Langmuir 1996, 12, 5515-5518
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Electrochemical Patterning of Self-Assembled Monolayers onto Microscopic Arrays of Gold Electrodes Fabricated by Laser Ablation Leonard M. Tender,† Robinson L. Worley,† Hongyou Fan,† and Gabriel P. Lopez*,†,‡ Department of Chemical and Nuclear Engineering and Department of Chemistry, The University of New Mexico, 209 Farris Engineering Center, Albuquerque, New Mexico 87131-1341 Received June 24, 1996. In Final Form: September 12, 1996X This report demonstrates proof-of-concept application of two established electrochemical techniques affecting self-assembled monolayerssthe thermodynamic control of monolayer assembly at gold and the electrochemical desorption of self-assembled monolayers from goldsto pattern monolayers onto gold microelectrodes. When combined with a simple laser-based microfabrication technique, using commercially available microscope adaptations, to form arrays of individually-addressable gold microelectrodes from continuous thin gold films on glass, two new patterning methodologies result that enable fabrication of monolayer-based affinity arrays for potential use in chemical and biosensors. The described microfabrication technique is central to the success of these patterning methodologies because it allows SAM patterning of metal arrays immediately after their fabrication, so as to avoid problems associated with forming SAMs on commercially-supplied arrays. In this paper, each patterning methodology is used to fabricate an interdigitated microelectrode array in which one set of electrodes is modified with an active antibody and the other set of electrodes is resistant to nonspecific protein adsorption.
1. Introduction Recently there has been considerable interest in the microscopic patterning of self-assembled monolayers (SAMs) on metal surfaces1 and of biological molecules adsorbed to patterned SAMs on metal surfaces2,3 for applications that include chemical sensing,4 microlithography,5 and drug screening.6 Our interest in patterning lay in a desire to develop methods amenable to the fabrication of microscopic arrays on gold surfaces in which elements consist of gold regions modified with SAMs formed from one (or more) of many different ω-substituted alkanethiols as the basis for miniaturized multianalyte biosensors.7 Ideal methods enabling fabrication of such arrays would be well-suited to patterning a large number of distinct, microscopic elements with no geometric constraints, would not require micromanipulation of the alkanethiols (e.g., microwriting,7 microstamping,2,8 and micropipetting), would not require multiple photolithographic steps5,9,10 (so as to avoid potential problems associated with multiple mask alignments and loss of biological activity), and would use readily available instrumentation. We demonstrate herein proof-of-concept †
Department of Chemical and Nuclear Engineering. Department of Chemistry. X Abstract published in Advance ACS Abstracts, October 15, 1996. ‡
(1) Kumar, A.; Abbott, N. L.; Enoch, K. E.; Biebuyck, H. A.; Whitesides, G. M. Acc. Chem. Res. 1995, 28, 219. (2) Lopez, G. P.; Biebuyck, H. A.; Harter, R.; Kumar, A.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10774. (3) Delmarche, E.; Sundarababu, G.; Biebuyck, H.; Michel, B.; Gerber, Ch.; Singrist, H.; Wolf, H.; Ringsdorf, H.; Xanthopoulos, N.; Mathieu, H. J. Langmuir 1996, 12, 1997. (4) Mrksich, M.; Grunwell, J. R.; Whitesides, G. M. J. Am. Chem. Soc. 1995, 117, 12009. (5) Chan, K. C.; Kim, T.; Shoer, J. K.; Crooks, R. M. J. Am. Chem. Soc. 1995, 117, 5875. (6) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 252, 767. (7) Perez-Luna, V.; Tender, L. M.; Opperman, K. A.; Hampton, P. B.; Harris, R. B.; Lopez, G. P. Manuscript in preparation. (8) Lopez, G. P.; Biebuyck, H. A.; Frisbie, D.; Whitesides, G. M. Science 1993, 260, 647. (9) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem. Int. Ed. Engl. 1995, 34, 91. (10) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117, 12050.
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application of two established electrochemical techniques affecting SAMs of alkanethiolates to pattern onto gold substrates prototype arrays of SAMssthe thermodynamic control of monolayer self-assembly at gold11 and the electrochemical desorption of SAMs from gold.12 When these techniques are combined with a simple laser-based microfabrication technique (described below) to form arrays of individually- addressable gold microelectrodes from continuous thin gold films on glass, two new patterning methodologies emerge that embody the aforementioned ideals. The success of these patterning methodologies is tied directly to the microfabrication technique because it avoids problems associated with SAM-inhibiting contaminant adsorption by allowing formation of SAMs on the array’s SAM immediately after fabrication. 2. Experimental Section 2.1. Materials. (1-Mercaptoundec-11-yl)hexa(ethylene glycol), HO(CH2CH2O)6(CH2)11SH, referred to here as “EG6SH” was synthesized according to Pale-Grosdemage et al.13 Hexadecanethiol, CH3(CH2)15SH, referred to here as “C16SH” (Aldrich) was purified by column chromatography. Anti-BSA (monoclonal antibody to bovine serum albumin, mouse IgG2a isotype, Sigma), BSA-FITC (bovine serum albumin labeled with fluorescein isothiocyanate, Molecular Probes), and avidin-FITC (Molecular Probes) were diluted to 10 µg/mL in pH 7.2 phosphate-buffered saline: “PBS” (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 140 mM NaCl, and 3 mM KCl in deionized H2O, pH adjusted by dropwise addition of HCl). Gold substrates consisted of 200-1000 Å of Au evaporated under vacuum (chamber pressure ) 5 × 10-7 Torr) at a rate of 1 Å/s onto 10-50 Å, respectively, of Cr (adhesion layer) which had been evaporated at 0.5 Å/s onto piranha etch (7/3 by volume H2SO4/30% aqueous H2O2)-cleaned microscope glass slides. Electrochemistry was conducted in a singlecompartment cell with a platinum counter electrode and a Ag/ AgCl/3 M KCl reference electrode using a Pine Instruments AFRDE5 potentiostat. (11) Weisshaar, D. E.; Lamp, B. D.; Porter, M. D. J. Am. Chem. Soc. 1992, 114, 5860. (12) Widrig, C. A.; Chinkap, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335. (13) Pale-Grosdemage, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12.
© 1996 American Chemical Society
5516 Langmuir, Vol. 12, No. 23, 1996 2.2. Methods. 2.2.1. Laser-Based Microfabrication. Microfabrication by laser ablation of thin metal films (up to 1000 Å thick) evaporated onto glass, silicon, or polymers into arrays of individual electrically-addressable microscopic electrodes is readily accomplished with commercially-available microscope adaptations. Our system consists of a Nikon Diaphot 300 inverted microscope adapted with a computer-controlled, pulsed nitrogenpumped dye laser emitting at 390 nm with a maximum intensity of 15 µJ/pulse14 focused directly through the microscope objective lens. The use of a pulsed UV laser aligned in this manner has found numerous applications as a microdissecting tool in biological sciences at the cellular15 and subcellular levels16 including, for example, the only means of microdissection within a living cell.17,18 We have found that this tool easily ablates thin gold films from glass, forming circular regions of exposed glass with diameters of 5-20 µm when using a 20× dry objective, 1-5 µm when using a 40× dry objective, and less than 1 µm when using a 100× oil immersion lens.19 When combined with a submicron resolution, high-speed, computer-controlled X-Y-Z stage,20 it is relatively easy to precisely move the gold surface while ablating to fabricate complex arrays of individuallyaddressable gold microelectrodes for use, for example, as substrates for our microscopic affinity arrays. Advantages of this microfabrication technique over conventional microlithographic techniques (typically performed out-of-house) to form such arrays include the unlimited freedom in design with near immediate feedback from application, the avoidance of SAMinhibiting contamination formation of the gold surface due to the speed in which arrays can be fabricated from freshlyevaporated metal films and utilized, and the avoidance of residual photoresist or other packaging encountered when using commercially-supplied microscopic metal arrays that may inhibit or prevent SAM formation. Figure 1 illustrates a scheme employed for the laser fabrication of interdigitated microband electrode arrays, such as those employed in surface acoustic wave (SAW) sensors21 and in a wide range of electrochemical investigations,22 which we use as substrates for prototype affinity arrays. Here, a 1/4 in. × 1 in. glass microscope slide coated with a metal film is bisected lengthwise into two electrically-addressable halves (emphasized by shading), labeled A and B, by a 1-20 µm thick band of exposed glass (depending upon laser power, objective power, and laser focusing) formed by laser ablation. The magnified circular region represents a portion of the gold film ablated in a pattern forming a series of n interdigitated microband pairs that alternate in electrical contact with halves A and B of the gold surface. The freedom in design afforded by this microfabrication technique allows any practical number of n interdigitated bands to be created with fixed or varying lengths (from microns to centimeters), fixed or varying widths (from microns to centimeters), and fixed or varying separations (from microns to centimeters) to meet specified needs. In addition, by substantially reducing the laser output power, we are presently exploring the use of this instrumentation to laser-desorb SAMs23 from gold surfaces for (14) Laser Scissors, Cell Robotics, Inc., Albuquerque, NM. (15) Berns, M. W.; Wright, W. H.; Weigand-Steubing, R. Rev. Cytol. 1991, 129, 1. (16) Greulich, J.; Voelkel, S. J. Microsc.1992, 167, 127. (17) Berns, M. W.; Alist, J.; Edwards, J.; Strahs, K.; Girton, J.; McNeill, P.; Rattner, J. B.; Kitzes, M.; Hammer-Wilson, M.; Liaw, L. H.; Siemans, A.; Koonce, M.; Petersons, S.; Brenner, S., Burt, J.; Walter, R.; Bryant, P. J.; van Dyk, D.; Coulombe, J.; Cahill, T.; Berns, G. S. Science 1981, 213, 505. (18) Liang, H.; Wright, W. H.; Cheng, S.; He, W.; Berns, M. W. Exp. Cell Res. 1993, 204, 110. (19) Laser ablation was typically conducted with gold surfaces facing up. Use of an oil immersion lens, therefore, will not contaminate gold films. For a given objective, the diameter of the ablated region can be precisely tuned by adjusting the laser power and/or by defocusing the laser slightly out of the gold plane. (20) SmartStage, Cell Robotics Inc., Albuquerque, NM; 0.1 µm resolution at 200 µm/s. (21) Janata, J. Principles of Chemical Sensors; Plenum Press: New York, 1989. (22) (a) Sheppard, N. F.; Tucker, R. C.; Wu, C. Anal. Chem. 1993, 65, 1199. (b) Naoui, K.; Ueyama, K.; Osaka, T. J. Electrochem. Soc. 1990, 137 (2), 494. (c) Bard, A. J.; Crayston, J. A.; Kittleson, G. P.; Shea, T. V.; Wrighton, M. S. Anal. Chem. 1986, 58 (11), 2321. (23) Takehara, K.; Yamada, S.; Ide, Y. J. Electroanal. Chem. 1992, 333, 339.
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Figure 1. Generalized scheme for the laser ablation-based fabrication of an interdigitized microband electrode array from continuous metal films evaporated onto glass (200-1000 Å of Au on a 10-50 Å Cr underlayer). Here a macroscopic gold surface is bisected lengthwise into two electrically isolated halves (emphasized by shading) labeled A and B by a band of exposed glass (1-20 µm thick depending upon laser power, objective power, and laser focusing) formed by laser ablation. The area within the magnified circular region results from rastering the glass/metal surface back-and-forth above the laser while ablating so as to microfabricate n pairs of interdigitized microbands (labeled A and B) that alternate in electrical contact with halves A and B. subsequent remodification with SAMs formed from different alkanethiols as an alternate method to pattern SAM-modified gold. 2.2.2. Thermodynamic Control of Monolayer Self-Assembly. Following the protocol of Porter et al., 11 application of a voltage of -1.2 V vs Ag/AgCl/3 M KCl to a gold electrode while in a 1-10 mM C16SH in 0.5 M ethanolic KOH solution will block monolayer self-assembly. We consistently observe that when a thin film gold electrode is (1) immersed into 1-10 mM C16SH, 0.5 M ethanolic KOH while biased at -1.2 V, (2) held in solution while biased at -1.2 V for 1 min (sufficient time for C16S monolayer self-assembly at an unbiased electrode), (3) removed from solution while biased at -1.2 V, (4) rinsed thoroughly with water and ethanol while biased at -1.2 V, (5) disconnected from potential control, and (6) immersed into a 1-10 mM ethanolic EG6SH solution for 1 min, its wettability (advancing water contact angles of 37°) and XPS spectra24 are consistent with those of a wellordered EG6S monolayer on a fresh gold electrode. Patterning of C16S and EG6S SAMs onto the alternating bands of an interdigitated array on the basis of the thermodynamic control of monolayer formation is accomplished in the following manner. Following the steps described above, the end of the gold film on glass sample containing the pattern depicted in Figure 1 is immersed into a solution of 1 mM C16SH in 0.5 M ethanolic KOH for 1 min while biasing half B to -1.2 V vs a Ag/AgCl/3 M KCl reference electrode by electrical contact to half B at the exposed end. This results in the formation of a C16S monolayer onto the unbiased alternating bands in electrical contact with half A while leaving the bands in contact with half B unmodified. After copious rinsing with water and ethanol and immersion for 1 min into a 1 mM ethanolic EG6SH solution, bands in contact with half B are subsequently modified with a EG6S monolayer.25 2.2.3. Electrochemical Desorption of SAMs From Gold. We have observed that it is straightforward to remove EG6S monolayers from gold films by application of a voltage cycling between -1.0 and -1.5 V vs Ag/AgCl/3 M KCl in PBS at 500 (24) High-resolution C(1s) XPS analysis conducted on a Surface Science Instruments (SSI) X-Probe ESCA Instrument at the University of Washington Surface Analysis Recharge Center using an Al KR 1,2 monochromatized X-ray source to stimulate photoemission. See ref 13 for detailed description of high-resolution C(1s) XPS analysis of methylterminated and oligo(ethylene glycol)-terminated monolayers on gold.
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Langmuir, Vol. 12, No. 23, 1996 5517
mV/s for less than 1 min. This is a modification of the protocol of Porter et al.12 for electrochemical stripping of n-alkanethiols in 0.5 M ethanolic KOH that we have devised to extend electrochemical patterning schemes based on electrochemical SAM desorption to pattern SAMs formed from ω-substituted alkanethiols terminated with biologically-active functional groups.7 Typically, the addition of 1 mM K3Fe(CN)6 is used as an in situ indicator of the extent of monolayer desorption (i.e., as the SAM is desorbed, the Fe(CN)63-/4- voltammetry, observed by cycling between -0.1 V and +0.4 V, approaches that of an unmodified gold electrode of the same area).26 Fe(CN)63-/4voltammetry and XPS spectra of gold electrodes modified with C16S monolayers subjected to this modified stripping protocol strongly suggest that a significant portion of the C16S monolayer remains adsorbed to the electrode, even after cleavage of the Au-S bond, presumably due the insolubility of C16SH in PBS (compared to that of the oligo(ethylene glycol)-terminated EG6SH). We consistently observe that gold electrodes from which EG6S monolayers are electrochemically stripped in PBS and remodified with C16S monolayers by immersion into 1-10 mM C16SH ethanolic solutions for 1 min demonstrate wettabilities (advancing water contact angles of 115°) and XPS spectra consistent with those of well-ordered C16S monolayers on fresh gold electrodes. Electrochemical patterning of C16S and EG6S SAMs onto the alternating bands of an interdigitated array based on the electrochemical desorption of SAMs from gold is accomplished in the following manner. After laser ablation of the array, the gold surface is immersed for 1 h in a 1 mM ethanolic EG6SH solution to form EG6S monolayers on bands A and B. Immersion of the end of the gold-on-glass sample containing the array into PBS and application of a voltage cycling between -1.0 and -1.5 V vs Ag/AgCl/3 M KCl to bands labeled A (by electrical contact to half A at the exposed end) selectively desorbs the EG6S monolayer from bands labeled A. After copious rinsing with water and ethanol, immersion of the entire array into a 1 mM C16SH ethanolic solution for 1 min results in the formation of a C16S SAM on bands A.
3. Results and Discussion 3.1. Patterning of SAMs onto Microscopic Interdigitated Arrays by Thermodynamic Control of Monolayer Formation. Figure 2 illustrates the results of electrochemically patterning, by thermodynamic control of SAM formation, two different SAMs onto a microscopic interdigitated array fabricated by laser ablation of a gold film (200 Å Au on a 10 Å Cr adhesion layer) as described and the subsequent patterning of biomolecules onto one set of the bands. Shown in Figure 2 is a confocal fluorescence micrograph27 of a portion of the interdigitated array patterned with SAMs formed from C16SH and EG6SH onto the alternating bands (and other electricallyconnected regions) labeled A and B, respectively. The alkanethiols patterned were chosen because their SAMs demonstrate contrasting behavior to nonspecific adsorption by biomolecules (i.e., EG6S SAMs resist biomolecule adsorption whereas C16S SAMs promote biomolecule adsorption).3 Figure 2 was recorded after the entire array was (1) immersed for 2 h in a solution of anti-BSA (10 µg/mL in PBS, pH 7.2), (2) rinsed thoroughly with PBS without exposing the array to air (so as not to lose antibody activity), and (3) immersed for 10 min in a solution of (25) Contamination of monolayers previously formed on other elements may occur by displacement of monolayer constituents by alkanethiols in solution. Such cross-contamination may be minimized, however, by using low concentrations of alkanethiols and/or using short immersion times and/or using analogous disulfides. See for example: Biebuyck, H. A.; Bain, C. D.; Whitesides, G. M. Langmuir 1994, 10, 1825. (26) Creager, S. E.; Collard, D. M.; Fox, M. A. Langmuir 1990, 6, 1617. (27) Confocal fluorescence microscopy performed on a BioRad MRC600 instrument at the Imaging Center, University of New Mexico, Department of Biology, using the standard BioRad fluorescein excitation filter and cube set.
Figure 2. Confocal fluorescence micrograph (fluorescein excitation and emission) of a section of an interdigitized gold microband array after exposure of the entire array to BSAFITC (10 µg/mL in PBS, pH 7.2). Regions labeled A were modified with a C16S monolayer and nonspecifically adsorbed anti-BSA, and regions labeled B were modified with a EG6S monolayer. The bright areas reflect binding of the fluorescentlylabeled protein by its patterned antibody. SAM patterning was accomplished by (1) blocking C16S SAM formation at regions labeled A (by application of a strong reducing potential to regions A) while the entire array was immersed in a C16SH ethanolic solution (2) and then immersing the entire array (no bias applied to regions A or B) into an EG6SH ethanolic solution so as to form an EG6S monolayer on regions labeled B. Anti-BSA was patterned onto regions labeled A by its preferential nonspecific adsorption onto the C16S monolayer (and not to the EG6S monolayer) when the entire array is immersed into a solution of anti-BSA (10 µg/mL in PBS, pH 7.2).
BSA-FITC (10 µg/mL in PBS, pH 7.2). The observed contrast in Figure 2 results from the preferential nonspecific adsorption of the antibody onto the C16SH-modified bands labeled A and the subsequent specific binding of the labeled BSA by its patterned antibody. A confocal fluorescence micrograph of a similarly patterned array (i.e., bands A modified with a C16S SAM and then with nonspecifically adsorbed anti-BSA, and bands B modified with an EG6S SAM) showed no significant fluorescence at bands A or B after a 10 min immersion of the entire array into a solution of avidinFITC (10 µg/mL in PBS, pH 7.2). This result supports the conclusion that the observed fluorescence in Figure 2 is due to specific binding of the labeled protein by its antibody and not to nonspecific adsorption of the labeled protein to any exposed C16S SAM. It is important to note that the extension of this technique to patterning more than two different monolayers (say n) should be straightforward. After microfabricating an array of n individually-addressable microelectrodes, exposure of the entire array to a 0.5 M ethanolic solution of an alkanethiol should result in a SAM of that alkanethiolate only on the first element if the other elements are biased to a sufficiently reductive potential or have been previously modified with another SAM.25 Then, by sequentially releasing potential control of the elements as the array is exposed to 0.5 M ethanolic solutions of different alkanethiols, it should be possible to build up a microscopic array consisting of n gold elements modified with SAMs of n different alkanethiolates. Furthermore, the compactness of arrays patterned in this manner is only limited by the microfabrication technique (i.e., feature dimensions on the order of
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Figure 3. Confocal fluorescence micrograph (fluorescein excitation and emission) of an interdigitized array, patterned by a different electrochemical technique than that used in Figure 2. Here monolayer patterning was accomplished by (1) forming EG6S monolayers on the entire array, (2) then electrochemically stripping an EG6S monolayer from bands labeled A by application of a strong reducing potential, (3) and then immersing the entire array in a C16SH ethanolic solution. The entire array was then immersed into a solution of anti-BSA (10 µg/mL in PBS, pH 7.2) for 2 h and then into a solution of BSA-FITC (10 µg/mL in PBS, pH 7.2) for 10 min, as was done for Figure 2.
1 µm) because it is not necessary to selectively expose or deliver to each electrode its respective alkanethiol. Rather, the electrodes are all exposed to the same alkanethiols and patterning is accomplished by electrochemical manipulation of the targeted elements. 3.2. Patterning of SAMs onto Microscopic Interdigitated Arrays by Electrochemical Stripping of Monolayers. Figure 3 illustrates the results of electrochemically patterning C16S and EG6S monolayers onto the alternating interdigitated bands (labeled A and B, respectively) of a microscopic array fabricated by laser ablation of a gold film (200 Å Au on a 10 Å Cr adhesion layer) as described above and the subsequent patterning of biomolecules onto the set of bands labeled A. The patterning of the two different SAMs onto bands A and
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B was accomplished here, however, by electrochemical desorption. Figure 3 is a confocal fluorescence micrograph of the patterned interdigitated array that was recorded after the entire array was (1) immersed for 2 h in a solution of anti-BSA (10 µg/mL in PBS, pH 7.2), (2) rinsed thoroughly with PBS without exposing the array to air, and (3) immersed for 10 min in a solution of BSA-FITC (10 µg/mL in PBS, pH 7.2). Again, the contrast in Figure 3 results from the preferential nonspecific adsorption of the antibody onto the C16S-modified bands labeled A and the subsequent specific binding of the labeled BSA by its patterned antibody. The extension of electrochemical desorption of SAMs to pattern SAMs of n different ω-substituted alkanethiols onto n individually-addressable microscopic gold elements should also be straightforward. Here, sequential stripping the EG6S SAM from elements and exposing the entire array to new alkanethiol-containing solutions should allow the build up of arrays consisting of n different SAMmodified gold microelectrodes. As described above, the compactness of arrays modified in this manner is only limited by the microfabrication technique. 4. Conclusion We have demonstrated two new microscopic chemical patterning methodologies that embody ideals associated with the fabrication of SAM-based affinity arrays for potential use in chemical and biosensors. We are presently investigating application of patterning techniques on the basis of electrochemical monolayer desorption to create microscopic, multielement arrays of gold microelectrodes that are modified with SAMs possessing biologically-active terminal groups that bind specifically to target analyte species. Central to the creation of such arrays is the ability not only to desorb SAMs in environments innocuous to biological activity (as demonstrated) but to subsequently form new SAMs onto their targeted array elements under similar innocuous conditions. Acknowledgment. This research was funded by ONR Multidisciplinary University Research Initiative Grant N00014-95-1-1315, by ONR Grant N00014-95-1-0901, by ONR Defense University Research Instrumentation Program Grant N00014-95-1-0255, and by NSF Grant HRD-9450475 LA960627O
