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Nanostructured substrates for high density protein arrays
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Microelectronic Engineering 85 (2008) 1370–1374 www.elsevier.com/locate/mee
Nanostructured substrates for high density protein arrays Frank A. Zoller a,b, Celestino Padeste a,*, Yasin Ekinci a,c, Harun H. Solak a, Andreas Engel b a Laboratory for Micro- and Nanotechnology, Paul Scherrer Institut, 5232 Villigen, PSI, Switzerland Maurice E. Mu¨ller Institute, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland c Laboratory for Metal Physics and Technology, ETH Zu¨rich, Wolfgang-Pauli-Str. 10, 8093 Zu¨rich, Switzerland b
Received 25 September 2007; received in revised form 14 December 2007; accepted 23 December 2007 Available online 12 January 2008
Abstract Nearly defect-free arrays of several 104 gold dots of 12 ± 3 nm in diameter inside 10 nm deep cavities were fabricated for immobilization of proteins. Extreme ultraviolet interference lithography (EUV-IL) at the XIL-beamline of the Swiss Light Source was used to produce 140 nm period arrays of 50 nm holes in a 40 nm thick PMMA layer on oxidized silicon wafers. The size of the openings was reduced by glancing angle deposition (GLAD) of metals such as chromium and silver. Reactive ion etching of the underlying substrate, followed by deposition of a few nanometers of gold, lift-off and thermal annealing resulted in perfectly ordered arrays of small gold nanoparticles with well-defined size distribution. The combination of passivation of the silica surface with polyethylene glycol (PEG) derivatives and functionalization of gold with thiols enables the preparation of large area arrays of well separated functional protein molecules. Ó 2008 Elsevier B.V. All rights reserved. Keywords: EUV interference lithography; Glancing angle deposition; Gold nanodot array; Selective protein immobilization
1. Introduction Arrays of active proteins anchored to flat surfaces are steadily gaining importance on the one hand for the highly parallel analysis of biomaterials [1] and on the other hand as model systems to study the interactions of living cells with biomolecules. The density of protein patterns has drastically increased in the recent years and the quality of the created patterns has been improved by careful design of the used immobilization methods and surface protection chemistry. The ultimate limit of integration of protein arrays is reached with structures of the same dimensions as the size of the immobilized proteins. A number of structuring techniques appear suitable to create protein patterns at such high density. Electron beam lithography as well as focused ion beam lithography allows feature sizes in the 10 nm range [2] with the hand*
Corresponding author. Tel.: +41 56 310 21 41. E-mail address: [email protected] (C. Padeste).
0167-9317/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2007.12.061
icap of being comparably slow due to their serial writing process. Two to three times better resolution, at the expense of the application of an even much slower method, can be realized when an AFM tip is used to write the pattern, e.g. in dip-pen nanolithography [3], nanopen reading and writing [4], nanoshaving [5], or nanografting [6]. Large area nanopatterning of functional proteins was demonstrated using colloidal lithography but the size of areas with perfect ordering is usually limited [7,8]. Direct printing of macromolecules using elastomeric stamps [9] is a popular technique for the creation of protein patterns but has not yet reached single molecule resolution. In this work we describe the development of interference lithographic patterning using extreme ultraviolet (EUV) light as a high-throughout and top-down structuring technique [10] towards these dimensions using glancing angle deposition (GLAD [11]) and thermal annealing [12] of metals for feature size reduction. This approach has reproducibly yielded gold dots of 12 ± 3 nm diameter located on a
F.A. Zoller et al. / Microelectronic Engineering 85 (2008) 1370–1374
well defined lattice. The process sequence explored is schematically shown in Fig. 1.
1371
3. Results and discussion 3.1. EUV interference lithography
2. Experimental Highest resolution periodic line structures produced to date with EUV-IL are in the range of 12.5 nm half pitch [15]. In this work it was intended to produce sites for protein immobilization in the diameter range of 10 nm, but separated by at least 100 nm in order to avoid interactions between the immobilized proteins and to facilitate independent addressing of individual molecules. Due to the intensity distribution in the interference region of the four beams used to create the patterns the minimum size of holes achievable with 140 nm period structures was in the range of 50 nm (Fig. 2a).
Arrays of 50 nm diameter holes in 40 nm thick PMMA, spin-coated onto thermally oxidized silicon wafers, were produced with EUV-IL at the XIL beamline of the Swiss Light Source as described elsewhere [10]. For the deposition of metals a Balzers BAE 250 (Oerlikon Balzers, Lichtenstein) evaporation system was used. For GLAD the substrates were tilted to a highly oblique angle between 60° and 80° relative to the incident flux and were continuously rotated about an axis normal to the substrate at 120 rpm. The deposition rate was set between 0.2 and 5 nm/s. Reactive ion etching (RIE) was carried out with a Plasmalab System 100 (Oxford Instruments, Witney, Oxon, UK), using SF6 (40 sccm) or a combination of CHF3 (40 sccm) and O2 (3 sccm) at a pressure of 100 mTorr, using 80–100 W forward power at 300 ± 2 K. After RIE, gold was deposited in the Balzers BAE 250 with the substrate at an orthogonal orientation with respect to the gold flux at 0.7–1.5 nm/s. Chromium was wet etched in a solution of ceric ammonium nitrate (5% in diluted acetic acid) at pH 6. The lift-off was performed in dichloromethane under sonication. Thermal annealing was performed in vacuum (<2 10 5 mbar) at 512 °C for 20 min or in air at atmospheric pressure at 600–800 °C for 10 min. For passivation of the SiO2 surfaces the samples were incubated for 30 min in a solution of 0.5 mg/ml of PLLPEG (PLL(20)-g[3.5]-PEG(2), Surface Solutions, Zu¨rich, Switzerland [13]) in 10 mM HEPES/150 mM NaCl buffer (pH 7.4). Alternatively Hydro-Stellan Star-PEG (N612K, SusTech, Darmstadt, Germany [14]) was applied by spincoating. The effectiveness of the PEG-coatings was evaluated using fluorescence microscopy after incubation of the samples with 100 lg/ml fluorescent labelled avidin.
3.2. Oblique metal evaporation The size of the holes produced with EUV-IL was constricted by GLAD of chromium or silver onto the substrates which were rotating around an axis perpendicular to the surface (Fig. 1b). The metal, the evaporation angle and the deposition rates are the main parameters controlling the GLAD. Chromium (Fig. 2b) led to the best constriction of the holes but the layers had a grainy appearance. Due to higher fluidity, silver formed much smoother layers, but did not provide as much constriction of the holes (Fig. 2c). Finally it was found that GLAD masks with a chromium/silver layer provided a good size constriction and smooth surface (Fig. 2d). First, chromium was evaporated, causing the major constriction of the opening. Second, silver was deposited yielding masks with smooth and homogenous appearance. The bi-metallic masks gave the best uniformity in the subsequent RIE and gold deposition steps. Silver was best evaporated with a speed of 4–5 nm/s. In the case of chromium a high deposition rate of 5 nm/s led to a layer of needle-like crystals. In contrast the shape of the clusters formed by a rate of only a few hundred pm/s
EUV-IL patterned PMMA
a
d
gold evaporation
glancing angle metal deposition
b
e
lift-off
reactive ion etching
c
f
passivation & protein binding
Fig. 1. Scheme of the process sequence for creating ordered arrays of anchored protein: (a) A hole array is patterned in PMMA with EUV-IL. (b) A metal mask is deposited at a shallow angle under rotation of the substrate. (c) A reactive ion etching step is used to form cavities in which gold is deposited (d). (e) After lift-off the gold is annealed to form single gold dots inside the cavities. (f) The gold and the SiO2 surface are chemically modified to obtain the protein binding sites in a protein-resistant surface.
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F.A. Zoller et al. / Microelectronic Engineering 85 (2008) 1370–1374
Fig. 2. Scanning electron microscope images showing the effect of glancing angle metal deposition on photoresist structures: (a) original structure; (b) after deposition of chromium, (c) silver and (d) a chromium/silver double layer.
appeared spheroidal and small. The disadvantage of a low deposition speed was the strong rise of the temperature of the substrate resulting in deformation of the photoresist structures. For chromium the best compromise between the constitution of the mask and the protection of the photoresist was achieved at a value of 1 nm/s. The angle at which the evaporated metal atoms impinge on the substrate has a major influence on the degree of constriction. On the one hand, oblique angles in the range of 80° or higher hardly closed the openings. On the other hand, angles smaller than about 60° for chromium and 70° for silver caused severe lift-off problems, since the evaporated metal formed contacts with the surface of the wafer.
3.4. Gold deposition and lift-off To form the final gold islands, a 5–10 nm thick gold layer was deposited onto the sample at an angle of 90°. The subsequent lift-off by dissolving the PMMA in an organic solvent turned out to be impossible, as the solvent did not pass through the holes in the metal layer and therefore could not reach the PMMA. This problem was solved by using wet chemical chromium etching for 30–60 s. The etching solution also dissolved the PMMA but did not affect the gold. The etching step needed to be controlled carefully since gold clusters deposited on the mask could be removed into the solvent and re-deposited on the sample surface.
3.3. Reactive ion etching 3.5. Thermal annealing In order to ensure a flat surface for final AFM analysis, first a short reactive ion etching step was carried out to etch holes in the substrate using either CHF3/O2 or SF6 as the etching gases. In both cases the Cr layer on the PMMA structure proved to be a very stable etch resist, but the narrow holes lowered the etching rates of the SiO2 by a factor of 2–3. As determined by AFM after lift-off, the maximum achievable rates were 10 nm/s for CHF3/O2 and 7 nm/s for SF6. As expected, CHF3/O2 etched more anisotropically. However the CHF3 passivation steps resulted in the formation of a contamination, possibly fluorocarbon residues, at the side of the etched structures that could only be removed by extended exposure to O2 plasma after the lift-off.
A typical appearance of the gold deposits after successful lift-off is shown in Fig. 3b. Below a nominal thickness of 5 nm the deposited metal did not form a continuous layer, but rather clusters were formed and spread out in the area of the holes in the chromium mask. Since the gold-covered area was greater than the intended final size of the gold islands, samples were subjected to thermal annealing. Condensation of the clusters into bigger agglomerates and reshaping towards a spherical appearance was achieved by thermal treatment in vacuum or air at temperatures of at least 500 °C or 600 °C, respectively. With the thermal annealing, the diameter of the gold islands could be shrunk
F.A. Zoller et al. / Microelectronic Engineering 85 (2008) 1370–1374
1373
Fig. 4. SEM and AFM images of gold dot arrays.
Fig. 3. (a) 3D view of a chromium mask. (b, c) the same structure after RIE etching, gold deposition and lift-off; before (b) and after (c) annealing of the gold islands.
by a factor of the 2–3 while their height increased by a factor of 3. The characterization of the final structures by AFM and SEM (Fig. 4) showed large arrays of perfectly ordered gold dots of 12 ± 3 nm in diameter. A low density of defects (<1%) was detected in arrays of up to several 104 binding sites, each located in an etched hole of about 10 nm depth. 3.6. Surface Modification In order to allow for selective binding of proteins to the gold islands only, the silicon dioxide surface was protected against non-specific protein binding using poly-ethylene glycol (PEG) derivatives; either as poly-L-lysine copolymers (PLL-PEG) binding firmly to the oxide via the poly-lysine [13], or as star-shaped PEG applied via spincoating [14].
Incubation of the substrates in the PLL-PEG solution for 30 min resulted in a dense coating, which efficiently prevented the binding of fluorescent labelled avidin. The rms roughness of the coating as determined by AFM, was the same as the rms roughness of the silicon oxide surface below, indicating that a film of uniform thickness was formed on the surface. The Star-PEG molecules are endowed with reactive end groups which interlink the deposited material but deactivate under extended contact with humidity. The StarPEG layer thickness can be adapted between 2 and 25 nm by changing the spin-coating parameters. This effect could be used as an alternative to the RIE etching step to level out the topology of the gold dots on the substrate. Levelling effects also caused a decrease in surface roughness after spin-coating of the Star-PEG. Qualitative determination of the fluorescence intensity showed a slightly less efficient protein binding protection of the Star-PEG compared to PLL-PEG. The binding of proteins to the surface of the gold islands can be achieved either using thiols as linkers, or by direct anchoring of designed proteins via terminal cysteins. Sitedirected protein binding on the nanostructured substrates will be the subject of further studies. 4. Conclusion and outlook Perfectly ordered, dense and virtually defect-free lattices of several 104 anchor sites for the fixation of single or just a
1374
F.A. Zoller et al. / Microelectronic Engineering 85 (2008) 1370–1374
few proteins were produced. The height difference between the gold island and the substrate could be levelled out by an RIE step applied before metal deposition. The flatness of the substrates reached in this way and the predetermination of the exact position of the protein immobilization sites are of great advantage when characterizing the binding of proteins to the gold islands by scanning probe techniques. References [1] A. Lueking, D.J. Cahill, S. Mu¨llner, Drug Discovery Today 10 (2005) 789. [2] W. Hu, K. Sarveswaran, M. Lieberman, G.H. Bernstein, J. Vac. Sci. Technol. B 22 (2004) 1711. [3] R.D. Piner, J. Zhu, F. Xu, S.H. Hong, C.A. Mirkin, Science 283 (1999) 661.
[4] G.-Y. Liu, N.A. Amro, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 5165. [5] S. Xu, G.-Y. Liu, Langmuir 13 (1997) 127. [6] K. Wadu-Mesthrige, N.A. Amro, J.C. Garno, S. Xu, G. Liu, Biophys. J. 80 (2001) 1891. [7] H. Agheli, J. Malmstro¨m, E.M. Larsson, M. Textor, D.S. Sutherland, Nano Lett. 6 (2006) 1165. [8] P. Hanarp, M. Ka¨ll, D.S. Sutherland, J. Phys. Chem. B 107 (2003) 5768. [9] A. Kumar, G.M. Whitesides, Appl. Phys. Lett. 63 (1993) 2002. [10] H.H. Solak, J. Phys. D: Appl. Phys. 39 (2006) R171–R188. [11] K. Robbie, M.J. Brett, J. Vac. Sci. Technol. A 15 (1997) 1460. [12] B.J.Y. Tan, C.H. Sow, T.S. Koh, K.C. Chin, A.T.S. Wee, C.K. Ong, J. Phys. Chem. B 109 (2005) 11100. [13] N.-P. Huang, R. Michel, J. Vo¨ro¨s, M. Textor, R. Hofer, A. Rossi, D.L. Elbert, J.A. Hubbell, N.D. Spencer, Langmuir 17 (2001) 489. [14] J. Groll, K. Albrecht, P. Gasteier, S. Riethmueller, U. Ziener, M. Moeller, Chembiochem 6 (2005) 1782. [15] H.H. Solak, Y. Ekinci, P. Ka¨ser, S.G. Park, J. Vac. Sci. Technol. B 25 (2007) 91.
Microelectronic Engineering 85 (2008) 1370–1374 www.elsevier.com/locate/mee
Nanostructured substrates for high density protein arrays Frank A. Zoller a,b, Celestino Padeste a,*, Yasin Ekinci a,c, Harun H. Solak a, Andreas Engel b a Laboratory for Micro- and Nanotechnology, Paul Scherrer Institut, 5232 Villigen, PSI, Switzerland Maurice E. Mu¨ller Institute, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland c Laboratory for Metal Physics and Technology, ETH Zu¨rich, Wolfgang-Pauli-Str. 10, 8093 Zu¨rich, Switzerland b
Received 25 September 2007; received in revised form 14 December 2007; accepted 23 December 2007 Available online 12 January 2008
Abstract Nearly defect-free arrays of several 104 gold dots of 12 ± 3 nm in diameter inside 10 nm deep cavities were fabricated for immobilization of proteins. Extreme ultraviolet interference lithography (EUV-IL) at the XIL-beamline of the Swiss Light Source was used to produce 140 nm period arrays of 50 nm holes in a 40 nm thick PMMA layer on oxidized silicon wafers. The size of the openings was reduced by glancing angle deposition (GLAD) of metals such as chromium and silver. Reactive ion etching of the underlying substrate, followed by deposition of a few nanometers of gold, lift-off and thermal annealing resulted in perfectly ordered arrays of small gold nanoparticles with well-defined size distribution. The combination of passivation of the silica surface with polyethylene glycol (PEG) derivatives and functionalization of gold with thiols enables the preparation of large area arrays of well separated functional protein molecules. Ó 2008 Elsevier B.V. All rights reserved. Keywords: EUV interference lithography; Glancing angle deposition; Gold nanodot array; Selective protein immobilization
1. Introduction Arrays of active proteins anchored to flat surfaces are steadily gaining importance on the one hand for the highly parallel analysis of biomaterials [1] and on the other hand as model systems to study the interactions of living cells with biomolecules. The density of protein patterns has drastically increased in the recent years and the quality of the created patterns has been improved by careful design of the used immobilization methods and surface protection chemistry. The ultimate limit of integration of protein arrays is reached with structures of the same dimensions as the size of the immobilized proteins. A number of structuring techniques appear suitable to create protein patterns at such high density. Electron beam lithography as well as focused ion beam lithography allows feature sizes in the 10 nm range [2] with the hand*
Corresponding author. Tel.: +41 56 310 21 41. E-mail address: [email protected] (C. Padeste).
0167-9317/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2007.12.061
icap of being comparably slow due to their serial writing process. Two to three times better resolution, at the expense of the application of an even much slower method, can be realized when an AFM tip is used to write the pattern, e.g. in dip-pen nanolithography [3], nanopen reading and writing [4], nanoshaving [5], or nanografting [6]. Large area nanopatterning of functional proteins was demonstrated using colloidal lithography but the size of areas with perfect ordering is usually limited [7,8]. Direct printing of macromolecules using elastomeric stamps [9] is a popular technique for the creation of protein patterns but has not yet reached single molecule resolution. In this work we describe the development of interference lithographic patterning using extreme ultraviolet (EUV) light as a high-throughout and top-down structuring technique [10] towards these dimensions using glancing angle deposition (GLAD [11]) and thermal annealing [12] of metals for feature size reduction. This approach has reproducibly yielded gold dots of 12 ± 3 nm diameter located on a
F.A. Zoller et al. / Microelectronic Engineering 85 (2008) 1370–1374
well defined lattice. The process sequence explored is schematically shown in Fig. 1.
1371
3. Results and discussion 3.1. EUV interference lithography
2. Experimental Highest resolution periodic line structures produced to date with EUV-IL are in the range of 12.5 nm half pitch [15]. In this work it was intended to produce sites for protein immobilization in the diameter range of 10 nm, but separated by at least 100 nm in order to avoid interactions between the immobilized proteins and to facilitate independent addressing of individual molecules. Due to the intensity distribution in the interference region of the four beams used to create the patterns the minimum size of holes achievable with 140 nm period structures was in the range of 50 nm (Fig. 2a).
Arrays of 50 nm diameter holes in 40 nm thick PMMA, spin-coated onto thermally oxidized silicon wafers, were produced with EUV-IL at the XIL beamline of the Swiss Light Source as described elsewhere [10]. For the deposition of metals a Balzers BAE 250 (Oerlikon Balzers, Lichtenstein) evaporation system was used. For GLAD the substrates were tilted to a highly oblique angle between 60° and 80° relative to the incident flux and were continuously rotated about an axis normal to the substrate at 120 rpm. The deposition rate was set between 0.2 and 5 nm/s. Reactive ion etching (RIE) was carried out with a Plasmalab System 100 (Oxford Instruments, Witney, Oxon, UK), using SF6 (40 sccm) or a combination of CHF3 (40 sccm) and O2 (3 sccm) at a pressure of 100 mTorr, using 80–100 W forward power at 300 ± 2 K. After RIE, gold was deposited in the Balzers BAE 250 with the substrate at an orthogonal orientation with respect to the gold flux at 0.7–1.5 nm/s. Chromium was wet etched in a solution of ceric ammonium nitrate (5% in diluted acetic acid) at pH 6. The lift-off was performed in dichloromethane under sonication. Thermal annealing was performed in vacuum (<2 10 5 mbar) at 512 °C for 20 min or in air at atmospheric pressure at 600–800 °C for 10 min. For passivation of the SiO2 surfaces the samples were incubated for 30 min in a solution of 0.5 mg/ml of PLLPEG (PLL(20)-g[3.5]-PEG(2), Surface Solutions, Zu¨rich, Switzerland [13]) in 10 mM HEPES/150 mM NaCl buffer (pH 7.4). Alternatively Hydro-Stellan Star-PEG (N612K, SusTech, Darmstadt, Germany [14]) was applied by spincoating. The effectiveness of the PEG-coatings was evaluated using fluorescence microscopy after incubation of the samples with 100 lg/ml fluorescent labelled avidin.
3.2. Oblique metal evaporation The size of the holes produced with EUV-IL was constricted by GLAD of chromium or silver onto the substrates which were rotating around an axis perpendicular to the surface (Fig. 1b). The metal, the evaporation angle and the deposition rates are the main parameters controlling the GLAD. Chromium (Fig. 2b) led to the best constriction of the holes but the layers had a grainy appearance. Due to higher fluidity, silver formed much smoother layers, but did not provide as much constriction of the holes (Fig. 2c). Finally it was found that GLAD masks with a chromium/silver layer provided a good size constriction and smooth surface (Fig. 2d). First, chromium was evaporated, causing the major constriction of the opening. Second, silver was deposited yielding masks with smooth and homogenous appearance. The bi-metallic masks gave the best uniformity in the subsequent RIE and gold deposition steps. Silver was best evaporated with a speed of 4–5 nm/s. In the case of chromium a high deposition rate of 5 nm/s led to a layer of needle-like crystals. In contrast the shape of the clusters formed by a rate of only a few hundred pm/s
EUV-IL patterned PMMA
a
d
gold evaporation
glancing angle metal deposition
b
e
lift-off
reactive ion etching
c
f
passivation & protein binding
Fig. 1. Scheme of the process sequence for creating ordered arrays of anchored protein: (a) A hole array is patterned in PMMA with EUV-IL. (b) A metal mask is deposited at a shallow angle under rotation of the substrate. (c) A reactive ion etching step is used to form cavities in which gold is deposited (d). (e) After lift-off the gold is annealed to form single gold dots inside the cavities. (f) The gold and the SiO2 surface are chemically modified to obtain the protein binding sites in a protein-resistant surface.
1372
F.A. Zoller et al. / Microelectronic Engineering 85 (2008) 1370–1374
Fig. 2. Scanning electron microscope images showing the effect of glancing angle metal deposition on photoresist structures: (a) original structure; (b) after deposition of chromium, (c) silver and (d) a chromium/silver double layer.
appeared spheroidal and small. The disadvantage of a low deposition speed was the strong rise of the temperature of the substrate resulting in deformation of the photoresist structures. For chromium the best compromise between the constitution of the mask and the protection of the photoresist was achieved at a value of 1 nm/s. The angle at which the evaporated metal atoms impinge on the substrate has a major influence on the degree of constriction. On the one hand, oblique angles in the range of 80° or higher hardly closed the openings. On the other hand, angles smaller than about 60° for chromium and 70° for silver caused severe lift-off problems, since the evaporated metal formed contacts with the surface of the wafer.
3.4. Gold deposition and lift-off To form the final gold islands, a 5–10 nm thick gold layer was deposited onto the sample at an angle of 90°. The subsequent lift-off by dissolving the PMMA in an organic solvent turned out to be impossible, as the solvent did not pass through the holes in the metal layer and therefore could not reach the PMMA. This problem was solved by using wet chemical chromium etching for 30–60 s. The etching solution also dissolved the PMMA but did not affect the gold. The etching step needed to be controlled carefully since gold clusters deposited on the mask could be removed into the solvent and re-deposited on the sample surface.
3.3. Reactive ion etching 3.5. Thermal annealing In order to ensure a flat surface for final AFM analysis, first a short reactive ion etching step was carried out to etch holes in the substrate using either CHF3/O2 or SF6 as the etching gases. In both cases the Cr layer on the PMMA structure proved to be a very stable etch resist, but the narrow holes lowered the etching rates of the SiO2 by a factor of 2–3. As determined by AFM after lift-off, the maximum achievable rates were 10 nm/s for CHF3/O2 and 7 nm/s for SF6. As expected, CHF3/O2 etched more anisotropically. However the CHF3 passivation steps resulted in the formation of a contamination, possibly fluorocarbon residues, at the side of the etched structures that could only be removed by extended exposure to O2 plasma after the lift-off.
A typical appearance of the gold deposits after successful lift-off is shown in Fig. 3b. Below a nominal thickness of 5 nm the deposited metal did not form a continuous layer, but rather clusters were formed and spread out in the area of the holes in the chromium mask. Since the gold-covered area was greater than the intended final size of the gold islands, samples were subjected to thermal annealing. Condensation of the clusters into bigger agglomerates and reshaping towards a spherical appearance was achieved by thermal treatment in vacuum or air at temperatures of at least 500 °C or 600 °C, respectively. With the thermal annealing, the diameter of the gold islands could be shrunk
F.A. Zoller et al. / Microelectronic Engineering 85 (2008) 1370–1374
1373
Fig. 4. SEM and AFM images of gold dot arrays.
Fig. 3. (a) 3D view of a chromium mask. (b, c) the same structure after RIE etching, gold deposition and lift-off; before (b) and after (c) annealing of the gold islands.
by a factor of the 2–3 while their height increased by a factor of 3. The characterization of the final structures by AFM and SEM (Fig. 4) showed large arrays of perfectly ordered gold dots of 12 ± 3 nm in diameter. A low density of defects (<1%) was detected in arrays of up to several 104 binding sites, each located in an etched hole of about 10 nm depth. 3.6. Surface Modification In order to allow for selective binding of proteins to the gold islands only, the silicon dioxide surface was protected against non-specific protein binding using poly-ethylene glycol (PEG) derivatives; either as poly-L-lysine copolymers (PLL-PEG) binding firmly to the oxide via the poly-lysine [13], or as star-shaped PEG applied via spincoating [14].
Incubation of the substrates in the PLL-PEG solution for 30 min resulted in a dense coating, which efficiently prevented the binding of fluorescent labelled avidin. The rms roughness of the coating as determined by AFM, was the same as the rms roughness of the silicon oxide surface below, indicating that a film of uniform thickness was formed on the surface. The Star-PEG molecules are endowed with reactive end groups which interlink the deposited material but deactivate under extended contact with humidity. The StarPEG layer thickness can be adapted between 2 and 25 nm by changing the spin-coating parameters. This effect could be used as an alternative to the RIE etching step to level out the topology of the gold dots on the substrate. Levelling effects also caused a decrease in surface roughness after spin-coating of the Star-PEG. Qualitative determination of the fluorescence intensity showed a slightly less efficient protein binding protection of the Star-PEG compared to PLL-PEG. The binding of proteins to the surface of the gold islands can be achieved either using thiols as linkers, or by direct anchoring of designed proteins via terminal cysteins. Sitedirected protein binding on the nanostructured substrates will be the subject of further studies. 4. Conclusion and outlook Perfectly ordered, dense and virtually defect-free lattices of several 104 anchor sites for the fixation of single or just a
1374
F.A. Zoller et al. / Microelectronic Engineering 85 (2008) 1370–1374
few proteins were produced. The height difference between the gold island and the substrate could be levelled out by an RIE step applied before metal deposition. The flatness of the substrates reached in this way and the predetermination of the exact position of the protein immobilization sites are of great advantage when characterizing the binding of proteins to the gold islands by scanning probe techniques. References [1] A. Lueking, D.J. Cahill, S. Mu¨llner, Drug Discovery Today 10 (2005) 789. [2] W. Hu, K. Sarveswaran, M. Lieberman, G.H. Bernstein, J. Vac. Sci. Technol. B 22 (2004) 1711. [3] R.D. Piner, J. Zhu, F. Xu, S.H. Hong, C.A. Mirkin, Science 283 (1999) 661.
[4] G.-Y. Liu, N.A. Amro, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 5165. [5] S. Xu, G.-Y. Liu, Langmuir 13 (1997) 127. [6] K. Wadu-Mesthrige, N.A. Amro, J.C. Garno, S. Xu, G. Liu, Biophys. J. 80 (2001) 1891. [7] H. Agheli, J. Malmstro¨m, E.M. Larsson, M. Textor, D.S. Sutherland, Nano Lett. 6 (2006) 1165. [8] P. Hanarp, M. Ka¨ll, D.S. Sutherland, J. Phys. Chem. B 107 (2003) 5768. [9] A. Kumar, G.M. Whitesides, Appl. Phys. Lett. 63 (1993) 2002. [10] H.H. Solak, J. Phys. D: Appl. Phys. 39 (2006) R171–R188. [11] K. Robbie, M.J. Brett, J. Vac. Sci. Technol. A 15 (1997) 1460. [12] B.J.Y. Tan, C.H. Sow, T.S. Koh, K.C. Chin, A.T.S. Wee, C.K. Ong, J. Phys. Chem. B 109 (2005) 11100. [13] N.-P. Huang, R. Michel, J. Vo¨ro¨s, M. Textor, R. Hofer, A. Rossi, D.L. Elbert, J.A. Hubbell, N.D. Spencer, Langmuir 17 (2001) 489. [14] J. Groll, K. Albrecht, P. Gasteier, S. Riethmueller, U. Ziener, M. Moeller, Chembiochem 6 (2005) 1782. [15] H.H. Solak, Y. Ekinci, P. Ka¨ser, S.G. Park, J. Vac. Sci. Technol. B 25 (2007) 91.