Nanowell-Array Surfaces Prepared by Argon Plasma Etching through

May 25, 2005 - These nanowell arrays are prepared via a plasma-etch method using a nanopore alumina film as the etch mask. A replica of the pore struc...
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Langmuir 2005, 21, 8429-8438

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Nanowell-Array Surfaces Prepared by Argon Plasma Etching through a Nanopore Alumina Mask Myungchan Kang, Shufang Yu, Naichao Li, and Charles R. Martin* Department of Chemistry and Center for Research at the Bio/Nano Interface, University of Florida, Gainesville, Florida 32611-7200 Received January 18, 2005. In Final Form: April 14, 2005 A method for preparing a glass surface containing an ordered array of nanowells is described. These nanowell arrays are prepared via a plasma-etch method using a nanopore alumina film as the etch mask. A replica of the pore structure of the alumina mask is etched into the glass. We demonstrate that chemical information in the form of negatively charged latex nanoparticles can be selectively stored within these nanowells and not indiscriminately deposited on the surface surrounding the nanowells. To accomplish this, the chemistry of the glass surfaces within these nanowells (walls and bottoms) must be different from the chemistry of the surface surrounding the nanowells. Two different procedures were developed to make the inside vs. surrounding surface chemistries different. Atomic force microscopy (AFM) was used to image the nanowells and, via friction-force measurements, to prove that the inner nanowell surfaces can be made chemically different from the surface surrounding the nanowells.

Introduction Spatially storing chemical information in an array pattern across a surface is useful for a number of different technologies. For example, array-based gene and protein sensing relies on spotting microscopic samples of biomaterials across a substrate surface.1-3 The number of genes or proteins that can be detected per square centimeter of sensor surface is determined by the size of the spots, and recent advances such as dip-pen nanolithography4 have pushed the spot size from micrometers to nanometers. Nano- or microscale two-dimensional arrays generated by dip-pen nanolithography have been used for selective adsorption of proteins,5 cells,5 magnetic nanoparticles,6 charged polystyrene particles,7 oligonucleotides-functionalized gold particles,8 and conducting polymers.9 The array-based fiber-optic sensor technology developed by Walt et al. is another example.10-14 They used fiberoptic imaging bundles consisting of thousands of fused optical fibers, which were optically connected, to obtain individually addressable parallel sensing platforms. They chemically etched the ends of the fiber cores, and in one example,12,14 DNA-functionalized microspheres were randomly fixed in the microwells on the core ends. Image * Author to whom correspondence should be addressed. E-mail: [email protected]. (1) Friend, S. H.; Stoughton, R. B. Sci. Am. 2002, 286, 44. (2) Espina, V.; Mehta, A. I.; Winters, M. E.; Calvert, V.; Wulfkuhle, J.; Petricoin III, E. F.; Liotta, L. A. Proteomics 2003, 3, 2091. (3) Schena, M. Microarray analysis, 1st ed.; J. Wiley: Hoboken, NJ, 2003; Chapter 7. (4) Ginger, D. S.; Zhang, H.; Mirkin, C. A. Angew. Chem., Int. Ed. 2004, 43, 30. (5) Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702. (6) Liu, X.; Fu, L.; Hong, S.; Dravid, V. P.; Mirkin, C. A. Adv. Mater. 2002, 14, 231. (7) Demers, L. M.; Mirkin, C. A. Angew. Chem., Int. Ed. 2001, 40, 3069. (8) Zhang, H.; Li, Z.; Mirkin, C. A. Adv. Mater. 2002, 14, 1472. (9) Lim, J.-H.; Mirkin, C. A. Adv. Mater. 2002, 14, 1474. (10) Pantano, P.; Walt, D. R. Chem. Mater. 1996, 8, 2832. (11) Szunerits, S.; Walt, D. R. ChemPhysChem 2003, 4, 186. (12) Epstein, J. R.; Leung, A. P. K.; Lee, K.-H.; Walt, D. R. Biosens. Bioelectron. 2003, 18, 541. (13) Epstein, J. R.; Walt, D. R. Chem. Soc. Rev. 2003, 32, 203. (14) Ferguson, J. A.; Steemers, F. J.; Walt, D. R. Anal. Chem. 2000, 72, 5618.

processing techniques were used to decode each microsphere and register the entire array. In this case, the number of array elements is determined by the diameter of the optical fibers, which can be as small as 250 nm.10 We describe here a new approach for spatially storing chemical information in an ordered array on a surface. The substrate is an array of nanowells etched into a glass surface.15 These nanowell arrays are prepared via a plasma-etch method using a nanopore alumina film16 as the etch mask. A replica of the pore structure of the alumina mask is etched into the glass. We demonstrate that chemical information in the form of negatively charged latex nanoparticles can be selectively stored within these nanowells and not indiscriminately deposited on the surface surrounding the nanowells. To accomplish this, the chemistry of the glass surfaces within these nanowells (walls and bottoms) must be different from the chemistry of the surface surrounding the nanowells. Two different procedures were developed to make the inside vs. surrounding surface chemistries different. Atomic force microscopy (AFM) was used to image the surfaces and detect the different inside vs. surrounding surface chemistries. Experimental Section Materials. Al foil (99.99%) was obtained from Alfa Aesar, and microscope premium finest glass slides were obtained from Fisher. Four types of surfactant-free, functionalized polystyrene latex particles were purchased from Interfacial Dynamics Corporationsanionic sulfate latexes (41 ( 6 or 75 ( 6 nm) and cationic amidine latexes (40 ( 10 or 78 ( 11 nm). 1-Octadecanethiol (ODT, Aldrich), NaCl (Fisher Scientific), 3-(aminopropyl)triethoxysilane (APTS, Gelest), octadecyltrichlorosilane (OTS, Aldrich), heptane (Aldrich), and anhydrous toluene (SigmaAldrich) were used as received. The alumina mask was an inhouse-prepared nanopore alumina film. The electrochemical anodization method used to prepare these films has been described previously.16 An anodization time of 15 min and voltage of 50 V were used. These films have two distinct surfacessthe surface that faced the solution during the anodization and the surface that faced the Al substrate during the anodization. The (15) Kang, M.; Yu, S.; Li, N.; Martin, C. R. Small 2005, 1, 69. (16) Li, N.; Mitchell, D. T.; Lee, K.-P.; Martin, C. R. J. Electrochem. Soc. 2003, 150, A979.

10.1021/la050146h CCC: $30.25 © 2005 American Chemical Society Published on Web 05/25/2005

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Figure 1. Cross-sectional schematics of the three different types of nanowell arrays investigated here. (A) A bare (noncoated) glass surface was plasma-etched through the alumina mask. (B) A Au/Pd-coated glass surface was plasma-etched through the mask, and the Au/Pd left on the surface surrounding the nanowells was then coated with a monolayer of ODT. (C) An APTS-coated glass surface was plasma-etched through the mask, and then OTS was coated on the exposed glass within the nanowells. later surface is often called the “barrier layer” surface because it initially has a barrier layer film which must be removed during processing.16,17 Fabrication of the Nanowell Arrays. Samples of the glass microscope slides that were ∼0.5 cm × ∼0.5 cm were used as the substrates for preparing the nanowell arrays. Prior to etching the arrays, the glass slides were cleaned by immersion in piranha solution (a mixture of 3:1 (v/v) 98% H2SO4 and 30% H2O2) for 30 min. After being rinsed five to six times with copious amounts of deionized water and ethanol, the glass slides were blown dry with nitrogen and were used immediately. The nanowell arrays were prepared by placing the alumina mask on top of the substrate to be etched with the barrier-layer side of the mask facing up. The masked substrate was then inserted into the vacuum chamber of a reactive-ion etching apparatus (Samco model RIE1C), and an Ar plasma was used to etch the substrate surface through the mask. The Ar plasma parameters were as follows 13.56 MHz, 140 W, 10 Pa Ar, Ar flow rate ) 12 sccm. The etch time was varied in order to vary the depth of the nanowells obtained. Three different types of nanowell arrays were investigated (Figure 1). The first, and simplest, was obtained by etching bare glass through the mask, such that the surfaces both within the nanowells (“inside” surfaces) and the surfaces surrounding the nanowells (“surrounding” surfaces) were bare glass (Figure 1A). In the second type of nanowell array, the inside surfaces were bare glass, and the surrounding surface was ODT-coated Au/Pd (Figure 1B). In the third array type, the inside surfaces were OTS-coated glass and the surrounding surface was APTS-coated glass (Figure 1C). To prepare the nanowell arrays shown in Figure 1B, the glass surface was first sputtered coated with a thin film of Au/Pd using a Desk II Cold Sputter instrument (Denton Vacuum, LLC). The Ar pressure was 75 mTorr, the sputtering current was 45 mA, and the sputtering time was 60 s. When these Au/Pd-coated surfaces were etched through the alumina mask, the portions of the Au/Pd film beneath the pores were etched away, and with continued etching, nanowells were etched into the underlying glass. Because the portions of the Au/Pd film that were not beneath a pore were not etched away, the surrounding surface remained Au/Pd and the inside nanowell surfaces were glass (Figure 1B). After etching, the Au/Pd film was rendered hydrophobic by coating with the thiol ODT, by immersion of the sample into a 1 mM ethanolic solution of ODT for 10 min, and then by rinsing in ethanol. Water contact angles on a Au/Pdcoated flat glass surface before and after thiol-modification were 16° and 96°, respectively. (17) Masuda, H.; Satoh, M. Jpn. J. Appl. Phys. 1996, 35, L126.

Kang et al. To measure the thickness of the Au/Pd film, the Au/Pd layer was removed by dissolution in a mixture of 1:1 (v/v) concentrated HCl and 30% H2O2. The thickness of the Au/Pd layer was determined by using AFM to measure the difference in the nanowell depth before and after removing the Au/Pd film. The thickness of Au/Pd film was determined to be ca. 30 nm. To prepare the nanowell arrays shown in Figure 1C, the glass surface was first coated with a film of APTS by immersing the sample for 20 min into a solution that was 5% silane, 90% ethanol, and 5% acetate buffer (50 mM, pH 5.2). The APTS film was then cured by heating in an oven at 120 °C for 2 h. The APTS-coated surface was then etched through the alumina mask. As per the Au/Pd case, the APTS beneath the pores was etched away to yield bare glass on the inside nanowell surfaces and APTS-coated glass on the surrounding surface. As per Figure 1C, the inside glass surfaces were then coated with OTS by immersion of the sample into a 1% OTS solution in toluene for 5 min followed by rinsing with toluene and then water. Deposition of Latex Particles. As discussed in the Introduction, one way to store chemical information at the nanowellarray surface entails inserting latex particles within the nanowells. The nanowell arrays shown in Figure 1B were used for these studies. Both cationic and anionic latex particles were investigated, and in both cases, particles with two different sizes (∼40 and ∼75 nm) were used. For the smaller particles, a solution that contained ∼3 × 1012 particles/mL was used, whereas the concentration of the larger particles was ∼4 × 1011 particles/mL. As will be discussed in detail below, these solutions either contained no added NaCl or had NaCl concentrations varying from 0.1 to 10 mM. The nanowell-array was immersed into the desired latex solution for 1 h (nanowell surface facing up) with stirring. The solution was then inserted into an ultrasonic bath and ultrasonicated for 1-2 min to remove any physisorbed particles. After being rinsed in water, the sample was rinsed in heptane to reduce the capillary forces before drying in a stream of nitrogen. Analogous latex-absorption experiments were also conducted at flat glass surfaces. AFM Studies. All AFM studies were performed using a Multimode NanoScope IIIa AFM (Digital Instruments). The x and y piezoelectric crystals were calibrated using a 1 µm × 1 µm period platinum-coated calibration grid. The z direction was calibrated by measuring the wavelength of the optical interference patterns resulting from reflection between the tip and a reflective substrate following Jaschke and Butt’s work.18 For enhanced resolution of tapping mode images, oxide-sharpened SSS tips (super sharp silicon tips, Nanosensors) were used. The image density was 512 pixels × 512 pixels, and the scan rate was 1 Hz. Double-layer force measurements were conducted using the methods described in detail in the literature.19-21 Two hundredmicrometer long V-shaped Si3N4 cantilevers (DI, manufacture suggested normal spring constant, k ) 0.12 N/m) were used, as the spring constants of the torsional modes are large for this geometry.21 Force-separation curves were obtained by recording the voltage from the split photodiode detector and the substrate displacement as given by the applied piezovoltages. All force curves were analyzed using a set of custom procedures written for Labview data analysis software (National Instruments).22 To reduce piezoelement hysteresis and to avoid squeezing action of the tip, we used a scan frequency for force measurements of 0.977 Hz. The manufacturer-suggested spring constant (0.12 nN/ nm) was used to convert the tip deflection to force without further complex and demanding spring constant measurements.23-27 (18) Jaschke, M.; Butt, H.-J. Rev. Sci. Instrum. 1995, 66, 1258. (19) Kang, M.; Gewirth, A. A. J. Phys. Chem. B 2002, 106, 12211. (20) Hillier, A. C.; Kim, S.; Bard, A. J. J. Phys. Chem. 1996, 100, 18808. (21) Noy, A.; Frisbie, C. D.; Rozsnyai, L. F.; Wrighton, M. S.; Lieber, C. M. J. Am. Chem. Soc. 1995, 117, 7945. (22) Kang, M. Ph.D. Thesis, University of Illinois, Urbana, IL, 2002. (23) Cleveland, J. P.; Manne, S.; Bocek, E.; Hansma, P. K. Rev. Sci. Instrum. 1993, 64, 403. (24) Senden, T. J.; Ducker, W. A. Langmuir 1994, 10, 1003. (25) Torii, A.; Sasaki, M.; Hane, K.; Okuma, S. Meas. Sci. Technol. 1996, 7, 179. (26) Hutter, J. L.; Bechhoefer, J. Rev. Sci. Instrum. 1993, 64, 1868. (27) Sader, J. E. Rev. Sci. Instrum. 1995, 66, 4583.

Nanowell-Array Surfaces Prepared by Plasma Etching A 10 µm tube scanner was used for the topographic and frictional measurements. All friction images and measurements were performed in contact mode using LFM tips (Nanosensors) under dry N2 gas in an environmental chamber (Digital Instruments). The normal force between the tip and sample was estimated using the spring constant provided by the manufacturer and the tip deflection sensitivity obtained from tip deflection vs z-displacement curves. All of the frictional measurements and images were obtained with the fast-scan axis perpendicular to the principal axis of the cantilever. Friction forces were calculated from plots of frictional signal vs. lateral displacements during 250 nm trace/retrace cycless friction loops along a single scan line repeatedly back and forth at 1000 nm/s. Friction forces were measured as a function of the systematically varied normal force. At least 10 measurements of friction forces were acquired for each normal force. The slowscan axis was disabled to ensure that the same area was sampled. For the purpose of comparison, all friction data were obtained using the same tip throughout the experiments. The LFM tip was washed in methanol prior to each measurement. Thus, variables such as the cantilever force constant and tip structure were held constant. To verify the tip composition during repeated measurements, a bare glass sample was used. Identical results were obtained from a bare glass sample surface before and after characterizing other samples. Scanning Electron Microscopy (SEM). SEM was used to measure the pore diameter, pore density, and thickness of the alumina mask. Data were obtained using a Hitachi S4000 FESEM. The pore diameter and pore density were determined using the WSxM image analysis package (Nanotec Electronica). This software gives the cross-sectional area of each pore in the image. This distribution of pore areas was used to calculate the corresponding distribution of pore diameters, assuming each pore is circular. An average pore diameter and a standard deviation of the pore diameters were calculated from these data. To improve the quality of the SEM image, the surface of this sample assembly was sputtered with a very thin Au/Pd film using a Desk II Cold Sputter instrument (Denton Vacuum, LLC).

Results and Discussion Surface Potential of Unetched vs Argon PlasmaEtched Glass. The latex particles are stored within the glass nanowells by electrostatic interactions between the particle and the glass surface. To understand the nature of these electrostatic interactions, we have used AFMbased double-layer force measurements to determine the surface charge of both the unetched and Ar-plasma etched glass. These measurements probe the electrostatic interaction between the charged (vide infra) AFM tip and the charged surface, as the tip penetrates the diffuse double layer at the surface.19-21 Flat glass surfaces, as opposed to the nanowell glass, were used for these studies. The latex particle solutions had a measured pH of 5.6, and both the unetched glass surface and the surface of the AFM tip are negatively charged at this pH.20,28 In contrast, Kitabayashi et al. showed that Ar-plasma etched glass surfaces were positively charged, when the surface charge was measured in a vacuum.29 This has been attributed to the implantation of positively charged Ar ions into the glass. However, to our knowledge, there have been no measurements of the sign of the surface charge of Ar-plasma-etched glass surfaces with the surface immersed in an electrolyte solution. For this reason, we have used AFM force measurements to determine the sign of the surface charge for the unetched and plasma etch glass in the aqueous electrolyte solution used here. Figure 2 shows force curves for an unetched glass surface and for surfaces that had been Ar-plasma etched for 1 and (28) Raiteri, R.; Margesin, B.; Grattarola, M. Sens. Actuators, B 1998, 46, 126. (29) Kitabayashi, H.; Fujii, H.; Ooishi, T. Jpn. J. Appl. Phys. 1999, 38, 2964.

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Figure 2. Force curves between the silicon nitride AFM tip and flat (not nanowell) glass surfaces. (A) Surface was not plasma-etched. (B) Surface was plasma-etched for 1 min. (C) Surface was plasma-etched for 5 min. Samples were immersed into 1 mM NaCl, with pH ∼5.6, during the measurements.

5 min. In all cases, the sample was immersed in 1 mM NaCl solution, which has a calculated Debye length of 9.6 nm.20,28,30 Considering the unetched surface first, we see increasing electrostatic repulsion between the tip and the surface as the separation distance decreases from ∼40 to ∼10 nm. This results because both the tip and the surface of the unetched glass are negatively charged. At a separation distance of ∼10 nm, the tip jumps into contact with the surface due to the dominance of van der Waals attraction at such small distances. In contrast, for the surface that had been plasma-etched for 5 min, there is an attractive interaction between the tip and the surface. Since the tip is still negatively charged, these data clearly show that this surface is positively charged. There is essentially no electrostatic force between the tip and the surface that had been plasma etched for only one minute. This indicates that the quantity of positive charge introduced by this brief plasma-etching period has neutralized the native negative charge of the glass and that there is now no net charge on the surface. These conclusions about the signs of the surface charges were confirmed via latex-particle adsorption experiments (Figure 3). Figure 3A shows an AFM image of an unetched glass surface that had been exposed to the cationic latex particles using the method described in the Experimental Section. Because, when immersed in the latex solution, the surface was negatively charged, it is completely coated with cationic particles. In contrast, an unetched surface that was exposed to anionic latex particles shows no evidence for particle adsorption due to the repulsion between the like-charged particles and surface (Figure 3B). When these experiments were repeated with surfaces that had been Ar-plasma etched for 5 min, we see the opposite patternsthe negatively charged latex sticks (Figure 3C) and the positively charged particles do not (Figure 3D). These data confirm the conclusion reached from the AFM measurements that the 5-min plasmaetched surface is positively charged. Morphology of the Nanowells and Controlling Nanowell Depth. Figure 4 shows top- and edge-view SEM images of the alumina mask used in this work; the pore diameter is 82 ( 2 nm and the thickness is ∼1.2 µm. When used as a plasma-etch mask, the plasma propagates through the pores in the mask and etches a replica of the pore structure of the mask in the underlying glass sur(30) Cappella, B.; Dietler, G. Surf. Sci. Rep. 1999, 34, 5.

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Figure 3. Tapping-mode AFM images of flat (not nanowell) glass surfaces after exposure to latex nanoparticle suspensions (diam ) ∼40 nm) in 10 mM NaCl solution. For (A) and (B), the flat glass surface was not plasma-etched: (A) after exposure to the positively charged latex particles and (B) after exposure to the negatively charged latex particles. For (C) and (D), the flat glass surface was plasma-etched for 5 min: (C) after exposure to the negatively charged latex particles and (D) after exposure to the positively charged latex particles.

face. This general technology was developed by Masuda et al. to prepare nanoporous diamond films.31-33 We have used this method to prepare nanoporous carbon films.16 In both of these cases, the nanoporous films were used as electrodes for electrochemical energy storage. Tapping-mode AFM images show that the pore structure of the alumina mask is faithfully reproduced in the glass (Figure 5). The nanowell diameter obtained from such images (80 ( 4 nm) is identical to the pore diameter in the mask. The number of nanowells per square centimeter of glass surface was obtained by counting wells in 20 1 µm × 1 µm AFM images. A nanowell density of (7.5 ( 0.4) × 109 wells/cm2 was obtained. This is identical to the pore density of the alumina mask obtained by counting pores in electron micrographs of the mask surface (Figure 4A). Figure 6A shows cross-sectional AFM profiles of individual nanowells obtained after the indicated plasma(31) Masuda, H.; Yasui, K.; Watanabe, M.; Nishio, K.; Rao, T. N.; Fujishima, A. Chem. Lett. 2000, 1112. (32) Honda, K.; Rao, T. N.; Tryk, D. A.; Fujishima, A.; Watanabe, M.; Yasui, K.; Masuda, H. J. Electrochem. Soc. 2001, 148, A668. (33) Honda, K.; Rao, T. N.; Tryk, D. A.; Fujishima, A.; Watanabe, M.; Yasui, K.; Masuda, H. J. Electrochem. Soc. 2000, 147, 659.

etch times. The flat profile at the bottom of these wells indicates that the AFM tip was, indeed, imaging the nanowell bottom. This may be contrasted with nanowell profiles obtained from longer etch times, which show triangular bottoms, a clear indication that the tip is not truly imaging the bottom of the well (data are not shown here). Figure 6B shows that, over the time interval where the true profile of the nanowell can be imaged, nanowell depth is linearly related to etch time with a slope corresponding to 5.3 nm/min of etching (solid line, corr coeff ) 0.98). The error bars in Figure 6B are associated with averages of profiles such as those in Figure 6A for >300 nanowells at each etch time. This mask-based nanowell etching technology necessitates that the alumina mask survives the Ar plasma treatment over the entire etching time period, i.e., the mask must not get etched away before the nanowells are etched into the glass. With the 1.2 µm-thick masks used here, we were able to etch for as long as 50 min and still the mask remained intact. Electron microscopy showed that the nanowells obtained after this very long etch time were ∼200 nm deep, much deeper than the wells investigated here.

Nanowell-Array Surfaces Prepared by Plasma Etching

Figure 4. Scanning electron micrographs of the alumina mask: (A) top view and (B) edge view.

Figure 5. Tapping-mode AFM image of a nanowell glass surface (Figure 1A) prepared by plasma-etching through the alumina mask for 10 min.

Selective Adsorption of Polystyrene Particles. A key technology described here entails making the chemistry of the surface surrounding the nanowells different from the chemistry of the surfaces within the nanowells. This allows for discrete storage of spherical nanoparticles

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within the nanowells but not indiscriminate deposition on the surfaces surrounding the nanowells. This concept was accomplished by sputtering the glass surface with an ultrathin (∼30 nm) Au/Pd film prior to etching (Figure 1B). With this technique, the surrounding surface is covered with a thin layer of Au/Pd and the inside surfaces are glass. In addition, the “chemical contrast” between the inner and surrounding surfaces was increased by chemisorbing ODT to the Au/Pd. This renders the surrounding surface hydrophobic while the inside surfaces remain hydrophilic and positively charged (vide supra). To explore the effects of the Au/Pd and ODT-coated Au/ Pd films on the surface chemistry, flat (not plasma-etched) samples were exposed to both the positively and negatively charged latex particles (Figure 7). When the Au/Pd-coated glass surface is exposed to the cationic particles, the surface becomes completely coated with these particles (Figure 7A). This is because the Au/Pd film is negatively charged due to Cl- adsorption.34-36 As would be expected, the anionic particles do not adsorb to this negatively charged surface (Figure 7B). (The fine structure seen in this image is due to the surface morphology of the Au/Pd itself.) After the Au/Pd film is coated with ODT, neither cationic nor anionic particles adsorb because now the surface is noncharged and hydrophobic (Figure 7C and D). To demonstrate that these surface chemistries can be used to selectively deposit latex particles within the nanowells, Au/Pd-film-coated glass surfaces were plasmaetched for 5.5 min to yield nanowells with an AFMdetermined depth of 55 ( 8 nm (Figure 8). Because the Au/Pd film was 30 nm thick, this measurement indicates the nanowell within the glass is, in fact, 25 nm deep, which corresponds well to the nanowell depth obtained after 5.5 min of etching at a surface that was not coated with Au/ Pd (Figure 6B). This result suggests that the Au/Pd film is etched at a much faster rate than the glass. AFM images of these etched Au/Pd-coated surfaces show that the pore structure of the alumina mask is faithfully reproduced here as well (Figure 8). Figure 8 shows AFM images of Au/Pd-coated, plasmaetched, and then ODT-coated surfaces after exposure to aqueous suspensions of either anionically charged or cationically charged latex particles (diameter ≈ 40 nm). Because the inside glass surfaces are positively charged, exposure to the anionic particles results in insertion of these particles into a majority of the nanowells (Figure 8A). Because of the small particle size, some nanowells contain two or three particles. As would be expected, there are no particles on the ODT-coated Au/Pd surface surrounding the nanowells. Furthermore, exposure to the cationic latex particles results in no particle deposition on either the inside or surrounding surfaces (Figure 8B). This is because these particles are electrostatically repelled by the inside glass surfaces and have no affinity for the neutral and hydrophobic surrounding surface. Figure 9 shows results of analogous experiments with the larger (diameter ≈ 75 nm) latex particles. Because the particle size closely matches the nanowell diameter, only one anionic particle can be inserted within a nanowell (Figure 9A). Again, the cationic particles adsorb to neither the inside nor surrounding surfaces (Figure 9B). Figures 8 and 9 demonstrate a significant point of this workschemical information in the form of negatively (and (34) Nishizawa, M.; Menon, V. P.; Martin, C. R. Science 1995, 268, 700. (35) Shi, Z.; Lipkowski, J. J. Electroanal. Chem. 1996, 403, 225. (36) Kerner, Z.; Pajkossy, T. Electrochim. Acta 2002, 47, 2055.

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Figure 6. (A) AFM profiles of single nanowells prepared using the indicated etch times. (B) Depth of nanowell obtained from profiles such as those in (A) vs etch time.

Figure 7. Tapping-mode AFM images of flat (not nanowell) Au/Pd-coated glass surfaces after exposure to latex nanoparticle suspensions (diam ) ∼40 nm) in 10 mM NaCl solution. For (A) and (B), the Au/Pd film on glass surface was not coated with a monolayer of ODT: (A) after exposure to the positively charged latex particles and (B) after exposure to the negatively charged latex particles. For (C) and (D), the Au/Pd film on glass surface was coated with a monolayer of ODT: (C) after exposure to the positively charged latex particles and (D) after exposure to the negatively charged latex particles.

not positively) charged nanoparticles can be spatially distributed and stored by spontaneous insertion into the inside nanowell surfaces that are chemically distinct from the surrounding surface. In the examples discussed here, the information stored is very limited in that all of the nanoparticles stored within the nanowells are identical. However, the nanowell surface could be exposed to a solution containing numerous chemically distinct nanoparticles. In analogy to Walt’s work, this would result in

random placement of these nanoparticles within nanowells across the nanowell surface.12-14 That is, a particle randomly selected from the mix would deposit into each nanowell. The challenge then would be to “read out” the chemical information stored on these high-density nanowell-array surfaces, i.e., to read out the specific chemical identity of each particle in each well. One possibility would be to use near-field optical microscopy to sequentially detect a fluorescent signal stored on the nanoparticles, or

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Figure 8. Tapping-mode AFM images of nanowell arrays where the surface surrounding the nanowells is coated with ODTmodified Au/Pd and the inside nanowell surfaces are plasma-etched glass (see Figure 1B): (A) after immersion of the array in a 10 mM NaCl suspension of the negatively charged latex particles (diam ) ∼40 nm) and (B) after immersion in an analogous suspension of the positively charged particles.

Figure 9. Tapping-mode AFM images of nanowell arrays where the surface surrounding the nanowells is coated with ODTmodified Au/Pd and the inner nanowell surfaces are plasma-etched glass (see Figure 1B): (A) after immersion of the array in a 10 mM NaCl suspension of the negatively charged latex particles (diam ) ∼75 nm) and (B) after immersion in an analogous suspension of the positively charged particles.

other chemical/biochemical system, within the individual nanowells. In addition, making the diameter of the nanowells somewhat larger would make it possible to read out the nanowell surface using confocal microscopy or an array-based optical detector. Effect of Electrolyte Concentration on Latex Particle Deposition. In all of the studies presented above, the latex particles were suspended in an electrolyte solution of 10 mM NaCl. By adjusting the electrolyte concentration, the packing density of charged particles inside the nanowells can be systematically varied. This is because an electrical double layer surrounds each particle and the thickness of the double layer (as approximated by the Debye screening length) increases with decreasing electrolyte concentration.37,38 As a result, at lower electrolyte concentrations, there is greater repulsion

between latex particles so that the saturation coverage of particles at the surface is reduced.37,38 To demonstrate this concept, Au/Pd-coated, plasma-etched, and then ODTcoated nanowell surfaces were immersed into anionic latex particle (diameter ≈ 40 nm) suspensions with a series of different added electrolyte (NaCl) concentrations (Figure 10). Without any added electrolyte, only about 15% of the nanowell is filled with anionic particles (Figure 10A), as opposed to the >90% coverage obtained with 10 mM NaCl (Figure 8A). Furthermore, unlike the higher-electrolyteconcentration case (Figure 8A), none of the filled nanowells (37) Chen, K. M.; Jiang, X.; Kimerling, L. C.; Hammond, P. T. Langmuir 2000, 16, 7825. (38) Hanarp, P.; Sutherland, D. S.; Gold, J.; Kasemo, B. Colloids Surf., A 2003, 214, 23.

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Figure 10. Tapping-mode AFM images of nanowell arrays where the surface surrounding the nanowells is coated with ODTmodified Au/Pd and the inner nanowell surfaces are plasma-etched glass (see Figure 1B): after immersion of the array in a suspension of the negatively charged latex particles (diam ) ∼40 nm) with electrolyte concentration of (A) 0, (B) 0.1, and (C) 1 mM NaCl.

Figure 11. Friction force microscopy measurements on flat glass surfaces that were coated with either APTS or OTS. (A) Friction loops obtained at an applied normal load of 20 nN. Solid curves, corresponding to the largest positive and negative friction signals, are for the APTS-coated surface. Dashed curves are for the OTS-coated surface. (B) Plots of frictional signal vs applied normal load. 4 ) APTS-coated surface; 3 ) OTS-coated surface.

contain more than one particle even though the particle diameter is smaller than the nanowell diameter. This is again a consequence of enhanced interparticle repulsion when there is no added electrolyte. As shown in Figure 10B, the coverage of particles obtained in 0.1 mM NaCl is similar to that obtained with no salt. Hanarp et al. obtained similar results for adsorption of charged particles at flat glass surfaces.38 These result indicate that the lower limit of the saturation coverage38 has already been achieved in 0.1 mM NaCl solution. Higher coverages are, however, obtained in 1 mM NaCl (Figure 10C), and at this electrolyte concentration, more than one particle can, again, occupy a nanowell. Patterning of Different Silanes to the Inside vs Surrounding Nanowell Surfaces. As illustrated in Figure 1C, the plasma-etch technology developed here can also be used to prepare nanowell arrays in which the inside and outside surfaces have different attached silane films. This was accomplished by attaching the amineterminated silane APTS to the glass before plasma-etching through the alumina mask. Subsequent plasma-etching causes the APTS to be removed from the inside nanowell surfaces but not from the surrounding surface. The plasma-etch-exposed glass on the inside surfaces can then be reacted with a second silane, in this case OTS, to make the inside vs surrounding surfaces chemically distinct. Friction force microscopy (FFM) 39-45 was used to visualize (39) Noy, A.; Vezenov, D. V.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006. (40) Wong, S.-S.; Takano, H.; Porter, M. D. Anal. Chem. 1998, 70, 5209. (41) Green, J.-B. D.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys. Chem. 1995, 99, 10965.

the chemical contrast between the inside vs surrounding surfaces. FFM experiments were first done on APTS- and OTS-coated flat glasses surfaces. Figure 11A shows plots of frictional signal vs lateral displacement at an applied normal load of 20 nN on flat glass surfaces modified with either APTS or OTS. The top half-loop of each curve corresponds to the trace scan, and the bottom half-loop is the retrace curve. The difference between the trace and retrace average values was taken as the friction force.41 Figure 11A shows that there is higher frictional force at the -NH2-terminated APTS-coated surface than at the -CH3-terminated OTS surface. This is in agreement with prior studies, which showed that Si3N4 AFM tips have higher frictional forces at surfaces with higher surfaces energies.46 Figure 11B shows plots of friction force vs applied normal load for the APTS- and OTS-modified surfaces. Friction increases at a rate of 9.0 mV/nN for APTS and 0.88 mV/ nN for OTS. From these data, it is also evident that there is about a factor of 4 difference in the frictional properties of the APTS- vs OTS-coated surfaces at an applied normal force of 20 nN. This difference is sufficient enough to allow FFM-based molecular contrast imaging at 20 nN. (42) Takano, H.; Kenseth, J. R.; Wong, S.-S.; O’Brien, J. C.; Porter, M. D. Chem. Rev. 1999, 99, 2845. (43) Noy, A.; Vezenov, D. V.; Lieber, C. M. Annu. Rev. Mater. Sci. 1997, 27, 381. (44) Jones, V. W.; Kenseth, J. R.; Porter, M. D.; Mosher, C. L.; Henderson, E. Anal. Chem. 1998, 70, 1233. (45) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071. (46) Thomas, R. C.; Houston, J. E.; Crooks, R. M.; Kim, T.; Michalske, T. A. J. Am. Chem. Soc. 1995, 117, 3830.

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Figure 12. (A) AFM height and (B) friction images of the shallow (∼2 nm deep) nanowell arrays where the outside glass surface is coated with APTS and the inside nanowell surfaces are coated with OTS (see Figure 1C). (C) Height profile showing two of the shallow nanowells. (D) Corresponding friction loop obtained at an applied normal load of 20 nN.

Analogous FFM measurements were done at a nanowell array where the inside nanowell surfaces were coated with OTS and the surrounding surface was coated with APTS. However, when an AFM tip encounters steps across a sample surface, transient frictional spiking features can be generated by the tip tripping at the edges of the steps. Prior work has shown that features with heights of ∼2 nm do not show significant transient frictional spiking features.41,47 For this reason, nanowells with depths of only ∼2 nm were fabricated for the friction-mode imaging experiments. The plot of nanowell depth vs etch time (Figure 6B) was used as a guide to prepare such very shallow nanowells; an etch time of 30 s was used. Figure 12 shows AFM height and friction images simultaneously obtained for such a shallow nanowell array with OTS on the inside surfaces and APTS on the surrounding surface. The height-mode image (Figure 12A) clearly shows the shallow nanowells, and a cross-sectional profile (Figure 12C) shows, as expected, that the nanowells are ∼2 nm deep. The dark areas in the friction image (Figure 12B) correspond to regions of low friction or weak (47) Overney, R. M.; Meyer, E.; Frommer, J.; Guentherodt, H. J.; Fujihira, M.; Takano, H.; Gotoh, Y. Langmuir 1994, 10, 1281.

interaction between the AFM tip and the functional groups on the surface. The friction loop (Figure 12C) shows that these regions of low friction correspond to the inside nanowell surfaces where the OTS was immobilized. Likewise, regions of high friction were observed on the surrounding surface, where the APTS was immobilized. Conclusions Ordered arrays of nanowell were plasma-etched into glass surfaces, using a nanopore alumina membrane as the etch mask. The depth of the nanowells can be controlled by varying the etch time. The chemistry of the surfaces within the nanowells can be made different from the chemistry of the surface surrounding the nanowells. This was accomplished here by two routes. The first entailed coating the glass surface with a Au/Pd film prior to etching the nanowells. After being plasma-etched through the alumina mask, the surfaces within the nanowells are freshly exposed glass while the surrounding surface retained the Au/Pd film. The second method entailed coating the glass with one silane, etching to create the nanowells, and then coating the exposed inside nanowell surfaces with a second silane.

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We have also shown that plasma-etched glass has positive surface charge29 even after exposure of the etched glass to aqueous electrolyte solution. The positive surface charge on the inside nanowell surfaces can be used to selectively deposit negatively charged latex particles within the nanowells. In addition, we have shown that, when different silanes are applied to the inside vs. surrounding surfaces, FFM measurements can be used to visualize the different inside vs. surrounding surface chemistries. While the current system stores chemical information in the form of simple anionically charged latex particles, with the appropriate linker, more sophisticated forms of chemistry could be stored within the nanowells. For example, DNA chains could be attached to the inner nanowell surface and the complementary chains stored

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by hybridization. The pore diameter in the alumina mask can be controlled at will from ∼10 to ∼300 nm, and with the smallest-diameter pores, the pore density can be as high as 1011 cm-2. Hence, this approach should yield array densities higher than any other nanofabrication technology. In addition, the plasma-etch method is compatible with any number of materials including carbons, polymers, and metals. Hence, it should be possible to create nanowells of this type in a broad range of materials. Acknowledgment. This work was supported by the National Science Foundation through the NIRT for Biomedical Nanotube Technology. LA050146H