Surface Characterization of a Biochip Prototype for ... - ACS Publications

Time-of flight secondary ion mass spectrometry (ToF-SIMS) has been employed to characterize and evaluate the surface of a novel biochip prototype, con...
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Surface Characterization of a Biochip Prototype for Cell-Based Biosensor Applications S. A. Makohliso,†,‡,§,| D. Le´onard,†,∇ L. Giovangrandi,⊥ H. J. Mathieu,| M. Ilegems,| and P. Aebischer*,†,§ Centre for Gene Therapy, Centre Hospitalier Universitaire Vaudois, Pavillon 4, CH-1011 Lausanne, Switzerland, Department of Materials Science, LMCH, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland, Surface Analysis Group, Department of Materials Science, LMCH, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland, and Institute of Micro & Optoelectronics, Department of Physics, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland Received June 12, 1998. In Final Form: December 31, 1998 Time-of flight secondary ion mass spectrometry (ToF-SIMS) has been employed to characterize and evaluate the surface of a novel biochip prototype, consisting of an array of gold microelectrodes on which the laminin-derived oligopeptide CDPGYIGSR-NH2 was immobilized. The microelectrodes were isolated from each other via a thin film of amorphous Teflon (Teflon AF). Prior to studying biochip surfaces, characterization of gold surfaces (supported on oxidized silicon wafers) incorporating the oligopeptide was carried out, to serve as reference standards. With positive-mode ToF-SIMS, the whole peptide could be observed, and in addition, ions that were characteristic of the constituent amino acids of the oligopeptide could also be observed. The microfabrication process for biochip realization comprised several steps which included the use of compounds that could potentially contaminate the resultant surface. Therefore, it was important to investigate the chemical composition of these surfaces with the highest level of sensitivity. With ToF-SIMS imaging it was possible to detect oligopeptide-related ions only on the microelectrodes and nowhere else. Assessment of the spectral data from user-defined regions within the imaged areas revealed that the microelectrode and Teflon surfaces were devoid of any process-related contamination. In some cases, ToF-SIMS revealed some defects on the biochip surface, which would otherwise not be readily detectable, thereby offering some insight into areas that might require further optimization in the fabrication process.

Introduction The development of silicon-based devices incorporating bioactive molecules has been a subject of longstanding interest in the fields of biosensors and bioelectronics.1-3 The use of semiconductor fabrication technology has particularly facilitated the proliferation of this trend by providing, inter alia, capabilities to realize patterned deposition of biomolecules in miniature device configurations and other microsystems.3,4 In these applications, the outermost surface often forms the primary interface through which the device can associate with or interrogate its surrounding environment. Therefore, the uppermost layers govern the surface phenomena that may ensue upon device exposure to various envisaged applications.5,6 In the event that the application ultimately requires an * To whom correspondence should be addressed at the Centre Hospitalier Universitaire Vaudois. Telephone: +41-21-314 2461. Fax: +41-21-314 2468. E-mail: [email protected]. † These authors contributed equally to this work. ‡ Centre Hospitalier Universitaire Vaudois. § Department of Materials Science, LMCH, Swiss Federal Institute of Technology. ⊥ Department of Physics, Swiss Federal Institute of Technology. | Surface Analysis Group, Department of Materials Science, LMCH, Swiss Federal Institute of Technology. ∇ Current address: Institute of Physical Chemistry, DC-LCPPM, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland. (1) Kleinfeld, D.; Kahler, K. H.; Hockberger, P. E. J. Neurosci. 1988, 8, 4098. (2) Fodor, S. P.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767. (3) Connolly, P. Trends Biotechnol. 1994, 12, 123. (4) Flounders, A. W.; Brandon, D. L.; Bates, A. H. Biosens. Bioelectron. 1997, 12, 447. (5) Hagenhoff, B. Biosens. Bioelectron. 1995, 10, 885.

association of the synthetic system with a biological element, it is necessary that its surface is appropriately tailored to accommodate the foreign species. This may be achieved by a variety of surface modification techniques that alter device surface properties such that the desired surface/foreign-species interaction is attained.5,7,8 Introduction of undesired contaminants, however, is a realistic possibility, either during device fabrication or during the modification stage. These contaminants could conceivably compromise the outcome of the fabrication process significantly. Furthermore, if living cells are to contact this surface, even the smallest quantities of contamination material could pose an appreciable cytotoxic hazard.9 Therefore, a careful evaluation of the surface composition and properties of the device may be critical toward successful development of such systems. In addition to conferring useful biomaterial information about the surface, this effort may also provide additional insight that may culminate in fabrication process revision or optimization. In a prior study, a semiconductor-based fabrication process was developed to attain thin micropatterned films of an amorphous Teflon polymer, for purposes of biopatterning neural cell adhesion.10 However, the resulting surfaces failed to inhibit or biopattern cell adhesion, unless an additional substrate-baking treatment was included (6) Benninghoven, A.; Hagenhoff, B.; Niehuis, E. Anal. Chem. 1993, 65, 630A. (7) Go¨pel, W. Biosens. Bioelectron. 1995, 10, 853. (8) Ratner, B. D. Biosens. Bioelectron. 1995, 10, 797. (9) Black, J. Biological Performance of Materials, 2nd ed.; Marcel Dekker Inc.: New York, 1992; Chapter 16. (10) Makohliso, S. A.; Giovangrandi, L.; Le´onard, D.; Mathieu, H.-J.; Ilegems, M.; Aebischer, P. Biosens. Bioelectron. 1998, 13, 1227.

10.1021/la980688h CCC: $18.00 © 1999 American Chemical Society Published on Web 03/23/1999

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at the end. The loss of the surface inhibitory and, hence, biopatterning capabilities was attributed to the presence of very minute quantities of amino species. These species could be detected only with ToF-SIMS (time-of-flight secondary ion mass spectrometry) but not with ESCA (Electron Spectroscopy for chemical analysis). Therefore, from a biomaterial perspective these surfaces were not acceptable for their intended application without the additional treatment (i.e. baking), even though they would be in the microelectronics community. This example, therefore, provides an illustration of the additional prudence that is often required when designing and assessing surfaces intended for biomedical applications. The current report presents the results of an ongoing project toward the realization of a neural cell-based biochip sensing system, whose surface is engineered to possess two key features. The first feature is a surface strategy for the biopatterning or spatial control of tissue deposition and cell adhesion at the micron level. Thin films of amorphous Teflon (Teflon AF) were employed for this task, using the methodology developed in the above-mentioned study.10 The second feature is to optimize the cell/electronics interface, that is, promote and enhance coupling between the cell and the microelectrode surface. Toward this end, our primary candidates were the laminin-derived synthetic oligopeptides that have been previously demonstrated to enhance cell attachment and outgrowth via highly specific receptor/ligand interactions.11,12 We constructed a biochip surface consisting of an array of gold microelectrodes isolated from each other by a thin film of amorphous Teflon. As a final step, cell-adhesion-promoting molecules were grafted onto the gold microelectrode surfaces. The synthetic nonapeptide CDPGYIGSR-NH2, whose sequence resembles a cell-attachment-promoting subdomain of laminin, was utilized, and it was crucial that this biomolecule binds only to the gold electrodes and nowhere else. The intention was to immobilize this biomolecule directly onto gold via the thiol side group of its terminal cysteine (C) residue. This immobilization strategy is an advantageous simplification over previous oligopeptide-surface-grafting protocols,13,14 as it requires no surface pretreatment and coupling agents. The biochip fabrication process consisted of several steps that included the use of polymeric resins for substrate photolithographic patterning and fluorosilane compounds for improving fluoropolymer film adhesion. Some of the materials used for biochip construction (e.g. gold) are very prone to surface contamination,15,16 and some have been implicated in effecting cytotoxicity (e.g. aluminum).17,18 It was, therefore, important to carefully assess the resulting composite surface. Since this assessment required the highest level of detail feasible, time-of-flight secondary ion mass spectrometry (ToF-SIMS) proved to be the ideal tool for probing the surface chemical details of this system. ToF-SIMS has unsurpassed surface sensitivity, and its high-mass(11) Kleinman, H. K.; Sephel, G. C.; Tashiro, K.-I.; Weeks, B. S.; Burrous, B. A.; Adler, S. H.; Yamada, Y.; Martin, G. R. Ann. N. Y. Acad. Sci. 1990, 580, 302. (12) Graf, J.; Iwamoto, Y.; Sasaki, M.; Martin, G. R.; Kleinman, H. K.; Robey, F. A.; Yamada, Y. Cell 1987, 48, 989. (13) Ranieri, J. P.. Bellamkonda, R.; Bekos, E. J.; Vargo, T. G.; Gardella, J. A., Jr.; Aebischer, P. J. Biomed. Mater. Res. 1995, 29, 779. (14) Massia, S. P.; Hubbell, J. A. J. Cell Biol. 1991, 114, 1089. (15) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (16) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988, 4, 365. (17) Muller, J. P.; Bruinink, A. Acta Neuropathol. 1994, 88, 359. (18) Xie, C. X.; Mattson, M. P.; Lovell, M. A.; Yokel, R. A. Brain Res. 1996, 743, 271.

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resolution capabilities allow it to distinguish between structures of very similar molecular composition (i.e. isobar ions), thus making it particularly suitable for analyzing the complex molecular environment encountered in the present application.6,19-21 The ToF-SIMS imaging feature provides a capability of mapping the distribution of surface species with micron lateral resolution (or better)20,21 and was, therefore, particularly suited for this problem, as this application involves surfaces with microstructures of various material and chemical composition. With the help of parallel extraction of spectral data from user-defined regions within the imaged areas, it was possible to obtain the complete surface chemical composition of the various biochip areas studied. Experimental and Instrumentation Section Oligopeptide Immobilization on Reference Gold Surface. Gold reference surfaces were prepared by electron-beam or thermal evaporation of high-purity gold onto oxidized silicon wafers. Prior to gold evaporation, about 50 nm of titanium was deposited to serve as an adhesion promoter to subsequent gold deposition (300 nm). Substrates were stored in fluoroware containers until further use. Prior to oligopeptide surface immobilization, the wafers would be diced into smaller pieces of about 1 cm × 1 cm or any other convenient size and then briefly sonicated in pure ethanol (Merck, ACS grade). They would then be immersed into a 0.1 mM solution of oligopeptide in double-distilled, deionized water and left at room temperature overnight, followed by a thorough rinse with double-distilled, deionized water prior to use. The synthetic oligopeptide sequence used was the lamininderived CDPGYIGSR-NH2 (Anawa, Switzerland). Biochip Surface Fabrication. The fabrication process is summarized in Figure 1A. Using a positive contrast photoresist (Shipley S-1400-27), the gold electrode array pattern (including line contacts) was photolithographically imprinted onto a thermally oxidized silicon (100) wafer (Figure 1i). The next step involved depositing a thin layer of amorphous Teflon (Figure 1Aii). For one biochip (biochip-1), a layer of fluorosilane was deposited and bound onto the SiO2 regions of the surface prior to the deposition of the amorphous Teflon solution; for another biochip (biochip-2), the fluorosilane step was omitted. The purpose of the fluorination step was to improve Teflon film adhesion. Briefly, using 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PCR Inc., Gainsville, FL), a 1% fluorosilane solution in 2-propanol with 5% water was prepared. Just prior to use, a drop of dilute HCl (0.01 N) was added, and the wafer substrate was immersed for about 20 s. The solution was drained and the substrate rinsed with clean solvent and then dried at 100 °C for 15 min. Amorphous Teflon film fabrication was carried out according to a protocol in our earlier report.10 Briefly, Teflon AF1601 (Dupont Polymer, Wilmington, DE) solution (6% solids) was diluted down to a 3% solids solution using the perfluorinated solvent Fluorinet FC77 (3M, St Paul, MN). The solution was spin-coated on the fluorosilane-coated substrates at a speed of 3000 rpm for 20 s. They were then baked up to 110 °C in a stepwise fashion at a rate of 12 °C/min and kept at that temperature for 10 min, to remove the solvent. The temperature was then raised to 250 °C for about 5 min and finally to 340 °C for 5 min. The next step was to pattern-etch the Teflon layer in order to re-expose the electrodes. A thermally evaporated aluminum layer (150 nm) was deposited (Figure 1Aiii). Using photolithography (negative photoresist, Hoechst AZ 5214 E), the Al layer was selectively etched such that only the Teflon areas covering the electrode surfaces were exposed. The ingredients of the aluminum etching solution were 85% phosphoric acid, 65% nitric acid, and 96% acetic acid, in a ratio of 15:4:1, respectively. The substrate was then exposed to an O2 plasma (100 W, 7 min), to etch away the exposed amorphous Teflon. Subsequently, the remainder of (19) Mantus, D. S.; Ratner, B. D.; Carlson, B. A.; Moulder, J. F. Anal. Chem. 1993, 65, 1431. (20) Brummel, C. L.; Lee, I. N. W.; Zhou, Y.; Benkovic, S. J.; Winograd, N. Science 1994, 264, 399. (21) Bertrand, P.; Weng, L.-T. Mikrochim. Acta [Suppl.] 1996, 13, 167.

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Figure 1. (A, top) Schematic representation of the biochip microfabrication process. Features not drawn to scale. (B, bottom) An aerial view of the central region of the final biochip depicting the main component features of the surface. The ground pad, typically located at the top left-hand of the surface, has been excluded from the given field of view. Arrow 1 points to a microelectrode site (typically 40 µm × 40 µm) that should be entirely gold. Arrow 2 points to the intermediate region between the microelectrode site and the surrounding TeflonAF film, and the composition of this region is partly gold and partly Teflon-AF. Arrow 3 points to an electrode line extending to the periphery. Note that the electrode is actually buried under the Teflon-AF layer but is visible because the Teflon-AF layer is transparent. The additional ghost rings around the microelectrodes are image artifacts and not surface features. Scale bar ) 100 µm. the protective Al layer was stripped off with the etching solution, followed by a thorough rinse with double-distilled, deionized water and a 2-propanol wash, after which the substrate was finally baked at 180 °C for about 15 min (Figure 1Aiv). The final step (Figure 1Av) was to deposit the oligopeptides on the gold surfaces of the biochip. This was accomplished as described above (see Oligopeptide Immobilization on Reference Gold Surface), by covering the biochip surfaces with an oligopeptide solution, and allowing the reaction to proceed overnight. The oligopeptide solution was then drained, and the surfaces were thoroughly rinsed with distilled, deionized water and then blown dry with argon. ToF-SIMS analysis was carried out immediately thereafter. The geometric arrangement of the biochip surface structures consists of a square gold patch (2 mm × 2 mm) at the top left-

Makohliso et al. hand corner of the surface that could serve as the reference electrode in biochip electrical measurements and is henceforth termed the ground pad and a 4 × 4 array of 16 microelectrodes that is located at the center (Figure 1B). The typical dimensions of individual microelectrode sites were about 40 µm × 40 µm, with an interseparation distance of 100 µm. From each microelectrode site, there was a thin conducting gold line (electrode line) extending to the periphery, which was intended to provide unique electrical addressing of each microelectrode site in future bioelectronic applications. The typical thickness of the amorphous Teflon layer was about 0.4 µm-0.5 µm, meaning that the microelectrodes were receded into grooves of about 0.5 µm in depth. Time-of-Flight Secondary Ion Mass Spectroscopy. ToFSIMS spectra were obtained with the aid of a PHI-Evans TRIFT mass spectrometer.22 The sample surface was biased at (3 kV with respect to the grounded extraction electrode, for positiveand negative-mode analysis, respectively. A Ga+ source operating at 15 keV and giving a dc ion beam current of 850 pA was pulsed at a frequency of 5 kHz. The measured unbunched pulse widths were about 8 ns. The analysis time was 5 min. The estimated rastered area was a square of about 84 × 84 µm2 for the gold substrates and 126 × 126 µm2 for biochip analysis, giving rise to total primary ion doses of 9.0 × 1011 and 4.0 × 1011 ions/cm2 for the gold surface and biochip analyses, respectively. These values fall within the dose range required for static SIMS conditions.23 A pulsed low-energy (∼20 eV) electron flood gun was employed for charge neutralization. Spectra and images were acquired under high-mass-resolution conditions (bunched primary ion beam). The mass resolution obtained on a Si wafer under these operating conditions was m/∆m > 3500 at mass ) 28 (Si+/-). The lateral distribution of surface ion species on the biochips was obtained via the microprobe imaging mode. In this mode, the ion images are obtained from the (x,y) raster position of the primary ion beam on the sample surface. The region-ofinterest (ROI) analysis facility allows one to selectively obtain spectral data from user-defined areas of interest of a selected image. We carried out the ROI analysis in “full raster” mode; that is, the full raster images were acquired, but only the spectra from the predefined regions are displayed. A raster size of 63 µm was used, and the acquisition time was 10 min. This gave a total primary ion dose of 3.2 × 1012 ions/cm2, which is a good trade-off between signal intensity and static SIMS conditions. The ground pad of the biochip would typically be analyzed first, with up to six random fields selected. Next, each of the electrodes would be scanned and a few studied in more detail with the ROI analysis. Typically, the three regions-of-interest were selected as follows: the first one at the center of the microelectrode, the second one on the Teflon region, and the third one at the border of the microelectrode and the Teflon layer (Figure 4c).

Results and Discussion ToF-SIMS Analysis of Reference Samples: The chemical formula and molecular weight of the oligopeptide are C40H64O13N13S and 966.447 amu, respectively. In the negative mode, both the unmodified and peptide-modified surfaces exhibited a nitrogen-containing peak at m/z ) 26 (CN-), and the peptide-modified surfaces exhibited an additional peak at m/z ) 42 (CNO-) (data not shown). For the plain gold sample, the observed peak (CN-) is interpreted as surface contamination, whereas, for the peptide-grafted sample, it is not certain if this is still contamination or a peptide-related fragment. On the other hand, the peak at m/z ) 42 (CNO-) exclusively found on peptide-grafted sample, is believed to be related to peptide presence. A comparison of these data with an earlier study in which SIMS spectra of amino acids were studied shows good agreement.19 In that study, the negative-ion spectra (22) Schueler, B. W. Microsc. Microanal. Microstruct. 1992, 3, 119. (23) Briggs, D.; Brown A.; Vickerman, J. C. Handbook of Static Secondary Ion Mass Spectrometry (SIMS); John Wiley: Chichester, U.K., 1992.

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Figure 2. Positive-ion ToF-SIMS spectra of the reference gold samples: (a) unmodified reference sample; (b) peptide-modified reference sample, indicating the emergence of new peaks such as m/z ) 70 and 136, associated with peptide presence. (c) Close-up positive-ion ToF-SIMS spectrum of the peptide-modified reference gold surface at around m/z ) 966, the molecular weight of the peptide.

of the amino acids typically contained both the CN- and the CNO- ions, thus raising a strong possibility that the CN- signal we observed on the peptide-modified surfaces was derived primarily from the immobilized peptide and may have probably displaced the contamination-related CN- species. Besides this, the negative-ion spectra did not yield any additional useful information. In the positive mode, the unmodified reference gold surface displayed the prevalence of several hydrocarbon peaks, as evident at m/z ) 43, 57, and 69 Da, for which the main contributing peaks were C3H7+ (43.055 amu), C4H9+ (57.070 amu), and C5H9+ (69.070 amu) (Figure 2a). In addition, nitrogen-containing hydrocarbon peaks could be observed, as seen, for example, at m/z ) 30, 46, 74, and 130 due to the major contributions of CH4N+ (30.034 amu), C2H8N+ (46.066 amu), C4H12N+ (74.097 amu), and C7H18N2+ (130.147 amu)/C8H20N+ (130.160 amu). The high sensitivity of ToF-SIMS is evident here, as even highresolution ESCA cannot detect any nitrogen on these surfaces (unpublished observations). Another clearly observable contamination at m/z ) 149 was due to the phthalate ion C8H5O3+ (149.024 amu). The gold peak

(196.967 amu) was also clearly detectable. Nevertheless, despite this adventitious contamination, peptide incorporation could still be achieved with adequate success. In general, the positive ion spectra of the peptide-grafted samples were typically characterized by the emergence of new peaks and in some cases an increase in the relative intensity of the nitrogen-containing peaks previously observed on plain/unmodified gold. On the basis of previous literature reports on typical fragments of various poly(amino acids) arising from a ToF-SIMS analysis,19,20 it was possible to identify the various constituent amino acids of the oligopeptide on our peptide-modified surfaces. The peak at m/z ) 70, mainly due to C4H8N+ (70.066 amu), representative of the poly(amino acid) proline (P), was particularly intense and characteristic of the peptidegrafted surface. Moreover, the rest of the active region of the peptide (YIGSR) could be observed. Tyrosine (Y) was identified via the peak at 136.076 amu (C8H10NO+); I, via the peak at 86.097 amu (C5H12N+); G, 28.019 amu (CH2N+) and 30.034 amu (CH4N+); S, 60.045 amu (C2H6NO+); and R, 174.112 amu (C6H14N4O2+) (Figure 2b). As expected, the intensity of the gold peak (196.967 Da) observed on

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Figure 3. Positive-ion ToF-SIMS imaging of a defective microelectrode from biochip-2. The gold ion (196.600-197.400 amu) is imaged prior to surface modification with peptide (a) and after peptide modification (b). (c) shows the distribution of a peptidederived ion after peptide modification (C4H8N+; 70.016-70.144 amu). Note that images b and c have a different intensity scale to enhance contrast.

the peptide-grafted surfaces had diminished in comparison to the unmodified surface, a consequence of peptide presence. Some of the contamination-related peaks observed on unmodified samples (e.g. at m/z ) 130, 149) also diminished or disappeared on the peptide-modified surfaces, suggesting that the oligopeptide immobilization process may have displaced some of the adventitious organic contamination. This would be in concert with earlier findings in which it was reported that organic contamination on gold surfaces is typically displaced by organosulfur compounds.16 Finally, it was also possible to observe the whole peptide (Figure 2c). Taken together, the above data suggest not only that the peptide is present but also that it remains intact and undegraded by the immobilization process. Biochip ToF-SIMS Analysis. The main difference between biochip-1 and biochip-2 was the omission of the fluorosilane pretreatment step in the fabrication process for biochip-2. The analysis was started off by comparing the ground pads of the devices just prior to peptide deposition. They displayed close similarity to the plain/ unmodified reference gold sample analyzed before, imply-

ing that no significant additional contamination was present at the end of the immobilization process besides what was already observed, and more importantly for biochip-1, no traces of the fluorosilane compound were detected. An analysis of randomly selected regions on each of the biochip’s ground pads showed consistency in surface composition. However, on one occasion an unexpected spot or blob (∼25 to 50 µm in diameter) was observed for both biochips. ToF-SIMS imaging revealed that these spots were essentially of Teflon composition (data not shown). These were interpreted as specks of Teflon dust/defects stuck on the gold whose origin remains unclear. It is believed that the presence of these defective spots could be due to a slight underetching of the surface, a problem that could be resolved by increasing either the plasma etching time or the etching power. Next, the microelectrode sites were analyzed using the ion-imaging technique. The ion images obtained were basically as expected. For example, the area within the electrode site borders typically displayed only ions related to gold or gold-associated contamination (e.g. hydrocarbons) and was devoid of any Teflon-related ions. However,

Prototype for Biosensor Applications

Figure 4. Typical positive-ion ToF-SIMS images of a microelectrode from biochip-1: (a) distribution of peptide-derived ion, C4H8N+ (70.016-70.144 amu); (b) ion derived from Teflon AF, CF+ (30.920-31.007 amu). (c) H+ image (0.601-1.300 amu) used for the “region-of-interest” (ROI) analysis, indicating the areas where spectral information was obtained.

an exception was observed with biochip-2, as a few microelectrodes showed some defects. On one microelectrode, gold was also observed along the electrode line (i.e. the conducting line that would connect the electrode to external circuitry), where it was supposed to be covered with Teflon (Figure 3a). After the completion of the virgin biochip analyses, the substrates were surface-modified with the oligopeptide overnight and were subsequently analyzed in a manner similar to that used for the virgin substrates, that is, the ground pads first and then the microelectrode sites. Essentially, the ground pads of both biochips showed the presence of the peptide (data not shown), as evidenced by a close similarity between these spectra and those of the peptide-modified reference gold sample from earlier on (Figure 2b). Both ground pads displayed the presence of the major peptide-identifying peaks (m/z ) 70 and 136) in comparable intensities to each other. As before, however, imaging SIMS revealed the occasional presence of defective spots or patches. Spectral analysis of user-defined regions within the image, that is, region-of-interest (ROI) analysis, showed that the central regions of these patches were devoid of peptide peaks but were of Teflon composition,

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Figure 5. Close-up positive-ion TOF-SIMS spectra at around m/z ) 70, where the peptide-derived ion (C4H8N+, 70.066 amu) occurs. Note that the peptide ion is essentially not present on ROI #2, the Teflon area (b), as is the case on ROI #1 (a) and ROI #3 (c).

in agreement with what was observed before oligopeptide immobilization. Next, ion imaging of the microelectrode arrays was carried out. This analysis revealed peptide presence only on the microelectrode sites (as intended) and not on the Teflon or elsewhere (Figure 4a). The distribution of Teflonderived ions, such as CF+, was either at the border of or outside the microelectrode sites (Figure 4b). In addition, ROI analysis of microelectrode surfaces (ROI #1, as defined in Figure 4c) corroborated this observation by also showing a similarity in spectral composition between the microelectrodes (Figure 5a) and the peptide-modified reference gold surface, thus demonstrating that the peptide is clearly detectable within this region. The spectral composition of the Teflon region outside the microelectrode site, that is, ROI #2 (as defined in Figure 4c), also displayed close similarity to amorphous Teflon used in our earlier study,10 and furthermore, no peptide-related species were detected on ROI #2 even though the entire biochip surface had been in contact with oligopeptide solution (Figure 5b). The interfacial region between the microelectrode site and the surrounding Teflon layer (i.e. ROI #3 as defined in Figure 4c) consisted of both gold and Teflon, and thus low quantities of peptide-related ions could also be detected here (Figure 5c). The absence of the peptide outside the

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microelectrode region (i.e. ROI #2) implies that peptide incorporation was indeed highly specific to gold areas and no inadvertent adsorption occurred elsewhere. This finding was particularly important, as the presence of oligopeptide on Teflon would hamper its major role, that is, to inhibit cell attachment outside microelectrode sites. It has been reported that even femtomolar surface quantities of the oligopeptide could be sufficient to trigger bioactivity or cell adhesion,14 and therefore the high surface sensitivity of ToF-SIMS was particularly key here to demonstrate the absence of the oligopeptide outside the microelectrodes. The defective microelectrode site of biochip-2 observed before again displayed gold-related ions, both within the microelectrode central region and along the electrode line. The intensity of the gold ions was higher, though, on the electrode line than on the microelectrode site (Figure 3b), and the peptide-related ions could be clearly seen within the microelectrode central region, while they had faded significantly along the electrode line (Figure 3c). An examination of the spectral information revealed a nonnegligible presence of Teflon-related species exclusively observed on the electrode line, as well as a higher intensity of poly(dimethylsiloxane) (PDMS) on the electrode line than on the microelectrode site. In this instance, the defects observed on some of the microelectrodes of biochip-2 are attributed to the omission of the fluorosilane pretreatment, whose purpose was to improve fluoropolymer film adhesion. Interestingly, when these defects were observed, they were always located around the border of the gold/SiO2 lateral interface. This could be a result of overall poor film adhesion, making some regions of the film more prone to lifting off during removal (by wet etching) of the protective aluminum layer, particularly around the regions of structural and material dissimilarity, for example, the gold/SiO2 interface. The fluorosilane pretreatment alleviates this problem by improving film adhesion on the SiO2 region, thereby probably improving overall adhesion sufficiently to stabilize the film even at the gold/SiO2 interface. Nevertheless, while the current fabrication process provides acceptable and reproducible results, we believe that the incorporation of an adhesion-promoting strategy for the fluoropolymer on the gold regions would provide additional security against any such problems. Furthermore, the recovery of biochip gold surfaces after exposure to the fabrication process is attributed to the final plasma-etching step, which probably sputters off any fabrication-related surface contamination on the ground pad and microelectrode sites, consequently making it feasible to incorporate the oligopeptide on these surfaces. The results from the defective microelectrode of biochip-2 seem to corroborate this hypothesis, wherein the oligopeptide could be adequately detected on the microelectrode site (previously exposed to plasma) but not on the electrode line, which was erroneously exposed but protected from plasma exposure, thereby suggesting that the failure for the biomolecule to incorporate onto the electrode line may have been due to the presence of additional surface contamination such as unetched Teflon and PDMS. Since an aluminum layer was used during device patterning, we also checked for any aluminum-related species on all the surfaces. Aluminum derivatives are widely believed to be deleterious to cell viability and biocompatibility.17,18 A close examination of the various ROI spectra showed essentially no aluminum presence on the biochip surface (data not shown), implying that its removal was fully accomplished. An additional biochip surface was peptide-modified and analyzed about 4 weeks postfabrication. Analysis of both

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its ground pad and microelectrodes revealed that the extent of peptide incorporation on the surface was considerably low, perhaps even below minimal levels required for bioactivity. On the contrary, biochip-1, which was reanalyzed almost 8 weeks after its initial analysis, still displayed a reasonable peptide presence on its electrode surfaces even at this time point, even though other ambient surface contamination had also accumulated (data not shown). This result, therefore, may imply that if the surfaces are modified soon after fabrication, they may retain a peptide presence for longer periods, whereas if they are kept and modified later, peptide incorporation may not be adequately achieved. This may, thus, have implications toward batch processing and longterm handling of these surfaces. Summary and Conclusion The objective of this study was to investigate the surface chemical properties of a microstructured and multimolecular biochip surface, comprising an array of gold microelectrodes incorporating a cell-attachment-promoting oligopeptide (CDPGYIGSR-NH2), and a thin film of amorphous Teflon between the electrodes and everywhere else. Using high-resolution ToF-SIMS, it was demonstrated that the multistep biochip microfabrication process had introduced negligible or no surface contamination that could hinder surface incorporation of the oligopeptide or effect cytotoxicity. It was shown that the protocol employed for immobilizing the biomolecule was highly specific to gold surfaces and did not lead to inadvertent adsorption of the peptide on the amorphous Teflon. Biological assays are currently underway in order to test the bioactivity of these surfaces, as well as to corroborate the rest of the ToF-SIMS findings from this study. In some instances, the analysis also detected surface imperfections, which would have been difficult to observe with standard semiconductor inspection instruments. These observations were useful with regard to quality control and fabrication process evaluation. For example, the defects obtained upon omission of the fluorosilane step support the inclusion or retention of this step in the fabrication procedure, and the adventitious observation of an island of Teflon on the ground pad suggests that the final plasma-etching step may need further optimization. This, therefore, illustrates the additional insight provided by ToF-SIMS toward revision and optimization of procedures employed in device realization. Altogether, these results demonstrate a role for a surface engineering perspective in the development of biochip and biosensor technology. Finally, the use of DNA molecules and their subunits for purposes of realizing novel supramolecular materials and nanoelectronic applications has been gaining attention lately.24-26 Within that context, we envisage the future possibility of utilizing the oligopeptide template and the electrically addressable microelectrode sites as a foundation for engineering novel self-assembling or supramolecular biochip and biosensor technology. Acknowledgment. This work was in part supported by the Swiss Priority Program in Materials (modules 4.2A and 4.1B). LA980688H (24) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Science 1998, 391, 775. (25) Deming, T. J. Nature 1977, 390, 386. (26) Bethell, D.; Schiffrin, D. J. Nature 1996, 382, 581.