Characterization of Self-Assembled Monolayers for Biosensor

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Langmuir 2000, 16, 1793-1800

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Characterization of Self-Assembled Monolayers for Biosensor Applications R. M. Nyquist,*,†,‡ A. S. Eberhardt,‡ L. A. Silks III,§ Z. Li, X. Yang,§ and B. I. Swanson‡ Los Alamos National Laboratory, Los Alamos, New Mexico 87545 Received January 8, 1999. In Final Form: September 16, 1999 Mixed monolayers of thiol-terminated poly(ethylene glycol) (PEG) and thioacetyl GM1 glycolipid on Au(111) were examined utilizing atomic force microscopy, infrared spectroscopy, and grazing incidence X-ray diffraction to determine the composition, structure, and morphology and to characterize the specific and nonspecific interactions with protein. These monolayer architectures are robust and are readily controlled to provide a network of receptor GM1 in the PEG-terminated matrix. However, we also find significant levels of nonspecific and nonnative protein binding that render this simplistic model system unsuitable for highly sensitive biosensor device applications.

Introduction Biosensors are designed to transduce highly specific biomolecular interactions into amplifiable signals. As advances in this technology are made, biosensors are becoming more reliable, more sensitive, more portable, and less expensive. To this end, a wide variety of transduction schemes have been employed, ranging from mass loading as in quartz crystal microbalance1 and surface acoustic waveguides2 to plasmon resonance,3 as well as electrochemical4 or biochemical approaches involving ion channels5 or surface impedance measurements, like colorimetric6,7 or fluorescence quenching.8 However, a requisite of any biosensor is the immobilization of sensing molecules on the transducing surface, and hence, the molecular organization has great impact on the bulk properties of the device as well as on the specificity of the desired sensing event and its successful transduction. The architecture of such an immobilized film would ideally consist of an inert molecular matrix that orients and homogeneously distributes the intended signaling molecules. Subsequently, control of device features hinges on a firm understanding of the interactions of the molecules with each other as well as with the substrate. Selfassembled monolayers (SAMs) are extremely stable and easily prepared with virtually any desired functionality, * Corresponding author. † Present address: Center for Biophysics and Computational Biology, University of Illinois, Urbana-Champaign, 505 S. Goodwin Ave., Urbana, IL 61801. ‡ Chemical Science and Technology Division. § Bioscience and Biotechnology Division. (1) Rickert, J.; Weiss, T.; Go¨pel, W. Sens. Actuators, B 1996, 31, 4550. (2) Yang, X.; Shi, J.; Johnson, S.; Swanson, B. Langmuir 1988, 4, 1505-1508. (3) Stelzle, M.; Weissmu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (4) Dong, S.; Li, J. Bioelectrochem. Bioenerg. 1997, 42, 7-13. (5) Cornell, B. A.; Braachmaksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580583. (6) Charych, D.; Cheng, Q.; Reichert, A.; Kuziemko, G.; Stroh, M.; Nagy, J. O.; Spevak, W.; Stevens, R. C. Chem. Biol. 1996, 3, 113-120. (7) Charych, D.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585-588. (8) (a) Song, X.; Nolan, J. P.; Swanson, B. I. J. Am. Chem. Soc., 1998, 120, 4873-4874. (b) Song, X.; Swanson, B. I. J. Am. Chem. Soc. 1998, 120, 11514-11515. (c) Song, X.; Swanson, B. I Langmuir, 1999, 15, 4710-4712.

as well as robust enough to survive transfer through an air-solution interface.9 A systematic characterization of the morphology of SAM biosensor architecture thus sheds light on possible means of controlling simple biomimetic architectures. In this paper, a two-component, coassembled SAM of thiolate molecules on a Au(111) substrate is examined as a prototypical biosensor matrix. The first component is terminated in poly(ethylene glycol) (PEG), six units attached to an eleven-unit hydrocarbon chain thiolate. The PEG unit serves to inhibit protein adsorption10,11 and was utilized in this study as a nonreactive matrix for the receptor molecules. The second component is the glycosphingolipid GM1, a natural membrane component that serves as a highly specific receptor for the cholera enterotoxin B subunit. For this study the GM1 was modified to allow anchoring to the substrate through a gold-thiolate bond. The GM1-cholera toxin interaction was chosen because it is biologically well-characterized.12-14 However, this study is meant to be illustrative of a matrix suitable for any similar protein-receptor pair. The resulting monolayers were characterized with atomic force microscopy, infrared spectroscopy, and grazing incidence X-ray diffraction in order to assess the potential of this system for use in a biosensor device. With the combination of these three techniques, the domain structure and composition was ascertained. The reactivity of the system was also examined, including both specific and nonspecific binding of the toxin, as well as its reactivity with a protein that does not specifically bind GM1. Materials and Methods The thioacetyl GM1 was synthesized from lyso-GM1 (Matreya, Inc.) and the succinimide ester of the corresponding 16-(thioacetyl)hexadecanoic acid. The primary amine on the sphingosine (9) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: Boston, 1991; and references cited therein. (10) Du, H.; Chandaroy, P.; Hui, S. W. Biochim. Biophys. Acta 1997, 1326, 236-248. (11) Malmsten, M.; Van Alstine, J. M. J. Colloid Interface Sci. 1996, 177, 502-512. (12) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361-1369. (13) Reed, R. A.; Mattai, J.; Shipley, G. G. Biochemistry 1987, 26, 824-832. (14) Hirai, M.; Iwase, H.; Arai, S.; Takizawa, T.; Hayashi, K. Biophys. J. 1998, 74, 1380-1387.

10.1021/la990018r CCC: $19.00 © 2000 American Chemical Society Published on Web 01/04/2000

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Figure 1. Schematic of envisioned mixed PEG-GM1 monolayer. Part A shows the GM1 molecule with indicated regions of synthetic modification. A schematic of the idealized monolayer is shown in part B, with isolated GM1 bound to the gold substrate and supported in a matrix of PEG-terminated molecules. Part C is an STM image of the actual gold surface utilized, 500 nm on a side. The left picture is of the surface topography, 15 nm full scale from black to white. On the right is a current image, 60 pA full scale, showing the flat terraces and single atom steps comprising the surface. This overall image underlies the following images such that the surface variations due to the monolayer are convoluted into the gold morphology. This sample has a decanethiol monolayer adsorbed to protect the surface during the measurement, and some etch pits are visible on the plateaus. tail of lyso-GM1 readily reacts with the activated carboxylic acid to give the desired thioacetyl GM1 (Figure 1A). The resulting thioacetyl GM1 was characterized by 500 MHz 1H NMR (data not shown). In addition to the standard resonances characteristic of the GM1 molecule, a singlet resonance at 2.30 ppm signified

the presence of the thioacetyl methyl group. We also prepared deactylneuramininic-lyso-GM1 by treatment of the GM1 with potassium hydroxide in methanol, which gives rise to the cleavage of the neuraminic acid acetyl group and the steryl tail in 8090% yield. It is known that reacylation (with aromatic and

Self-Assembled Monolayers for Biosensors aliphatic activated carboxylic acids) of the deactylneuramininiclyso-GM1 is selective at the ceramide amino group. Using the Sonnino et al. procedure,24 the sphingosine amino group was reacted with an activated 16-(thioacetyl)hexadecanoic acid to give the deacetylneuramininic thiolacetyl-GM1.15 The PEGterminated undecane thiol (referred to simply as PEG in this paper) was synthesized according to the procedure of Palegrosdemange et al.16 Several different types of gold substrates were utilized, appropriate to the characterization method. For grazing incidence X-ray diffraction (GIXD), single crystal Au(111) substrates were received from Aremco Products, Inc., polished, and precleaned with a gold iodide etch, and then the surface was prepared in ultrahigh vacuum with argon ion sputtering, alternated with and followed by an anneal to 500 °C to heal the surface. The surfaces were subsequently cleaned for reuse with acetone and nitric acid, followed by a flame-annealing process in which the crystal was heated to ∼700 °C in air, using the open flame of a propane or hydrogen torch. The gold was found to be miscut from (111) orientation by less than 0.5° across the 12 mm surface, with lateral domains of ∼2000 Å diameter. For Fourier transform infrared spectroscopy (FTIR), large (∼2 × 10 cm) rectangles of gold were sputtered onto silicon wafers in an argon ion PerkinElmer sputterer system. Prior to sputtering, the silicon substrates were rinsed several times with acetone and methanol and cleaned in UV ozone. An adhesive layer of titanium was deposited, followed by 1500-2000 Å of gold, and the substrates were used with no further preparation. For atomic force and scanning tunneling microscopy (AFM and STM), gold was electron-beam evaporated onto freshly cleaved mica at a rate of 1-2 Å/s to a thickness of 2000 Å, while the substrate was held at 300 °C. For FTIR, GIXD, and AFM, the monolayers were formed by immersing the appropriate gold substrate overnight in an 8 µM solution of molecules in dehydrated ethanol absolute (Quantum Chemical). Mixed monolayers were coassembled from PEG-GM1 mixtures with a total concentration 8 µM. For Figures 5 and 6, 8 µM solutions of decanethiol (Acros Organics) were used. After deposition, all samples were rinsed with ethanol, dried in an argon stream, and characterized ambiently. Film thicknesses were estimated using a Rudolph Research AutoEl III null ellipsometer at 632.8 nm and a 70° angle of incidence. Protein adsorption studies were performed with either CTX or albumin in order to ascertain the levels of specific and nonspecific protein adsorption, respectively. In each case, monolayers were immersed in an aqueous protein solution at room temperature for 10 min. They were then removed from the aqueous solution, rinsed in a gentle stream of deionized water (Millipore 18 MΩ) and dried under an argon stream before characterization in air. The cholera toxin B subunit (Sigma) was diluted in a 0.05 M, pH 7.4 Tris buffer to a concentration of 80 nM. The albumin (Sigma, 96%) was dissolved in 0.05 M Tris buffer, pH 7.4, to concentrations of 100 nM (comparable to that of the CTX), and 10 µM. FTIR was carried out in a nitrogen-purged Bruker spectrophotometer in grazing incidence reflection mode, at an incident angle of 77°, polarized parallel to the plane of incidence, and a resolution of 2 cm-1. The sum of 400 scans was utilized for the transform. GIXD measurements were performed at the X22B beamline at the National Synchrotron Light Source, Brookhaven National Laboratory, on a Huber four-circle diffractometer as described in a previous publication.17 The diffractometer was operated in a symmetric mode, utilizing 1.13 Å X-rays at an incident beam size of 0.5 mm horizontally and 1.0 mm vertically, with a vertical resolution of 0.03 Å-1. The samples were made on-site, using the flame-annealing technique described, and immediately placed in an ultrahigh-purity helium environment to protect from beam damage, with a beryllium window allowing X-ray transmission. Data were normalized to monitor intensities, by the Lorentz factor, by the out-of-plane detector resolution, and to the active (15) Sonnino; S.; Kirschner, G.; Ghidoni, R.; Acquotti, D.; Tettamanti, G. J. Lipid Res. 1985, 26, 248-257. (16) Palegrosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 12-20. (17) Eberhardt, A.; Fenter, P.; Eisenberger, P. Surf. Sci. 1998, 397, L285-L290.

Langmuir, Vol. 16, No. 4, 2000 1795 sample area (that part of the sample that is both illuminated by the X-ray beam and visible to the detector).18,19 AFM and STM were performed ambiently utilizing an extended Multimode Nanoscope IIIa. AFM images were acquired in tapping mode, with a 200 µm silicon tip (FESP, Digital Instruments) and a setpoint of 3 V. The STM image was taken at a bias of 300 mV and 50 pA current, using a Pt-Ir tip from Digital Instruments.

Results and Discussion The ideal sensing matrix would consist of individual receptor molecules supported in an inert matrix that orients and spaces them to minimize kinetic and steric constraints. If the system is not well-ordered, inappropriately reactive regions of the receptor molecule can be exposed and therefore more nonspecific adsorption is expected. Some biosensor transduction schemes employ a thin conductive gold film,4 and although the electrical properties are generally good, the surfaces used are generally not atomically flat. Thus, the thin film matrix traverses the rough surface, which can affect the number of defects in the monolayer film. The discrepancy between ideality and reality is illustrated in Figure 1. Part B is a schematic of the idealized monolayer, while part C is an STM picture of an actual gold surface, showing the atomically flat ∼1000 Å plateaus and the single molecule steps leading into badly ordered canyons. The substrate topography is important for understanding the AFM images of SAMs discussed later in the text, as all the monolayers overlay this fundamental roughness. Mixed monolayers were coassembled from a series of solutions of varying molar fractions of PEG and GM1, holding the overall molecular concentration constant. Although the substrates are exposed to solutions of varying GM1 concentration, the composition of the SAMs depends heavily on the kinetics of coassembly and often does not reflect the concentration of the solution. The resulting SAMs were therefore characterized by FTIR, and the spectra are shown in Figure 2. The first plot contains five spectra in the CH stretch regime of monolayers adsorbed from five relative concentrations indicated in mole fraction. The spectra are fit well with four peaks, as expected. In the case of 100% PEG, the peaks at 2920 and 2861 cm-1 are assigned to the CH2 antisymmetric and symmetric stretches in the alkane chain, respectively. Those at 2940 (a shoulder to the left) and 2893 cm-1 are assigned to the same stretches in the O-CH2 part of the molecule, as the PEG molecule does not contain any methyl groups.20 The PEG monolayer gave rise to an additional large C-O-C stretching band at 1120 cm-1, not shown here, as well as an O-CH2 wag mode at 1350 cm-1 (visible in Figure 2C). As the percentage of GM1 in the mixed monolayers increased, the CH2 stretches shifted to 2924 and 2853 cm-1, while some residual intensity remained at 2946 and 2892 cm-1, either due to an O-CH2 stretch or some residual CH3 in the molecule. No other distinct peaks were observed for GM1, although an amide or carboxylate mode might be expected; possibly these are shielded by the gold substrate.21,22 The change in integrated intensity for two of these peaks is shown in Figure 2B, fit to a simple noninteractive binary (18) Robinson, I. K. Aust. J. Phys. 1988, 41, 359-367. (19) Robinson, I. K. In Handbook on Synchrotron Radiation; Brown, G., Moncton, D. E., Eds.; Elsevier: New York, 1991; Vol. 3, Chapter 7. (20) Lin-Vien, D.; Colthup, N. B.; Fately, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: San Diego, CA, 1991. (21) Roeges, N. B. A Guide to the Complete Interpretation of Infrared Spectra of Organic Structures; John Wiley & Sons: New York, 1994. (22) Mu¨ller, E.; Giehl, A.; Schwarzmann, G.; Sandhoff, K.; Blume, A. Biophys. J. 1996, 71, 1400-1421.

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Figure 2. FTIR of mixed component monolayers. Two regions of FTIR spectra are shown here, with fits to the indicated peaks as a function of the molar fractional percentage of GM1 relative to PEG in the solution. Part A is of the CH stretch region, illustrating the evolution of the four main vibration bands in this spectrum for coassembled monolayers. Two of the vibrational intensities are quantified in part B. Plots C and D are of systems that have subsequently been exposed to CTX, showing the broad amide bands. In plot D the total area under both large peaks (amides I and II) is indicated, fitted to the functional form described in the text.

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Langmuir model. This model assumes the simplest possible form for competitive adsorption: that the PEG and GM1 adsorb independently with Langmuir kinetics, in which the deposition rate is simply proportional to the number of available sites on the gold substrate, with the concentrations of components in solution, and with some intrinsic reactivity rate specific to each molecule.17,23,24 More complex kinetic approaches are also possible, of course, but are not pursued in this paper.25,26 This fit also presumes that the change in integrated intensity is reasonably proportional to the number of molecules on the surface, an assumption that seems more reasonable in the presence of phase segregation and thus a similar local environment at most coverages, but cannot be proven without detailed knowledge of the relative IR absorption coefficient of the different molecules in their specific local environment. Given this large number of assumptions, no quantitative evaluation was made of the relative reaction rates of the two molecules, particularly in light of results that imply that multicomponent alkanethiolate SAMs are rarely in equilibrium with the solution until one component has been replaced on the surface by the other in ongoing exchange reactions.27 A few qualitative conclusions can be drawn, however. In both curves of Figure 2B, the distribution on the surface appears to be roughly equal for both molecules at a solution containing only 10% GM1. The acetyl-GM1, therefore, has a stronger affinity for the gold surface and a faster adsorption rate than the thiolate PEG. The specific protein-GM1 interaction was also characterized by FTIR after immersion of the monolayer in a solution of CTX. Upon exposure to CTX, new vibrational modes appeared in the protein amide region, indicated in Figure 2C, near 1675 and 1540 cm-1. These values are significantly shifted from the expected values of 1650 and 1530 cm-1 for the CTX amide I and II bands, indicating that although the toxin has become bound to the matrix, it is likely that the toxin becomes denatured when the sample passes through the air-water interface.28 Indeed, passage through the air-water interface is harsh treatment for a protein. Yet, it is not unreasonable to expect a biosensor to operate under such conditions. As the fraction of GM1 in the coassembly solution is increased, the amount of CTX that binds to the film also increases. The total intensity underneath the two amide peaks is shown in Figure 2D and fit to the same functional form as that in Figure 2B, expressing the amount of CTX on the surface relative to the amount of GM1 in the coassembly solution. Although the similar curve shape implies a proportionality relationship between the amount of GM1 on the surface and the amount of adsorbed CTX, a simple one-to-one relationship is unlikely. The amount of CTX reacted with the surface is proportional to the amount of GM1 in the monolayer preparation solution. Disappointingly, there is a small but significant amount of toxin adsorbed to the nonreactive 100% PEG monolayer. The domain structure as revealed by the AFM images reveals possible explanations for this nonspecific protein adsorption. Nonetheless, we have achieved reasonable control over the composition of these layers. (23) LeVan, M. D.; Vermeulen, T. J. Phys. Chem. 1981, 85, 32473250. (24) Forster, R. J.; Faulkner, L. R. Anal. Chem. 1995, 67, 12321239. (25) Qui-ones, I.; Guiochon, G. J. Chromatogr., A 1996, 734, 83-96. (26) Rowe, G. K.; Creager, S. E. Langmuir 1994, 10, 1186-1192. (27) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M.; Deutch, J. J. Phys. Chem. 1994, 98, 563-571. (28) Surewicz, W. K.; Leddy, J. J.; Mantsch, H. H. Biochemistry 1990, 29, 8106-8111.

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We turn now to correlation of the composition as determined by FTIR with the structure of the films. To get a rough estimate of the structure, we performed elliposometry to determine the average height of the film above the bare gold. These average heights of the coassembled monolayers depend greatly on the conformations of the molecules within the layer. Although the ellipsometric thicknesses are rough averages over macroscopic length scales and do not provide an unambiguous determination of the molecular ordering, they are helpful in combination with GIXD and AFM in assessing the possibilities for molecular ordering. We estimate from bond lengths that the fully extended height of the GM1 molecule used here is approximately 60 Å, while that of the PEG is about 32 Å. However, the average heights of well-ordered monolayers are expected to be slightly less than the fully stretched height. Indeed, the ellipsometric thickness of the 100% PEG monolayer was found to be 25 ( 2 Å, suggesting it may form a well-ordered film. The mixed monolayers varied in thickness from 25 to 35 Å, and therefore the mixed monolayers are also likely to be fairly well-ordered. However, the ellipsometric thickness of the 100% GM1 layer was only 26 ( 2 Å, suggesting that the pure GM1 monolayers may be disordered. This conjecture is not physically unreasonable; from a steric point of view, the large GM1 headgroup prohibits close packing in pure GM1 films. The fact that the mixed monolayers form thicker films despite the longer length of the GM1 molecules suggests that the GM1 is supported in a wellordered PEG matrix, particularly at low GM1 concentrations. The structure of the films suggested by the ellipsometry is corroborated by our GIXD and AFM results. While the ellipsometry provides macroscopic averages of the film heights, GIXD provides a sense of the packing at the molecular level on the atomically flat steps of the gold substrate. Figure 3 shows the GIXD signal from a completely and mostly PEG monolayer. The surface is found to be organized similarly to a monolayer of longchain alkanethiol, with the same broken hexagonal symmetry, indicated in the inset to Figure 3A, and the same high degree of order both in and out of plane.29 Since the monolayer is close to two-dimensional, the scattering intensity consists of diffuse rods perpendicular to the surface. The variation of intensity along these rods indicates the out-of-plane orientation of the molecules, that is, the tilt angle and direction. Figure 3A shows two transverse scans through the first-order hexagonal position indicated in the inset, while the inset shows a similar scan through the rectangular superlattice position. The width of the transverse scan provides a measure of the lateral coherence length on the surface, about 30 nm, again very similar to simple alkanethiolates. This remains unchanged with the addition of a small amount of GM1 into the matrix and the reaction with CTX. Figure 3B shows the integrated intensity fit from a series of transverse scans, like the one shown above, along the diffuse intensity perpendicular to the crystal surface. These curves are fit to a two-peak Gaussian. The peak positions indicate that the molecular tilt is very similar to a 14-carbon alkanethiol with a tilt angle of 30-35°, oriented within 15° of the next nearest neighbor direction.30 This is consistent with the 25 Å film thickness seen by ellipsometry. When the monolayer is exposed to CTX, with or without incorporated GM1, the relative intensities of the two peaks change, possibly due to change in (29) Fenter, P.; Eberhardt, A.; Eisenberger, P. Science 1994, 266, 1216-1218. (30) Fenter, P.; Eberhardt, A.; Liang, K. S.; Eisenberger, P. J. Chem. Phys. 1997, 106, 1600-1608.

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Figure 3. GIXD of mixed monolayers. The in- and out-of-plane scattering intensities are shown for the indicated points in reciprocal space. A schematic of the observed two-dimensional diffraction pattern is inset in part A, with the scans that are presented indicated by arrows. Part A is a transverse scan through a first-order hexagonal diffraction peak, (qx,qy,qz) ) (1.25, 0.725, 0.8 Å-1), fit to a Lorentzian line shape with width 0.8°, corresponding to a coherence length of 30 nm. The left inset is a scan through a rectangular superlattice peak at (0.63, 1.45, 0.8 Å-1). Part B shows several rod scans, the variation in intensity along a diffuse rod perpendicular to the sample surface, and positioned in-plane in the same peak as part A (1.25, 0.725 Å-1, qz), extending out of the page. The peak positions are indicated with a fit to two Gaussian line shapes. Each point in part B corresponds to the area under a transverse scan as in part A, but positioned at different qz.

scattering intensity and out-of-plane order, but the lateral order and tilt remain identical within error. Our results are also consistent with the notion that the two components are phase-segregated and the GM1 regions disordered, implying that mostly the PEG and possibly parts of the alkane chain from the GM1 are visible to diffraction and that the ordered regions of the PEG are not significantly affected by the CTX. Are there indeed phase-separated, poorly ordered, GM1rich domains, localized on the gold surface? From the GIXD results, the PEG and mixed monolayers seem fairly wellordered, and this order seems unaffected by exposure to the CTX. Why, then, do the FTIR results display nonspecific protein binding on the pure PEG monolayers? We have used atomic force microscopy of the monolayers to answer these questions. While others have focused on the AFM of GM1 and CTX at the level of molecular resolution to examine details of the structure of individual molecules and their interactions,31 that is not our aim. Rather, we wish to observe the mesoscopic domain structure that traverses the substrate topography shown in Figure 1C. (31) Yang, J.; Tamm, L. K.; Tillack, T. W.; Shao, Z. J. Mol. Biol. 1993, 229, 286-290.

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Figure 4. AFM of pure and mixed monolayers. This is a representational series of images, three sets of four images, 450 nm on a side. The left images are the surface topography, 15 nm full scale, and the right shows the phase response, 45° full scale. Pairs A and B show 100% PEG monolayer before and after exposure to CTX, respectively. Pairs C and D are the same for a 100% GM1 system; E and F, for a mixed monolayer with 2 mol % GM1 in solution, again as-deposited and exposed to CTX, respectively. The phase varies over a large dynamic range, reaching complete saturation in part F, while the topography images are overlaid onto the gold topography, as illustrated in Figure 1C.

We use the AFM images to provide information about the length scale on which the molecules aggregate or segregate and where the domains are in relation to the gold plateaus and canyons. Figure 4 presents a set of representative AFM images at the 450 nm length scale. In all cases the left image shows the surface topography at 15 nm dark to bright. Thus, on this length scale, we see the combination of

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monolayer and gold morphology expected from Figure 1B. The right image shows the phase response of the tapping mode AFM tip. Phase is a convolution of topography and the elasticity or stickiness of the surface,32,33 and changes in phase contrast can result from the tip encountering domains of different end functionality or different packing. Three pairs of images are presented, 100% PEG, 100% GM1, and 2% GM1 mixed monolayers, respectively; in each case, the first set showing monolayers prior to exposure to CTX and the second set showing monolayers after exposure to CTX. In the first pair of Figure 4A,B, the gold morphology dominates, with a small amount of texture ascribed to PEG ordering. After exposure to CTX, the PEG monolayer displays phase contrast only in the less ordered canyons between the atomically flat and ordered plateaus. The small features visible here are not individual proteins, which would appear as flattened spheres with diameter 30-55 Å and height 35 Å,34 but small aggregates of diameter 100-200 Å, and height 3040 Å above the surrounding background. The nonspecific binding of CTX to the pure PEG films, seen by FTIR, results from the CTX binding to regions where the monolayer is poorly ordered, as in the canyons between gold steps. A slightly longer molecule with more ethylene glycol units might help protect the surface in these regions but must remain short enough not to hinder the GM1. The next set of images, Figure 4C,D, are of the pure GM1 monolayer. As stated above, this layer is probably less well-ordered than PEG because the ellipsometric thickness is much shorter than molecular height. Also, some of the characteristic polarized FTIR peaks are very weak, which could be consistent with shielding from the gold substrate as the molecules adsorb somewhat laterally. The AFM images also bear this disorder in that the phase of the monolayer is almost featureless, implying a uniformly soft surface, characteristic of a disordered system. Unlike the pure PEG films, after exposure to CTX, the plateau surface as well as the canyons between the plateaus appear to be completely covered with randomly oriented toxin aggregates. Strong elasticity contrast results from the tip encountering domains of toxin and disordered GM1-rich domains. In the mixed systems of Figure 4E,F, many small regions are visible in the film, implying that the GM1 has phasesegregated from the PEG. Indeed, GM1-rich domains have been observed in mixed supported monolayers of GM1 and lipids,35 as well as SAMs.36,37 Within a single plateau, height variations of 10-15 Å imply that the GM1 is not everywhere fully extended; not surprising in a phasesegregated system. In this image, the small variations in phase due to the gold morphology, which are visible in the other phase images, are completely washed out. The phase contrast between CTX and monolayer completely saturates the image at this scale. Therefore, as seen in the GIXD data, the GM1-rich domains on the surface are likely to be disordered. On the plateau surface of the mixed monolayer, there is phase-segregated domain structure in the absence and presence of CTX. From Figure 4F, it is evident that, on the plateau surface, CTX forms a (32) Bar, G.; Rubin, S.; Parikh, A. N.; Swanson, B. I.; Zawodzinski, T. A.; Whangbo, M. H.; Langmuir 1997, 13, 373-377. (33) Overney, R. M.; Meyer, E.; Frommer, J.; Guntherodt, H. J.; Fujihira, M.; Takano, H.; Gotoh, Y. Langmuir 1994, 10, 1281-1286. (34) Cabral-Lilly, D.; Sosinsky, G. E.; Reed, R. A.; McDermott, M. R.; Shipley, G. G. Biophys. J. 1994, 66, 935-941. (35) Vie´, V.; Vanmau, N.; Lesniewska, E.; Goudonnet, J. P.; Heitz, F.; Legrimellec, C.; Langmuir 1998, 14, 4574-4583. (36) Stranick, S. J.; Atre, S. V.; Parikh, A. N.; Wood, M. C.; Allara, D. L.; Winograd, N.; Weiss, P. S. Nanotechnology 1996, 7, 438-442. (37) Tamada, K.; Hara, M.; Sasabe, H.; Knoll, W. Langmuir 1997, 13, 1558-1566.

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Figure 5. Nonspecific adsorption: AFM. A patterned monolayer is shown in which the surface is covered with a methylterminated alkanethiol using microcontact printing techniques. Only the circles are left bare and subsequently backfilled with the PEG-terminated thiol. The images are 36 µm on a side, the left showing topography with full scale contrast 10 nm and the right phase contrast, with 20° full scale contrast. Parts A and B show the monolayer before and after exposure to CTX, respectively. Part B is slightly distorted by lateral compression due to thermal drift during the measurement.

percolated network across the surface, connecting the small domains visible (Figure 4E), suggesting that GM1rich domains may extend in a thin, winding network homogeneously spread across the surface. The AFM images imply that the disordered regions of PEG are susceptible to nonspecific CTX adsorption. The toxin could distort the PEG monolayer to allow more CTX to adsorb than actually binds to GM1,38,39 but our GIXD results suggest not. On a nonatomically flat substrate such as gold, it is difficult to make perfect films. Figure 5 provides an illustration of this problem and underlining utility of PEG despite these problems. Shown is a microcontact printed surface in which the regions between the circles have been printed with decanethiol solution using an elastomer stamp and the circles subsequently filled in with PEG.40,41 Initially the slight height variation only indicates the relative sizes of these molecules, but after exposure to CTX, the PEG region is quite clearly visible as the protein has absorbed dramatically, and preferentially, on the methyl-terminated film.42 Different surfaces and their propensity to bind the CTX are quantitatively compared in the FTIR spectra of Figure 6A. The amide I and II bands are shown after exposure to CTX of bare gold, methyl-terminated monolayers, PEG, and GM1 films. The GM1 surface is clearly still the most reactive, followed closely by bare gold and the methylterminated decanethiol monolayer. As seen earlier, the complete PEG monolayer does attract some CTX. The lower gray PEG and GM1 lines in Figure 6A correspond to the FTIR absorbance in the absence of CTX. It seems (38) Mosser, G.; Brisson, A. J. Struct. Biol. 1991, 106, 191-198. (39) Ve´nien-Bryan, C.; Lenne, P. F.; Zakri, C.; Renault, A.; Brisson, A.; Legrand, J. F.; Berge, B. Biophys. J. 1998, 74, 2649-2657. (40) For details of the preparation procedure, see: Eberhardt, A. S.; Nyquist, R. M.; Parikh, A. N.; Zawodzinski, T.; Swanson, B. I. Langmuir 1999, 15, 1595-1598. (41) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498-1511. (42) Seigel, R. R.; Harder, P.; Dahint, R.; Grunze, M.; Josse, F.; Mrksich, M.; Whitesides, G. M. Anal. Chem. 1997, 69, 3321-3328.

Figure 6. Nonspecific adsorption: FTIR. These plots show the amide region for several different types of monolayers. Part A, from the top of the inset, indicates the spectrum for 100% GM1, bare gold, 100% decanethiol, and 100% PEG, all exposed to CTX. The gray lines at the bottom are the PEG and GM1 monolayers before exposure to CTX, for reference. In part B, the solid lines show GM1 before and after exposure to CTX, at the bottom and top of the plot, respectively, provided for reference. Between are bare gold and GM1 monolayers exposed to albumin at two different concentrations to measure nonspecific protein adsorption.

evident that CTX binds nonspecifically to disordered regions of the film. We also investigated the amount of binding of albumin to these films. Since there are no specific interactions between GM1 and albumin, all of the binding from albumin should be nonspecific. In Figure 6B, the GM1 exposed to toxin is used as a reference point to measure binding of albumin on bare and GM1-covered gold. Two different concentrations of albumin are used. The lower one, marked GM1+ALB, is at a concentration comparable to that of the CTX. Exposure to this solution results in only a trace FTIR signal. A significant signal is observed only when the concentration is increased another factor of 100, indicated as cALB. It is reassuring to note that even if GM1 is not well-ordered and exposes other functional groups to the surrounding medium, much of the specificity of its interaction with CTX is conserved. Conclusion Coassembled mixed monolayers of PEG and GM1 termination attached to the substrate through goldthiolate bonds were characterized to assess the usefulness of this prototypical system in a biosensor device. Control of the relative surface concentrations in the mixed monolayers is straightforwardly achieved through coassembly. The modified GM1 was found to have approximately 10 times higher affinity for the surface than the PEG thiolate. On the atomically flat gold terraces, the PEG surface order approaches that of simple alkanethiolates as examined by GIXD, and the monolayers were

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found to be very well-ordered at low GM1 concentrations. There is also partial phase segregation of the components, leading in this case to a network of receptor molecules on the gold terraces. Increased morphological control could also be achieved by combining replacement kinetics with the current approach.42,43 However, the rough surface of the gold precludes the feasibility of forming completely defect-free films via kinetic control. Because of these inevitable defects, these thin-film SAMs lack the ability to enable the protein to readily bind in its native, undenatured state. The levels of nonspecific adsorption observed for this system render the current configuration a less than optimal choice for sensors that strive for high sensitivity detection. While the films are robust and can be easily and consistently fabricated, they do not create an environment that enables the native, specific interactions desired for biosensing. Although GIXD shows that the in-plane order of mostly PEG monolayers does change, that the presence of CTX or GM1 may distort the out-of-plane ordering of the system, rendering it more vulnerable to further nonspecific toxin binding, and hence suggesting that the system simply may not mimic the cell membrane well enough to provide the same kind of specific interaction that takes place in vivo. This system also needs to be studied in situ, to verify that the FTIR signal of denatured toxin arises from the harsh air environment and not from the gold substrate itself. This system represents one of the simplest possible mimetic architectures for biosensor technology. Other, more biologically mimetic film schemes are currently being studied. For example, thin films of lipids affixed to gold, (43) Nyquist, R. M.; Eberhardt, A. S., Swanson, B. I. Manuscript in preparation.

Nyquist et al.

silicon, glass, or mica substrates mimic the cell membrane and conveniently integrate proteins or receptor molecules.44-47 These mimetic layers are readily prepared utilizing Langmuir-Blodgett48,49 or vesicle fusion50,51 techniques but are relatively fragile and are in most cases destroyed by passage through the air-water interface, making characterization difficult and limiting their use in portable, durable sensor devices. Hopefully, the lessons learned in the characterization of this system will guide the construction of more complex mimetic systems that may foster exclusively the native protein-receptor interactions. Acknowledgment. We thank J.-F. Bardeau, A. N. Parikh, and A.P. Shreve for helpful discussions. This work was carried out in part at the National Synchrotron Light Source, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences. R.M.N. acknowledges the support of the National Institutes of Health through Grant 5T3 GM08496-04. Work at LANL was supported by LDRD funding and performed under the auspices of the U.S. Department of Energy. LA990018R (44) Tien, H. T.; Wurster, S. H.; Ottova, A. L. Bioelectrochem. Bioenerg. 1997, 42, 77-94. (45) Sackmann, E. Science 1996, 271, 43-48. (46) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A-1127A. (47) Tamm, L. K.; McConnell, H. Biophys. J. 1985, 47, 105-113. (48) Ottova, A.; Tvorazek, V.; Racek, J.; Sabo, J.; Ziegler, W.; Hianik, T.; Tien, H. T. Supramol. Sci. 1997, 4, 101-112. (49) Zasadzinski, J. A.; Viswanathan, R.; Madsen, L.; Garnes, J.; Schwartz, D. K. Science 1994, 263, 1726-1733. (50) Plant, A. L. Langmuir 1993, 9, 2764-2767. (51) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316.