. Exposure to this wavelength is expected to render amino groups on the surface (see Figure 1b ). In order to identify the areas where deprotection took place, the sample was fi rst treated with biotin-NHS (Figure 1b , step (1)) which is expected to covalently bind to the free amino groups. In the second step the sample was treated with a solution of Cy5-labelled streptavidin (Figure 1b , step (2)) -which binds to the biotin- and then a fl uorescence microscopy image was recorded (Figure 1d ). The areas which were irradiated could be identifi ed because they showed higher fl uorescence intensity. This indicates that the photolysis reaction succeeded and that the decoration process allowed identifi cation of deprotected areas.

2.1.3. Two-Photon Photocleavage

As the next step, the cleavage of the NVoc by a two-photon process is performed in order to quantify the response of the NVoc cleavage at different energy doses in the absence of metallic nanoparticles.

Two-photon deprotection of the R-TEG-NH-NVoc surface layer (samples that will be refereed to as “silane only”) was tested by exposing the substrate to a Ti:Sa laser at 780 nm. In order to identify the energy threshold for the cleavage, several squares of 45 μ m × 45 μ m were written by raster scanning forming an array using different average intensities (I) (from ∼ 4 to 148 mW, which correspond to an energy per pulse in the range from 0.06 to 2 nJ pulse − 1 ) and irradiation times (t) (between 2 and 62 s). In this way, each square corresponds to a two-photon dose defi ned as D 2p = I 2 t. [ 22 ] After irradiation, the sample was labeled with the biotin-SAv system in order to deco-rate the free amino groups on the surface.

Figure 2 shows the obtained fl uorescence pattern. A clear transition from a low dose zone where no fl uorescent pattern was detected (top left), to a high dose zone with strong fl uores-cent squares (bottom right) could be observed. With the aim of evaluating what kind of process was responsible for the NVoc cleavage, the fl uorescence contrast (between illuminated area and the surrounding regions) was plotted versus I · t (one-photon), I 2 · t (two-photon) and I 3 · t (three-photon) (see

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Supporting Information, Figure S2) analogous to the analysis described by Alvarez et al. [ 22 ] The graph of contrast versus I 2 · t is by far the best description of the experimental data, indicating a two-photon deprotection.

The threshold dose values for the two-photon cleavage (D 2p−t ) are obtained from the dose used to irradiate those squares where there is a minimum perceptible but clear increase of the fl uorescence with respect to the fl uorescence background. These squares are marked in Figure 2 by arrows. The threshold for the different intensity and time combinations is consist-ently between 2000–5000% 2 s (see fi gure caption for exact values). This variation within a factor of around two is accept-able, since in this article changes by orders of magnitude are investigated.

2.2. Photocleavage in the Presence of Gold-Nanoparticles

2.2.1. Photocleavage in the Presence of Gold-Discs

The next step is to study the effect of the incorporation of metallic nanoparticles to the silane layer samples on the NVoc photocleavage threshold doses. First, a system with gold-disc structures will be investigated.

The inset in Figure 3a presents a scanning electron microscopy (SEM) image for glass-supported gold-discs. The particles, prepared by nanosphere lithography, are ∼ 40 nm high and ∼ 200 nm in diameter and have a random distribution on the surface, typical for the fabrication process (see Experimental).

Figure 3a also shows their extinction spectrum which exhibits a main resonance peak around 780 nm. This peak can be associated to a dipole-like response, which is the simplest resonance mode that a structure can support. [ 26 ]

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Figure 3 . Extinction spectrum of a sample with glass-supported Au-discs (a). The inset shows an SEM image of the sample. Fluorescence micro-scopy image for a silanized sample with Au-discs, irradiated at 780 nm and decorated with biotin-NHS + Streptavidin-Cy5 (b). The values of D 2p−t indicated are D 2p−t = (3%) 2 · 31 s = 279% 2 s and D 2p−t = (12%) 2 · 2 s = (6%) 2 · 8 s = 288% 2 s. For the irradiated squares within the white frame an inversion of the fl uorescence contrast appears.

Glass substrates with the Au-nanostructures were modifi ed with R-TEG-NH-NVoc. These samples will be referred to as “disc-silane samples”. They were irradiated with the Ti:Sa laser (array writing) and then fl uorescently labeled (biotin-NHS + SAv-Cy5) (see Figure 3b ) as specifi ed before. The threshold values for deprotection in this case were D 2p−t ∼ 280% 2 s (squares marked with the arrows in Figure 3b ). A lowering of the threshold by around one order of magnitude in compar-ison with the ones observed on silane layers in absence of the nanoparticles is obtained. This indicates that there is a signifi -cant enhancement of the laser radiation mediated by the Au-nanostructures. Further images of the same experiment for other samples showing similar results for the D 2p−t are presented in the Supporting Information.

For the high dose values used in this experiment ( ∼ 20000% 2 s and higher), an inversion of the contrast in the fl uorescent pattern was observed (Figure 3b , region within the area marked with dashed lines) which was not seen for the “silane-only” samples. SEM and AFM measurements showed that a strong deforma-tion of the metallic nanoparticles had occurred at high expo-sure doses (Supporting Information Figure S4 and Figure S5).


Disc deformation and the inverted contrast in the fl uorescence at high exposure dose occurred at the same threshold value, between 9000 and 29000% 2 s, around 2 orders of magnitude higher than the threshold for NVoc deprotection. Therefore, we conclude that the change in the shape of the metal objects could be responsible for the contrast inversion for the fl uorescence image at the higher values of I 2 · t . A more detailed discussion about how the disc deformation might alter the fl uorescence contrast is given in the S.I. For the disc-silane samples, the focus of this manuscript lies entirely on doses below 500% 2 s where these problems do not occur.

2.2.2. Photocleavage in the Presence of Gold Crescents

Next, a system with glass-supported gold-crescents is dis-cussed. The crescents have a more complicated structure than the discs, they possess sharp corners and are prone to induce a higher near fi eld enhancement than the discs presented in the previous section. Therefore they are expected to render even lower threshold values for the two-photon deprotection proc-esses than the ones obtained up to this moment.

The inset in Figure 4a presents a typical scanning electron image for glass-supported Au-crescents. The particles have a random distribution on the surface with identical orientations, which is typical for the fabrication process by colloidal lithog-raphy. [ 27 ] They are ∼ 40 nm in height and ∼ 115 nm in diameter.

Figure 4a shows the corresponding nonpolarized extinction spectrum. A peak at 560 nm corresponding to very small gold particles is observed. [ 28 ] The two peaks at ∼ 850 and ∼ 1140 nm correspond to the standing waves along the contour of the crescents. A sketch of the corresponding charge distribution is given in Figure 4a . More details about the resonances of these nanostructures were reported by Rochholz et al. [ 29 ]

The same experiment as for the disc-silane samples (silani-zation, array writing and dye decoration) was also done for crescent-silane samples. The arrows in Figure 4b show the two-photon dose threshold, with visible increased fl uorescence from the squares (in comparison with the background). Values of D 2p−t between 20–30% 2 s were found.

2.3. Comparison Between Systems

In Figure 5 , the two-photon cleavage thresholds doses (D 2p−t ) for the three different systems studied (samples only with silane, samples with discs and silane, and samples with crescents and silane) are summarized. For each system the D 2p−t values for two different samples are presented. The multiple symbols for each sample (for every system) represents each of the threshold values indicated with the white arrows in the fl uorescence threshold images. The silane without nanoparticles presents thresholds D 2p−t above 1000% 2 s. With Au structures these values are clearly reduced by more than one order of magni-tude for the system with the discs (D 2p−t ∼ 280% 2 s), and by two orders of magnitude for the system with the crescents (D 2p−t ∼ 30% 2 s). This confi rms our hypothesis that the crescents are more effi cient in the enhancement of the photochemical depro-tection, most probably due to the presence of sharp tips.


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Figure 4 . Extinction spectrum of gold crescents on glass and sketch of the crescents with the corresponding charge distribution for resonance at ∼ 850 and at ∼ 1140 nm (a). The inset shows an electron microscopy image of the crescents. Fluorescence microscopy image for a silanized sample with Au-crescents, irradiated at 780 nm and decorated with biotin-NHS + Streptavidin-Cy5 (b). The values of D 2p−t marked are D 2p−t = (3%) 2 · 2 s = 18% 2 s and D 2p−t = (1%) 2 · 31 s = 31% 2 s. For the irradiated squares within the white frame a decrease of the fl uorescence contrast appears.

Figure 5 . Comparison of the doses for the two-photon thresholds (D 2p−t ) for silanized samples without metallic nanoparticles, with Au-discs and with the Au-crescents.

Small variations between the samples of the same system that were treated nominally identically might be due to differ-ences in the amount of silane on the surface, resulting in a better or worse contrast. It is important to note that identical values are found for different samples which were studied for each case.

2.4. Fluorescence Imaging of Individual Particles

The experiments described up to this moment could only provide an indirect proof that a localized photocleavage is happening around the metallic nanoparticles. For this reason, in order to check where upon irradiation with the Ti:Sa laser the depro-tection on the substrate with nanoparticles is taking place, fl uorescence images using a customized scanning confocal optical microscope [ 30 ] were taken ( λ exc = 633 nm) as this micro-scope provides a higher lateral resolution. The results are pre-sented in Figure 6 . In the upper row, microscopy images from

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the sample without metal structures (“silane only”) taken with the Zeiss microscope are shown (a, c, e, g). Below fl uorescence images from a sample with gold discs – the same sample as presented in Figure 3b -obtained with the customized confocal microscope are displayed. Both rows show sample regions which contain non-irradiated areas and a part or the complete 45 × 45 μ m 2 square which was exposed to different two photon doses and all substrates were decorated in the same way. The squares depicted in each column were irradiated to identical D 2p . In the lower row, only a part of the irradiated squares near the border is depicted, but enough to enable comparison to the non-irradiated surrounding. In the images of the disc-silane samples, the individual bright spots correspond to the indi-vidual metallic structures.

For a dose of 72% 2 s there is no contrast between inside and outside the irradiated area, which agrees with the observation in Figure 3b and Figure 2 that neither with nor without parti-cles any fl uorescence increase is seen. At a dose of 1100 s% 2 for the sample without nanoparticles (Figure 6c ), no contrast between irradiated and non-irradiated areas is observed, as this dose is still below the deprotection threshold (D 2p−t ) without metallic NP. At this dose, for the disc-silane sample (Figure 6d ), inside the square brighter dots and an unchanged background are seen. This is interpreted as a proof for the selective depro-tection of silanes next to the metal nanoparticles (and conse-quently more labeled-protein around the discs), mediated by the enhanced near-fi eld.

For a dose of 9000 s% 2 the regime where deprotection hap-pens already without nanoparticles is entered: a weak square is seen on the sample without discs (Figure 6e ). At this D 2p in the sample with the Au-discs (Figure 6 f) not only brighter dots are observed, but also the background fl uorescence between the individual objects inside the square increases with respect to non-irradiated areas: there was enough energy to deprotect the silane even without metallic nanoparticles.

For high irradiation doses (D 2p = 620000 s% 2 ) the same effects as for D 2p = 9000 s% 2 are found, but more pronounced: there is a higher fl uorescence from the nanoparticles and also more in the whole area in the irradiated square (Figure 6 h). This is also shown in the corresponding fl uorescence image of the sample without Au-NP (Figure 6 g), where strong fl uores-cence is seen.

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Figure 6 . Fluorescence images for silanized sample without (upper row) and with Au-discs (lower row) irradiated with different intensities and time conditions (two-photon dose indicated for each column). For clarity an intensity color scale was chosen in the lower row. The brighter spots in the lower row correspond to the discs. The insets in both sets of rows represents the scanned area with the fl uorescence microscope and the regions in red or green color are the scanned areas which were irradiated with the Ti-Sa laser.

2.5. Proof of Sub-Wavelength Lithography

Fluorescence decoration works nicely and shows good con-trast, but it does not allow sub-wavelength resolution like other methods such as atomic force microscopy or electron micro-scopy and therefore no proof of subwavelength structuring by fl uorescence is possible. For this reason, in order to dem-onstrate that upon Ti:Sa irradiation optical deprotection of the photosensitive group took place at sub-wavelength scales nearby the metallic nanoparticles, irradiated samples were dec-orated with Au colloids so that they selectively attach only to deprotected or to non-deprotected silane in order to be able to map where cleavage happened. This procedure is a less simple decoration method but decoration can be visualized with the scanning electron microscope. For this purpose, a crescent-silane sample was irradiated at 780 nm with different doses (D 2p ) and then incubated in an aqueous suspension of ∼ 20 nm mean diameter Au colloids with a citrate shell (further details in Experimental Section). In Figure 7 corresponding electron microscopy images are shown.

In Figure 7a the border between a strongly irradiated area (D 2p = 310000% 2 s) and the non-irradiated background is


shown. Colloids preferentially adsorb to the non-irradiated area where NVoc group is present on the surface (left part of the image), whereas the irradiated area where an amine has been generated (right part), is almost free from Au-colloids. The better affi nity of the colloids for the non-irradiated area means that there is an inverted contrast, rendering the decoration method not suitable for structuring, but as the method offers a good contrast between both types of regions is an appropriate one for the proof of principle of sub-wavelength structuring.

In regions where the irradiation dose was 0 or very low, Au-colloids are everywhere on the surface (indicating that there is no NVoc cleavage) even on the crescents. This is the case for Figure 7b and c. Doses around 60% 2 s correspond to the onset of an effect for the fl uorescence staining (Figure 4b ). For this dose no particles on top of the crescents are seen (Figure 7d ).

For doses around 500% 2 s we anticipate from the fl uores-cence investigations deprotection around the crescents and no deprotection of the rest of the silane on the substrate. Figure 7e shows a decoration which avoids a small area around the metal ( ∼ 60 nm length) and no sign of crescent destruction. This clearly indicates that the crescents enhanced the incident radia-tion and induced the deprotection of the silane in their close


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Figure 7 . SEM images from a silanized Au-crescents glass-supported sample irradiated in different areas with increasing doses and treated with Au-citrate colloids. The corresponding two-photon dose ( abbreviated in this fi gure as D ) for each region of the sample is indicated in the image.

environment. This proves that clear sub-wavelength dimen-sions are written optically in the silane monolayer.

For higher doses (Figure 7 f, g, and h) two effects are observed: First the overall particles coverage on the irradiated areas dimin-ishes with the dose and second the area around the Au-crescents where there are no particles becomes larger. On top of that, there is a clear crescent deformation for doses ≥ 9000% 2 s, matching also with the decrease in the fl uorescence intensity (Figure 4b ).

For very high two-photon doses (D 2p = 310000% 2 s) a full deprotection is anticipated. This behaviour is seen in the right part of Figure 7a , where no Au particles are bound.

These results obtained with the Au-colloid decoration are fully consistent with the ones obtained using the dye as label, and in addition prove that deprotection occurs in the near-fi eld on a scale of 60 nm.

3. Conclusions

Femtosecond laser irradiation of glass-supported gold nano-particles surrounded by a photolabile polysiloxane layer has

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demonstrated that the photoreaction only occurred in regions in the 10-nm proximity of the nanoparticles by tuning the two-photon dose. These results prove that this strategy enables photolithographic patterning beyond the optical dif-fraction limit. The cleavage dose of a photosensitive moiety by a two-photon process was reduced between 1 and 2 orders of magnitude in the presence of the nanoparticles. Using a photo-cleavable caging group, amino groups were photogenerated on the surface. This opens the possibility of covalent attachment of a big variety of chemical compounds on localized areas, and create sub-wavelength patterns, e.g., of active proteins or syn-thetic functional polymers.

4. Experimental Section Crescent-Shaped Structures : Crescent-shaped noble metal nanoparticles

were prepared via a nanosphere lithographic method. [ 27 ] A glass substrate (object slide, Menzel Glaeser, Germany) was cleaned fi rst by sonication for 10 min in an aqueous detergent solution (2% Hellmanex, Hellma), rinsed with ultrapure water (Milli-Q, Millipore), then sonicated in ethanol (Chromasolv, Sigma-Aldrich) for another 10 min, and blown dry with a nitrogen stream. Polystyrene nanospheres (Poly-styrene Nanobead: NIST, Polysciences, 1% w/v in aqueous suspension), 100 nm in nominal diameter, were diluted with water to 1:100 (v/v) and then were randomly dispersed on the glass with a micropipette. A ∼ 1 nm thick chromium fi lm followed by a ∼ 40 nm thick gold fi lm were evaporated (Auto 306, Edwards, Sussex UK) on the colloid-covered substrates, the samples being tilted 30 ° relative to the metal source. After the metal deposition the gold-coated samples were etched by exposing them to an argon ion beam (RR-I SQ76, Roth&Rau, Wüstenbrand, Germany) inciding perpendicularly to the substrate. After the etching process the colloidal mask was removed mechanically by means of an adhesive tape (Scotch Magic Tape 810: 19 mm × 33 m, 3 M France) and a second etching step was applied to ensure the removal of any extra sputtered material from the surface.

Disc-Shaped Structures : Disc-shaped noble metal nanoparticles were prepared according to a similar nanosphere lithographic method. [ 26 ] First the glass substrates were cleaned by sonication for 15 min in a detergent solution (2% Hellmanex, Hellma), rinsed with ultrapure water (Milli-Q, Millipore), then sonicated in ethanol (Chromasolv, Sigma-Aldrich) for 10 min, and dried in oven at 80 ° C for 30 min. Then a ∼ 0.6 nm thick chromium fi lm and a ∼ 40 nm thick gold fi lm were evaporated (Auto 306, Edwards, Sussex UK) on the glass substrates, the samples being perpendicular to the metal source. Polystyrene nanospheres (Poly-styrene Nanobead: NIST, Polysciences), 150 nm in nominal diameter, were randomly dispersed on the metal layer with a micropipette. A 1:100 (v/v) aqueous dilution of the original polystyrene nanosphere suspension was used. After the colloid deposition the gold-coated samples were etched by exposing them to an argon ion beam (RR-I SQ76, Roth&Rau, Wüstenbrand, Germany) inciding perpendicularly to the substrate. After the etching process, the colloidal mask was removed fi rst by means of an adhesive tape (Scotch Magic Tape 810: 19 mm × 33 m, 3 M France) and then by plasma treatment (Technics Plasma GmbH) for 5 min at 300 W (0.2 mbar O 2 ).

Synthesis of R-TEG-NH-NVoc Silane : Silane containing nitroveratryl (NVoc)-caged amine groups and protein repellent tetraethylene glycol units (this silane will be abbreviated as R-TEG-NH-NVoc) was synthesized according to the procedure described by Alonso et al. [ 23 ]

Silanisation : The glass slides decorated with discs and crescents were sonicated fi rst in a THF solution for 5 min, rinsed with ultrapure water (Milli-Q, Millipore), then sonicated in ethanol (Chromasolv, Sigma-Aldrich) for another 10 min, blown dry with a nitrogen stream, and fi nally activated by plasma treatment for 10 min at 300 W (gas ratio of 0.9 mbar Ar/0.1 mbar O 2 ). Immediately after these steps, the substrates were silanized by dipping them in a solution of the

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R-TEG-NH-NVoc protected silane in toluene (Aldrich) ( ∼ 40 μ L in 30 mL toluene) overnight in an oil bath at 80 ° C. Then the substrates were thoroughly rinsed with THF (Chromasolv, Sigma-Aldrich) and ethanol.

UV–Vis Spectra : UV–vis spectra were recorded on a Perkin Elmer Lambda 900 UV/VIS/NIR spectrometer.

UV lamp : For substrate irradiation, a 350 W mercury lamp from a MJB3-UV400 mask aligner from Süss Micro Tec (lines at 365, 405 and 435 nm) was used. The patterns for one-photon deprotection were created by illuminating at 365 nm through a chromium-coated quartz mask containing squares of 10 μ m × 10 μ m and 30 μ m spacing.

Dye Decoration : For dye decoration, the samples were immersed fi rst in a biotin N-hydroxysuccinimide ester (biotin-NHS) solution in DMF (Aldrich) with a concentration ∼ 60 mg/mL for 2 h, then rinsed and sonicated in DMF; then rinsed with THF and with ultrapure water. To the biotin, a streptavidin labeled with the cyanine dye Cy5 ( λ exc = 633 nm; λ em = 670 nm, Catalog Number 19–4317, Natutec) was bound in a phosphate buffered solution (PBS) at pH = 7.4 for 2 h, and then rinsed PBS and fi nally with ultrapure water.

Colloidal Gold Decoration : Gold particles were synthesized by heating a 0.25 mM aqueous solution of hydrogen tetrachloroaurate (III) trihydrate to the boiling point and then adding a 85 mM aqueous solution of trisodium citrate dehydrate following a procedure by Frens. [ 31 ] They have a mean diameter of 20 nm as determined by scanning electron microscopy. The substrates were immersed in an aqueous suspension of gold nanoparticles for 3 hours and afterwards rinsed with ultrapure water.

Confocal Microscopy : We used a commercial confocal laser scanning microscope setup (Carl Zeiss, Jena, Germany) consisting of the module LSM 510 and an inverted microscope model Axiovert 200. In all experiments described below we employed a Plan-Neofl uar 20x objective (Carl Zeiss, Jena, Germany) with a numerical aperture (NA) of 0.5 and a working distance of 2 mm. The Cy5 decorated samples were imaged by excitation at a HeNe laser wavelength of 633 nm and using a LP650 long pass emission fi lter.

Two-Photon Patterning : The two photon absorption patterning was done using a titanium:sapphire laser (Ti:Sa Mai Tai, Spectra Physics Inc., USA) coupled to the confocal microscope setup described above. This laser is tunable from λ = 780 nm to λ = 920 nm and provides ∼ 100 fs pulses at a repetition rate of 80 MHz. The laser light was tightly focused by the microscope objective on the polysiloxane-coated substrate to a spot with a diameter of ∼ 1 μ m. As the NVoc has its absorption maximum around 350 nm, the lowest available laser wavelength of 780 nm was selected for the 2-photon patterning experiments. The maximum time-averaged laser power in the object plane at this wavelength was about 148 mW, corresponding to a pulse energy of roughly 2 nJ. Using the galvanometric mirrors of the LSM 510, the focal spot was continuously raster-scanned (512 lines) on the substrate surface, creating squared illuminated patterns with a typical size of 45 μ m × 45 μ m. Both the total exposure time and the applied laser power were varied in order to study the effect of these parameters on the deprotection.

Atomic Force Microscopy : The topography and phase of the samples were investigated before and after the laser irradiation by atomic force microscopy (AFM) in air at room temperature with a commercial AFM (Nanoscope IIIa,Veeco) in tapping mode. Silicon cantilevers (Olympus) 160 μ m long, 50 μ m wide, and 4.6 μ m thick, with an integrated tip of a nominal spring constant of 42 N/m and a resonance frequency of 300 kHz were used. The tip was scanned at rates of about 0.4 Hz for scan sizes ∼ 2 μ m and minimal applied forces were used.

Scanning Electron Microscopy : Measurements were performed with a Zeiss-LEO Gemini 1530 with a resolution of 5 nm.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

© 2010 WILEY-VCH Verlag Gwileyonlinelibrary.com

Acknowledgements We thank Natalie Horn, and Noelia Bocchio for the help with the glass-supported gold-nanoparticles production.

Received: May 11, 2010 Published online: October 15, 2010

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