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Molecular Organization in SAMs Used for Neuronal Cell Growth Olena Palyvoda,*,† Andrey N. Bordenyuk,§ Achani K. Yatawara,§ Erik McCullen,† Chung-Chu Chen,| Alexander V. Benderskii,§ and Gregory W. Auner†,‡ Smart Sensors and Integrated Microsystems (SSIM), Electrical and Computer Engineering Department, Biomedical Engineering Department, and Department of Chemistry, Wayne State UniVersity, Detroit, Michigan 48202, and Medical Electronics and DeVice Technology Center, Industrial Technology Research Institute, Taiwan, ROC ReceiVed October 19, 2007. In Final Form: January 25, 2008 The attachment of cells onto solid supports is fundamental in the development of advanced biosensors or biochips. In this work, we characterize cortical neuron adhesion, growth, and distribution of an adhesive layer, depending on the molecular structure and composition . Neuronal networks are successfully grown on amino-terminated alkanethiol self-assembled monolayer (SAM) on a gold substrate without adhesion protein interfaces. Neuron adhesion efficiency was studied for amino-terminated, carboxy-terminated, and 1:1 mixed alkanethiol SAMs deposited on gold substrates. Atomic force microscopy and X-ray photoelectron spectroscopy were used to measure the roughness of gold substrate and thickness of SAM monolayers. Conformational ordering and ionic content of SAMs were characterized by vibrational sum frequency generation (VSFG) spectroscopy. Only pure amino-terminated SAMs provide efficient neuronal cell attachment. Ordering of the terminal amino groups does not affect efficiency of neuron adhesion. VSFG analysis shows that ordering of the terminal groups improves with decreasing surface roughness; however the number of gauche defects in alkane chains is independent of surface roughness. We monitor partial dissociation of carboxy groups in mixed SAMs that implies formation of NH3+ neighbors and appearance of catanionic structure. Such catanionic environment proved inefficient for neuron adhesion. Surface roughness of metal within the 0.7-2 nm range has little effect on the efficiency of neuron adhesion. This approach can be used to create new methods that help map structureproperty relationships of biohybrid systems.
Introduction The development of novel noninvasive strategies to directly interface the activities of cells with materials is important for enabling a broad class of hybrid microsystems that combine living and nonliving components.1,2 The highly developed ability of biological systems to recognize specifically designed features on the molecular scale can be utilized for the development of new investigation techniques for determination of the orientation and control of the conformation of surface active species. This is critical to the understanding of many industrial and biological processes, where surface properties and hence interfacial behavior are modified through the addition of surface active molecules to promote desired interactions. Self-Assembled monolayers have emerged as a promising approach for defining material features on length scales smaller than those conveniently accessible by lithography.1,3-5 SAMs formed with alkanethiols on gold have been widely studied due to their surface stability.6-18 Since they show specific sensitivity to irradiation, aliphatic and aromatic SAMs have been used as * Corresponding author. E-mail:
[email protected]. † Electrical and Computer Engineering Department, Wayne State University. ‡ Biomedical Engineering Department, Wayne State University. § Department of Chemistry, Wayne State University. | Industrial Technology Research Institute. (1) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Annu. ReV. Biomed. Eng. 2001, 3, 335-373. (2) Whitesides, G. M. W. A. MRS Bull. 2005, 31, 19-27. (3) Brinker, J. MRS Bull. 2004, 631-640. (4) Emmanuel, D.; Bruno, M.; Biebuyck, H. A.; Christoph, G. AdV.Mater. 1996, 8, 719-729. (5) Delamarche, E.; Bruno, M.; Biebuyck, H. A.; Gerber, C. AdV. Mater. 1996, 8, 719-729. (6) Kim, Y. T.; McCarley, R. L.; Bard, A. J. J. Phys. Chem. 1992, 96, 7416. (7) Fenter, P.; Eberhardt, A.; Eisenberger, P. Science. 1994, 266, 1216-1218. (8) McDermott, C. A.; McDemott, M. T.; Green, J. B.; Porter, M. D. J. Phys. Chem. 1995, 99, 13257.
positive and negative electron beam resist templates for growing polymer brushes with a resolution of 20 nm, for the manufacturing of biomolecular/biomolecular nanopatterns, and as spacers for the preparation of stable metal/organic monolayer/metal sandwich structures.19-23 The surface modifications can be easily introduced, for example the carboxy- and amino-terminated alkanethiol SAMs. These groups play major roles in a wide variety of chemical and biological phenomena such as membrane transport, molecular recognition, cell adhesion, cell signaling, DNA/protein interactions, and control of mineral growth.24,25 Molecular interactions at biological interfaces are very complicated due to the remarkably diverse character of biomolecules, and researchers have suggested implant applications, widely used for sensors, such as model substrates and bio-membrane mimetic studies of bio-molecules at surfaces and micrometer scale patterned surfaces.26-29 (9) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490. (10) Poirier, G. E. Langmuir. 1997, 13, 2019-2026. (11) Ron, H.; Cohen, H.; Matlis, S.; Rappaport, M.; Rubinstein, I. J. Phys. Chem. B. 1998, 102, 9861. (12) Byloos, M.; Al-Maznai, H.; Morin, M. J. Phys. Chem. B. 1999, 103, 6554. (13) Smith, A., D.; Wallwork, M. L.; Zhang, J.; Kirkham, J.; Robinson, C.; Marsh, A.; Wong, M. J. Phys. Chem. B. 2000, 104, 8862-8870. (14) Duan, L.; Garrett, S. J. J. Phys. Chem. B. 2001, 105, 9812. (15) Liu, D.; Szulczewski, G. J.; Kispert, L. D.; Primak, A.; Moore, T. A.; Moore, A. L.; Gust, D. J. Phys. Chem. B. 2002, 106, 2933. (16) Yang, X.; Perry, S. S. Langmuir. 2003, 19, 6135. (17) Ulman, A. Chem. ReV. 1996, 96, 1533. (18) Lavrich, D. J.; Wetterer, S. M.; Bernasek, S. L.; Scoles, G. J. Phys. Chem. B. 1998, 102, 3456. (19) Wang, H. C. S.; Li, L.; Jiang, S. Langmuir. 2005, 21, 2633-2636. (20) Vogt, A. K.; Brewer, G. J.; Offenhausser, A. Tissue Eng. 2005, 11, 17571767. (21) Suzuki, I.; Sugio, Y.; Moriguchi, H.; Hattori, A.; Yasuda, K.; Jimbo, Y. Nanobiotechnology 2004, 151, 116-121. (22) Schreiber, F. J. Phys.: Condens. Matter 2004, 16, R881-R900. (23) Schwartz, D. K. Annu. ReV. Phys. Chem. 2001, 52, 107-137. (24) Lynch, I.; Dawson, K. A.; Linse, S. Sci. STKE 2006, 2006, 14-18. (25) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 4th ed.; W.H. Freeman: New York, 2004.
10.1021/la7032675 CCC: $40.75 © 2008 American Chemical Society Published on Web 03/14/2008
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Intermolecular forces like hydrogen bonding, electrostatic interactions, and van der Waals forces in monolayers formed with amino- and carboxy-terminated alkanethiols are extensively studied using chemical force measurements13,30 and contact angle titrations31 as a function of pH. However, the key features, such as orientation and conformation of molecular groups at the interface play a major role in the above phenomena. Also, the chemical nature of the terminal functional group helps to modify the surface or the interface properties. The electrostatic interactions also play an important role in biochemical processes. It becomes increasingly clear that besides general effects of pH and ionic strength on enzymatic and binding processes many cases can be found where very specific arrangements of charged, dipolar, or hydrogen-bonding groups occur which perform a specific action.25 Examples include helices near ligand binding sites,32 metal ions in metalloproteins, salt bridges operating in virus assembly, allosteric regulation of hemoglobin, and hydrogen bonding in protein-DNA recognition.33 For example, it can be used for the detection and characterization for site-specific immobilization and conformation of single molecules or molecular aggregates.1,34,35 Neuron adhesion is a combination of biochemical and physiological processes, which involves many steps from recognizing suitable molecular groups to providing signals to bind these groups. The process is not very well understood. It is known that there are specific peptides providing specific binding and artificial polymers with a high content of amine groups, e.g., poly-D-lysine, providing nonspecific binding.36,37 Development of biological implants demands the creation of new substrates for development of neuronal networks. Such substrates must be biocompatible with neurons and tissues of the macroorganism and simultaneously be safe with respect to metabolization after being implanted in the organism.38 Moreover, it is important to understand the response of such substrates to physiological change in intraorganism conditions like secretion from various glands, which can change the chemical environment of the implant. Currently, substrates are based on gold films coated with SAM and with the proteins providing neuron adhesion. The disadvantage of such substrates is the presence of adhesive proteins, which can be metabolized causing destruction of the implant. Here, we present our efforts to develop biocompatible substrates based on SAMs on gold without an intermediate adhesive protein. We have modeled the ionic content of the biointerface by using different terminal groups of the SAM molecules and have studied the conformational structure of SAM with neuronal cell adhesion. (26) Slaughter, G. E.; Bieberich, E.; Wnek, G. E.; Wynne, K. J.; GuiseppiElie, A. Langmuir. 2004, 20, 7189-7200. (27) Gole, A.; Orendorff, C. J.; Murphy, C. J. Langmuir. 2004, 20, 71177122. (28) Masadome, T.; Ueda, A.; Kawakami, M. Anal. Lett. 2004, 37, 225-233. (29) Schaak, R. E.; Cable, R. E.; Leonard, B. M.; Norris, B. C. Langmuir. 2004, 20, 7293-7297. (30) Smith, A. D.; Wallwork, M. L.; Zhang, J.; Kirkham, J.; Robinson, C. Langmuir. 2001, 17, 1126-1131. (31) Lee, T. R.; Carey, R.; Biebuyck, H. A.; Whitesides, G. M. Langmuir. 1994, 10, 741-749. (32) Baker, E. N.; Drenth, J. Biological macromolecules and assemblies; Jurnak, F. A., McPherson, A., Eds.; Wiley: New York, 1987; Vol. 3, p 314. (33) Kandel, E. R.; Schwartz, J. H.; Jessel, T. M. Principles Of Neural Science, 4th ed.; McGraw-Hill: New York, 2000. (34) Whitesides, G. M. W. A. MRS Bull. 2006, 31. (35) Wilkinson, C. D. W. Eur. Cells and Mater. 2004, 8, 21-26. (36) Palyvoda, O.; Chen, C. Chu; Auner, G. William Biosens. Bioelectron. 2007, 22, 2346-2350. (37) Banker, G.; Goslin, K. Culturing NerVe Cells; MIT Press: Cambridge, MA, 1991. (38) Kandel, E. R.; Schwartz, J. H.; Jessel T. M. Principles Of Neural Science, 4th ed.; McGraw-Hill: New York, 2000.
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Materials and Methods 2.1. Preparation and Activation of Surface Hydroxyl Groups on Glass. Borofloat glass wafers (Mark Optics, Inc.) were cleaned with an aqueous detergent solution, rinsed with DI water, and then dried. The glass samples were then immersed in Piranha solution (containing 3 parts of concentrated sulfuric acid (Fluka) and one part of 30% hydrogen peroxide (Fluka)). The wafers were rinsed repeatedly with DI water, immersed in ethanol (Fluka), and dried under nitrogen atmosphere. 2.2. Gold Deposition. Three different types of thin films of gold were prepared by electron beam evaporation (Electron Beam Evaporator-BJD 1800), using Cr as an adhesion promoter and subsequent deposition of gold onto Schott Borofloat glass wafer (125 × 125 mm, 700 µm thick Mark Optics, Inc.) and mica V-1 quality surfaces (Electron Microscopy Sciences) in our clean room facilities (class 100). Evaporation was performed at a pressure of 1.0 × 10-7 Torr, and different deposition rates were used for three different gold surfaces. For simplicity, they were labeled as gold A, gold B, and gold C (gold deposited on mica). Gold A was prepared with 400 Å Cr layer (deposition rate 5 Å/sec) and subsequent deposition of 4000 Å Au layer (10 Å/sec). Gold B contains 50 Å Cr (1 Å/sec) and 500 Å Au (1 Å/sec). Gold C was prepared with 200 Å Cr (1 Å/sec) and 1000 Å Au (1 Å/sec). 2.3. SAM Coating Technique. Gold coated glass samples were cleaned with Piranha solution followed by distilled water and ethanol and allowed to dry. Three different 1 mM thiol solutions were prepared using ethanol (Fluka): 11-amino-1-undecanethiol, 10-carboxy-1decanethiol, and 1:1 mixture of 11-amino-1-undecanethiol and 10carboxy-1-decanethiol (Dojindo Molecular Technologies, the purity of SAMs is >90.0%). The gold coated samples were immersed in the freshly prepared solution for 24 h at room temperature to form the monolayers. The samples were rinsed with ethanol, ultrasonically cleaned in absolute ethanol for 2 min followed by rinsing with reagent grade ethanol, and then dried. Samples were stored in a N2 atmosphere until use. 2.4. Cell Culture Technique. All modified samples were immersed in 70% ethanol and air-dried in a sterile condition for 1 h. Embryonic day 18 (E18) rat (Sprague-Dawlley) cortical neurons were isolated as previously described37 by dissociation with 0.05% trypsin/EDTA (Gibco) and mechanical trituration in Ca2+ and Mg2+ free Hank’s buffered saline solution (HBSS). Cortical cells were gently resuspended in serum-free Gibco Neurobasal medium supplemented with 0.5 mM GlutaMax (Gibco), 25 µM glutamate (Gibco), B27 supplement (Gibco), penicillin (50 units/mL), and streptomycin (50 µg/mL) (Invitrogen). Cells were plated at a density of 75 × 103 cells/cm2 onto 1 cm × 1 cm samples. Plane glass and glass coated with poly-D-lysine (PDL, 50 µg/mL, Sigma) were used as positive and negative controls. After 24 h, the plating medium was replaced by Gibco Neurobasal medium without glutamate. Cultures were maintained at 37 °C in a 5% CO2 incubator for 6 days. 2.5. Immunocytochemistry for Neurons. For immunofluorescence, cells were fixed using 4% paraformaldehyde and permeabilised by 0.1% Triton X-100 (Sigma) in a phosphate-buffered saline (Gibco). Immunofluorescence was performed according to standard procedures using primary antibodies rabbit anti-rat neuron-specific enolase (NSE, Polysciences) directed against NSE (a neuron-specific marker protein) and FITC-labeled secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG(H + L) (molecular probes). All samples were counterstained with Phalloidin (Alexa Fluor, actin-specific marker), and nuclei were stained with Hoechst 33258 (Sigma). Samples were embedded in an antifade reagent (Invitrogen) and examined with a Nikon Eclipse TE2000-U microscope to identify neuron growth and distribution. For statistical analysis and reproducibility, verification of each experiment was repeated at least three times. 2.6. AFM Measurements. The surface morphology of different gold coated substrates and plane glass wafers were characterized by atomic force microscopy (AFM), operated in contact mode. Images were obtained at room temperature using a commercial microscope (Nanoscope III, Digital Instruments). 2.7. XPS Methods. X-ray photoelectron spectroscopy (XPS) was performed with a Perkin-Elmer 5500 system, using monochromatic
Molecular Organization in SAMs
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Figure 1. Immunocytochemistry images (×10) of the neurons on (1) gold thin film A coated with PDL, (2) gold thin film B coated with PDL, (3)gold coated on mica with PDL, (4) gold thin film A coated with amine (NH2)-terminated alkanethiolate SAM, (5) gold thin film B coated with amine (NH2)-terminated alkanethiolate SAM, (6) gold coated mica coated with amine (NH2)-terminated alkanethiolate SAM, (7) gold thin film coated with carboxylic acid (COOH)-terminated alkanethiolate SAM, and (8) gold thin film coated with a mixture of the carboxylic acid (COOH)-/amine (NH2)-terminated alkanethiolate SAM. Al KR radiation (1486.6 eV). Prior to the experiments, the binding energy scale was calibrated to the Au 4f peak at 83.8 eV and the Cu 2p at 932.4 eV. The fwhm for the Au 4f 7/2 peak was 0.88 eV. The experiments were performed at a power of 100 W, using a pass energy of 23.5 eV. Scans were performed for the Au 4f, C 1s, and O1s lines (and the N 1s and S 2p lines for the SAM coated samples) at a resolution of 0.10 eV/step. Data was collected at takeoff angles of 45, 60, and 75° (the takeoff angle is defined as the angle between the surface normal and the entrance slit of the detector. Initially, two similar plain Au samples were placed side by side, and the intensity of the Au 4f line was measured on each sample. The intensity varied by less than 4% between the two samples. Then, a SAM coated sample was placed side by side with a similar but uncoated sample, and the elemental region scans mentioned above were performed. For each sample, a Shirley-type background subtraction was performed, followed by fitting the Au 4f doublet with a GaussianLorentzian type function to calculate the Au peak area. This Au peak area is the intensity used in the calculations described below. 2.8. VSFG Spectroscopic Technique. A detailed description of our SFG setup was published elsewhere.39-41 Briefly, the vibrational sum frequency generation (VSFG) spectroscopy setup is based on a high power amplified femtosecond Ti-Sapphire laser system (Spectra Physics Spitfire sub-50 fs HP). Fifty percent of the 2 mJ fundamental output pulse (800 nm, fwhm < 35 fs, measured using a home-built noncollinear single shot autocorrelator) is used to pump the optical parametric amplifier (OPA 800C) followed by the signalidler difference frequency mixing in the 0.5 mm thick AgGaS2 crystal, producing 75 fs IR pulses (300 cm-1 spectral fwhm). The broadband VSFG scheme42,43 was employed, i.e., spectrally broad IR and spectrally narrow visible pulses were used to obtain SFG signals of high resolution in the frequency domain. Zero-chirp 4-f design pulse stretcher consisting of a grating, collimating lens, and a mirror equipped with a tunable slit was used to narrow the spectrum of the visible (800 nm) pulse from 430 cm-1 of the Spitfire’s output down to the desired spectral width. Since the line shapes in measured SFG spectra represent a convolution of visible pulses and the vibrational transition profile, the fwhm of visible pulse must be narrower than the fwhm of the SFG bands in order to perform deconvolution without ambiguity and find correct fwhm of the IR transitions. Thus, the fwhm of visible pulse was chosen to be 2 cm-1 for the CH stretch region and 10 cm-1 for the CdO region. The IR and visible beams were spatially and temporally overlapped at the sample surface with (39) Bordenyuk, A. N.; Jayathilake, H.; Benderskii, A. V. J. Phys. Chem. B. 2005, 109, 15941-15949. (40) Bordenyuk, A. N.; Benderskii, A. V J. Chem. Phys. 2005, 122, 134713. (41) Jayathilake, H. D.; Bordenyuk, A. N.; Weeraman, C.; Zhu, M.; Rosenblatt, C.; Benderskii, A. V. J. Chem. Phys. 2006, 125, 64706. (42) Richter, L. J.; Petralli-Mallow, T. P.; Stephenson, J. C. Opt. Lett. 1998, 23, 1594-1596. (43) Hommel, E. L.; Ma, G.; Allen, H. C. Anal. Sci. 2001, 17, 1325-1329.
the incidence angle for both beams at 65° from surface normal. The beam spot at the sample was ∼150 µm for both visible and IR beams. The laser power at the sample was 4 µJ/pulse for IR and 3-15 µJ/pulse for the visible, depending on the bandwidth, at 1 kHz repetition rate. The SFG signal reflected from the sample surface was recollimated, spatially and frequency filtered, passed through the 300 mm monochromator (Acton Spectra-Pro 300i), and detected using a liquid-nitrogen-cooled CCD (Princeton Instruments Spec10:100B, 100 × 1340 pixels). Polarization combination used for these experiments was PPP for SFG, visible, and IR beams, respectively. We have used CCD binning along the frequency axis in order to increase signal-to-noise ratio. The binning was chosen to be 2 pixels for CH stretch and 10 pixels for CO stretch regions providing spectrometer resolution of 1 cm-1 and 5 cm-1 in each region, respectively. The IR frequencies are calculated by subtracting the central frequency of the narrow visible pulse (measured using the same monochromator) from the SFG frequency. In addition, we calibrated the IR frequency scale using the known SFG surface spectrum of dimethyl sulfoxide (DMSO) and estimated our IR frequency calibration accuracy to be ( 2 cm-1. Both incident beams, IR and visible, are overlapped on the sample surface to generate the sum frequency signal. The SFG signal is enhanced when the incident infrared photon is resonant with a vibrational transition in the monolayer. Intensity of the SFG signal as a function of the IR frequency is (2) ISFG(ωIR) ∝ |χSFG (ωIR)|2 IIR(ωIR)Ivis(ωvis)
(1)
where IIR(ωIR) and Ivis(ωvis) are intensities of the IR and visible (2) is second order nonlinear susceptibility. The beams, and χSFG (2) χSFG(ωIR) can be represented as a series of coherently added Lorentzian lines, describing resonant IR transitions, and nonresonant terms, due to the contribution of gold substrate, using the following equation: bV
n
(2) χSFG (ωIR) ) aNReiφ +
∑ω V)1
IR
- ωV + iΓV
(2)
Here, the first term represents the nonresonant contribution of amplitude aNR, and φ is the phase difference between resonant and nonresonant components. The second term is the sum of the resonant transitions, with amplitude bV/ΓV, frequency ωV, and line width ΓV for the Vth vibrational mode.
Results Microphotographs, see Figure 1, present typical neuronal growth on tested surfaces. Our results show that the surfaces coated by PDL are biocompatible and nontoxic for neuronal
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Figure 2. AFM topographic images (image size ) 5 µm × 5 µm) recorded for plane glass, gold type A, gold type B, and gold type C deposited on the mica. The dimensions are shown in the cursor profiles.
correctly, with the sulfur atom at the greatest depth from the surface and the functional tail groups being closest to the surface. XPS data were used to estimate the thickness of the SAM layer. The thickness of the monolayer can be calculated from the relationship:
Id ) I0 exp
-d (λ cos θ)
(3)
where Id is the intensity of the Au signal attenuated by the SAM overlayer of thickness d, I0 is the intensity of the plane uncoated Au, λ is the attenuation length, and θ is the takeoff angle. The attenuation length for n-alkenethiol monolayers can be derived from the empirical relation:45 o
λ(A) ) 9.0 + 0.022KE(eV) Figure 3. Plot of Au intensity ratio versus 1/cos(θ) for the gold A and gold B samples.
cells, but only surfaces coated by amine (NH2)-terminated SAMs are good candidates for neuronal cell attachment. For plain glass, plain gold surfaces, and for the carboxy-terminated SAMs and the 1:1 mixture of the amino- and carboxy-terminated SAMs, significant cell adhesion was not observed (panels 7 and 8 of Figure 1). The rms surface roughness for plane surfaces was determined by AFM contact mode analysis. The typical values found, with the 5 × 5 µm2 fields of view, are shown in Figure 2. The average heights were 0.391 nm for glass surface, 1.918 nm for gold A, 0.714 nm for gold B, and 0.852 nm for gold deposited onto mica. Neuronal cell attachment is not significantly influenced by surface morphology within the 1-100 nm range of rms surface roughness, as measured by AFM. We have also examined the effects of surface morphology of the aminoterminated SAMs with respect to neuronal cell attachment using visual cell distribution analysis. Here we observe the growth and the morphology of the cortical neuronal cell networks to be the same as those on the control PDL-covered glass surfaces (panels 1-6 of Figure 1). Surface properties were verified by XPS (Figure 3), and the values obtained are in agreement with those reported in literature.44 Variable-angle XPS was used to obtain information on SAM composition and assembled alkanethiol orientation. Values obtained are in agreement with theoretical calculations, within the sensitivity of the instrument. Comparing atomic percentages at multiple depths of analysis indicated that SAMs were oriented (44) Bain, C. D.; Whitesides, G. M. Langmuir. 1989, 5, 1370-1378.
(4)
For the Au 4f7/2 photoelectrons, this gives an attenuation length of about λ ) 40 Å. Therefore, a plot of ln(Id/I0) versus 1/cosθ will give an estimate of the thickness. This plot is shown in Figure 3 for the Gold A and Gold B samples. The lines drawn through the points are a linear fit to the data. The slopes for the fits were 0.3 ( 0.02 and 0.43 ( 0.05 for the Gold A and B samples, respectively. This gives an estimate of the overlay thickness of d ) 12 ( 0.8 Å for Gold A and d ) 16.8 ( 2.0 Å for Gold B. For these types of SAMs on gold, the chains are thought to tilt at an angle of 30° to the surface normal with a thickness per C-C bond of 1.1 Å. This would give an overlay thickness of about 12.1 Å for our SAMs (n ) 11), which is within the fit error for both Gold A and B samples. The variations between the two data are thought to be due to local surface roughness variations as well as possible radiation damage to the samples from the X-ray source, which we tried to minimize by using a relatively low source power of 100 W. All SFG measurements were performed at room temperature for PPP polarization. We have separately studied the CH (28003000 cm-1), the CdO, (1450-2000 cm-1), and the OH and NH (3100-3550 cm-1) stretch regions. For amino-terminated and mixed SAMs, the NH stretch transitions were found to be strongly broadened presumably due to hydrogen bonding with neighbors and adsorbed water molecules and therefore are not informative about SAM structure. The same result was obtained for carboxyterminated and mixed SAMs for the OH stretch mode. For this reason, spectra of OH and NH regions are not presented here. Our SFG spectroscopic analysis is based on consideration of spectra for CH stretch and CdO (carbonyl) stretch regions. (45) Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y. T.; Parikh, A. N.; Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152-7167.
Molecular Organization in SAMs
Figure 4. VSFG spectra for (A) Carboxy-terminated SAM, (B) Amino-terminated SAM, and (C) 1:1 mix SAMs on gold Type A with PPP polarization.
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Figure 6. VSFG spectra for (A) carboxy-terminated SAM, (B) amino-terminated SAM, and (C) 1:1 mix SAMs on gold Type C with PPP polarization.
Figure 5. VSFG spectra for (A) carboxy-terminated SAM, (B) amino-terminated SAM, and (C) 1:1 mix SAMs on gold Type B with PPP polarization.
spectra taken 2 weeks after sample preparation. The trapped ethanol molecules in the ordered SAMs can become regularly oriented and contribute to the SFG response. SFG spectra recorded 1 month after deposition do not exhibit any presence of methyl stretch which confirms our supposition. As the intensity of these methyl transitions is small, their presence should not affect the results of our analysis. We distinguish four different transitions related to CH2 stretch modes, namely, CH2 symmetric stretch (d+) at ∼2855 cm-1, CH2 symmetric stretch of the group adjacent to the terminal group (d+w) at ∼2866 cm-1, CH2 asymmetric mode at (d-) at ∼2925 cm-1 and Fermi resonance (d+FR) at ∼2901 cm-1.46-48 For carboxy-terminated SAMs, all CH2 transitions are very weak and broad regardless of gold type, see panel A of Figures 4-6. Carboxy-terminated SAMs, see Figure 7, exhibit only one transition in the CdO stretch region. It is assigned to carbonyl stretch of the -COOH group, ∼1767 cm-1 (Figure 7C,D and Tables 4 and 5).49 The band is rather broad, probably due to the hydrogen bonding with adsorbed water vapor and neighboring molecules. It is barely seen and very broad for gold A and more easily distinguished being relatively narrow for gold B. This indicates the better ordered structure of the terminal carboxy groups for SAM deposited on gold B, which has a lower surface roughness. Spectra of mixed SAMs show three transitions in CO stretch region (Figure 7). Two of them can be readily assigned to the carbonyl stretch of the hydrogen-bonded CdO moiety of COOH group, ∼1765 cm-1 and the asymmetric CO stretch coming from the anionic COO- state, ∼1654 cm-1, due to the delocalization of the double bond (Figure 7A,B and Tables 4 and 5).49 The carbonyl stretch transition in mixed SAMs is stronger
The CH stretch region is dominated by CH2 stretch modes and Fermi resonance, see Figures 4-6. However, there are minor contributions that can be related to the CH3 stretch transitions. Their appearance can be attributed to either impurities or trace amounts of ethanol solvent trapped by the SAMs during the deposition process, since these bands disappear from the VSFG
(46) Himmelhaus, M.; Eisert, F.; Buck, M.; Grunze, M. J. Phys. Chem. B. 2000, 104, 576-584. (47) Macphail, R. A.; Strauss, H. L.; Snyder, R. G.; Elliger, C. A. J. Phys. Chem.. 1984, 88, 334-341. (48) Chow, B. C.; Ehler, T. T.; Furtak, T. E. Appl. Phys. B 2002, 74, 395-399. (49) Silverstein, R. M.; Bassler, G. C.; Morril, T. C. Spectrometric Identification of organic compounds; Wiley: New York, 1991.
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Figure 7. Normalized VSFG spectra for (A) 1:1 mix SAMs on gold Type A, (B) 1:1 mix SAMs on gold Type B, (C) 10-carboxy-1-decanethiol SAMs on gold Type A, and (D) 10-carboxy-1-decanethiol SAMs on gold Type B with PPP polarization in CdO region. Three transitions, (i) 1654 cm-1, (ii) ∼1700 cm-1, and (iii) ∼1765 cm-1, are observed for mix SAMs on both gold types A and B, and one transition is observed for carboxy-terminated SAMs on the above two gold types.
and narrower than that of the pure carboxy-terminated SAMs, despite the difference by a factor of 2 in the surface coverage of the carboxy groups, which should lead to a factor of 4 in the VSFG line intensity. Therefore, mixing of carboxy- and aminoterminated SAMs causes better ordering of the terminal groups. The presence of an ionized carboxylate group allows us to provide evidence for the existence of NH3+ groups, since amino group is the most efficient hydrogen acceptor in our system. In such a case, the nonionized amine and carboxyl groups of mixed SAM have a catanionic environment which may be a reason for better ordering of the terminal groups.
Discussion A fundamental understanding of substrate-directed control of cell function is critical to the rational design of surfaces relevant to biomaterials, tissue engineering scaffolds, and in vitro culture supports for biotechnological applications. In the case of chemically modified surfaces, polymers do not play a main role but promote specific cell attachment. Cell and tissue surfaces generally are net negatively charged.50 Widely used in cell culture technology as a host polymer for enhancing the cell attachment, poly-D-lysine (PDL) is an example of a simple class of synthetic polymers that can produce chiral secondary structures spontaneously that have a lot of amine groups. The native adhesion proteins (laminin, collagen, fibronectin, etc.) are found primarily in basement membranes, often tethered to collagen and fibrillar networks. Since each of these proteins contains multiple biologically active adhesion signal peptides, they collectively exert a growth regulatory function on cell migration, spreading, and (50) Lodish, H.; Scott, M. P.; Matsudaira, P.; Darnell, J.; Zipursky, L.; Kaiser, C. A.; Berk, A.; Krieger, M. Molecular Cell Biology; W. H. Freeman and Co.: Portland, OR, 2003.
differentiation. Also, the specificities of multifunction in cellular proteins responsible for cell attachment are derived from their three-dimensional configurations. Depending on the application, specifically engineered surfaces may either prevent or enhance cell/tissue growth with an appropriate host response.25,38,50 The main goal of this work was to gain insight into the formation and organization of the organic monolayer assembly. In the present work, using model substrates with well-controlled surface properties, we demonstrate that a simple surface chemistry could modulate cortical neuronal cell adhesion in a serum free medium. The model of cortical neurons was also chosen for many reasons. First, the neuron is polarized, possessing receptive dendrites on one end and axons with synaptic terminals at the other with high selectivity to the substrate. Second, the neuronal cell is electrically and chemically excitable and has a complete proliferation block. Third, nerve cells form specific signaling networks that mediate specific behaviors. Fourth, plasticity at chemical synapses that can give rise to further physiological changes that lead to anatomical changes, including pruning of preexisting connections and even growth of new connections,38 is of importance to the creation of neuronal prosthesis for neuronal recovery processes. The thiol chemistry was used to deposit a self-assembled monolayer (SAM) separately on gold surfaces with carboxyl (COOH)- and amine (NH2)-terminated SAMs to mimic negatively and positively charged surfaces, respectively. At the experimental physiological pH of 7.4, the COOHterminated SAMs present a negatively charged surface (COO-), while the NH2-terminated SAMs display a positively charged surface (NH3+). A combination of (COO-) and (NH3+) SAMs provides electrostatic interaction between charged or dipolar substituents and the reacting proton [Kirkwood-Westheimer theory].51
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Table 1. VSFG Experimental Results for 1:1 Mix-Terminated, Amino-Terminated, and Carboxy-Terminated SAMs on Gold Type A with PPP Polarization terminal group
transition
ω (cm-1)
b
gamma (Γ)
|b/Γ|2
aNR
phi (φ)
1:1 mix
d+ d+w d+FR d+FR /ddr+ r+FR rd+ d+w d+FR dr+ r+FR rd+ d+w d+FR d+FR /ddr+ r+FR r-
2855.1 2866.0 2899.2 2908.3 2926.5 2879.4 2945.6 2961.8 2855.4 2866.5 2905.2 2926.3 2878.6 2945.2 2960.5 2855.5 2865.3 2898.1 2907.3 2925.5 2879.0 2945.6 2960.8
-4.4075 -0.3472 -2.1217 -0.6283 -15.699 -1.9373 -0.5071 -2.7596 -7.4704 -0.1361 -9.0798 -7.1726 -0.4452 -0.1936 -0.3649 -1.0013 -0.6467 -0.6409 -0.2952 -11.483 -1.2246 -0.5647 -1.6048
8.0256 2.1887 6.4645 3.5349 16.029 6.7215 1.9741 5.9847 10.935 1.869 16.237 9.448 3.641 2.018 3.763 4.6937 3.9363 4.1677 3.1808 17.498 4.8304 2.8126 5.1938
0.3016 0.0252 0.1077 0.0316 0.9592 0.0831 0.0660 0.2126 0.4667 0.0053 0.3127 0.5763 0.0149 0.0092 0.0094 0.0455 0.0270 0.0236 0.0086 0.4307 0.0643 0.0403 0.0955
3.29
-1.891
2.81
-1.595
2.55
-1.681
amino
carboxy
For the present study, we have used vibrational sum frequency generation (VSFG) to investigate the conformational structure of the above-mentioned SAMs. VSFG spectroscopy is a highly surface specific nonlinear optical technique which allows detailed characterization of the molecular structure (orientation and conformation) at surfaces and interfaces39,52 including the orientation of the terminal groups.41,42,46,48,53 The surface specificity arises from the second-order nature of the response. Under the electric dipole approximation, the second-order nonlinear response is forbidden in media with inversion symmetry.54-56 As a result of this surface selection rule, the methylene groups, except groups adjacent to the terminal ones, of relatively long hydrocarbon chains in all-trans conformation do not contribute to the VSFG signal. This is due to the cancellation of dipole responses from neighboring methylene groups as a result of their interference. Therefore, in our study for the case of an all-trans conformation of alkane chains, we could expect the contribution from only methylene groups adjacent to the terminal group (d+w). However, if gauche defects are present in the chain, CH2 stretch vibrations (d+, d-, d+FR) at the defect position become VSFG-allowed due to the break in the inversion symmetry and appear in the VSFG spectrum. The intensities of the CH2 (Figures 4-6) transitions for carboxyterminated SAMs are comparable to the ones from methyl groups, which are present in negligible amount due to the residual ethanol. There are two possible scenarios that may result in observation of weak and broad transitions. First, the number of gauche defects is negligible, i.e., the SAM is in a highly ordered nearly all-trans conformation. Second, the SAM is so poorly ordered and the number of gauche defect is so high that they destructively interfere with one another forming a nearly centrosymmetric structure themselves. It is difficult to assume the first scenario, i.e., the presence of a nearly crystalline, highly ordered structure, with just a few gauche defects, in light of the surface roughness and (51) Bowden, K.; Grubbs, E. J. Chem. Soc. ReV. 1996, 25, 171-177. (52) Weeraman, C.; Yatawara, A. K.; Bordenyuk, A. N.; Benderskii, A. V. J. Amer. Chem. Soc. 2006, 128 14244-14245. (53) Nishi, N.; Hobara, D.; Yamamoto, M.; Kakiuchi, T. J. Chem. Phys. 2003, 118, 1904-1911. (54) Shen, Y. R. 1984, New York, Wiley. (55) Bloembergen, N. Opt. Acta 1966, 13, 311. (56) Bloembergen, N.; Simmon, H. J.; Lee, C. H. Phys. ReV. 1969, 181, 1261.
the analysis of the CH stretch frequencies (vide infra). CH2 stretch transitions are more intense and narrower for amino-terminated SAM, which may testify to the better ordered conformational structure of amino-terminated SAM compared to carboxyterminated one. The amino-terminated SAM has a smaller size of the terminal group than the carboxy-terminated SAM. In addition, carboxy groups readily form lateral hydrogen bonds which may affect ordering of the underlying alkane chains. Thus, we suggest that the poor organization of carboxy-alkanethiol SAMs on gold is probably due to the big terminal groups which can easily form hydrogen bonds and hence disturb the structure of alkane chains. As we showed in the Results section, surface roughness (Figure 2) is an important parameter affecting the number of functional terminal groups situated at the surface of the monolayer, not buried within the monolayer, and that are directly accessible by the binding sites of the cell. The intensities of CO stretch transitions in mixed SAMs (Figure 7A,B) do not show considerable variation for gold surfaces having different roughness unlike for pure carboxy-terminated SAMs, Figure 7C,D. This may indicate a strong stabilizing effect of the catanionic surface on the orientational ordering of the terminal groups. Catanionic surface does not seem to be affecting the conformational order of the amino-terminated chains because the parameters of CH2 transitions do not differ for the amino-terminated and the mixed SAMs, panels B and C of Figures 4-6. However, it improves considerably the ordering of chains with carboxy-terminated molecules because the CH2 transitions for the mixed SAMs are more intense and narrow than for the carboxy-terminated SAMs, Figure 7. In our investigation of SAMs, the parameters of CH2 transitions are comparable for all three types of gold substrates, Figures 4-6 and Tables 1-3. Thus, improving roughness of the gold films by a factor of 3, from 0.7 to 2 nm, does not seem to be a determining factor for the conformational structure of alkane chains with amino, carboxy, and mixed terminal groups. Nevertheless, for the SAM samples, the alkane chains have a lot of gauche defects, resulting in the appearance of CH2 transitions in SFG spectra in contrast to pure alkanethiols.42 It was previously shown57 that CH2 stretch frequencies are different for alkanethiols in the liquid and crystalline phases,
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Table 2. VSFG Experimental Results for 1:1 Mix-Terminated, Amino-Terminated and Carboxy-Terminated SAMs on Gold Type B with PPP Polarization terminal group
transition
ω (cm-1)
b
gamma (Γ)
|b/Γ|2
aNR
phi (φ)
1:1 mix
d+ d+w d+FR d+FR /ddr+ r+FR rd+ d+w d+FR dr+ r+FR rd+ d+w d+FR d+FR /ddr+ r+FR r-
2854.8
-8.2419
10.766
0.5861
2.57
-1.545
2899.7 2914.3 2927.4
-10.375 -1.3433 -9.7955
19.159 4.0132 11.562
0.2932 0.1120 0.7178
2962.9 2855.4 2866.1 2905.5 2927.6 2878.7 2945.4 2962.0 2855.7
-2.9031 -8.0189 -0.1254 -9.7189 -7.684 -0.4969 -0.4086 -0.7152 -0.1865
9.2565 9.99 1.987 16.921 8.589 3.612 2.809 3.873 1.903
0.0984 0.6443 0.0040 0.3299 0.8004 0.0189 0.0212 0.0341 0.0096
2.37
-1.703
1.85
-1.761
2899.2 2921.0 2926.8 2876.8 2947.4 2966.0
-0.3504 -0.1652 -6.643 -0.2195 -0.1217 -0.3102
3.8468 1.8064 17.981 5.1957 1.872 1.8699
0.0083 0.0084 0.1365 0.0018 0.0042 0.0275
amino
carboxy
Table 3. VSFG Experimental Results for 1:1 Mix-Terminated, Amino-Terminated and Carboxy-Terminated SAMs on Gold Type C with PPP Polarization terminal group
transition
1:1 mix
d+
amino
carboxy
d+w d+FR d+FR /ddr+ r+FR rd+ d+w d+FR dr+ r+FR rd+ d+ w d+FR d+FR /ddr+ r+FR r-
ω (cm-1)
b
gamma (Γ)
|b/Γ|2
aNR
phi (φ)
2856.6 2864.5 2904.5 2912.6 2930.7
-3.8543 -1.516 -1.1143 -0.06721 -18.648
6.294 4.0318 6.103 2.0218 15.812
0.3750 0.1414 0.0333 0.0011 1.3909
2.527
-2.215
2967.4 2854.9 2864.6 2905.1 2927.4 2878.0 2943.7 2962.7 2855.8 2867.4 2903.8 2915.0 2929.3 2883.7 2943.5 2961.0
-1.2933 -7.285 -0.9495 -8.740 -11.041 -1.851 -1.2195 -4.0291 -2.6098 -0.9801 -0.8587 -1.5192 -5.9726 -4.6414 -2.2805 -0.1077
5.0064 7.820 3.368 13.918 9.470 5.977 2.174 7.282 7.769 3.2492 5.1898 4.4225 8.9134 11.737 4.7481 1.9676
0.0667 0.8679 0.0795 0.3944 1.3593 0.0959 0.3147 0.3061 0.1128 0.910 0.0274 0.1180 0.4490 0.1564 0.2307 0.0030
2.81
-2.014
2.43
-2.036
namely, d+ and d- were found at 2851 and 2918 cm-1, respectively, for the crystalline phase vs at 2855 and 2924 cm-1 for the liquid phase (∼4-6 cm-1 shift). If we consider the SFG frequency calibration accuracy for our experimental setup to be (2 cm-1, the values obtained for CH2 stretch transitions (Tables 1-3) indicate that the SAM chains are in liquidlike, i.e., disordered, phase. This is in contrast to pure alkanethiol SAMs, e.g., dodecanethiols and octadecanethiols, which are usually found in the highly oredred 2D crystalline phase42,57 and for which weak d+ and d- transitions at 2850 and 2918 cm-1, respectively, are observed. When the SAM is in the crystalline phase, it has all-trans conformations of the alkane chain. As a result of this, CH2 vibrations are VSFG-inactive.53 If only terminal conformational gauche defects are present or there are no gauche defects at all in the hydrocarbon chains, we (57) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568.
should observe only weak d+w. However, we observe strong chain CH2 stretch modes: d+, d-, and d+FR at ∼2855, ∼2927, and ∼2905 cm-1, respectively, as well as terminal d+w transition. This indicates that a significant number of chain and terminal gauche defects are present in the SAM. While hydrogen-bonded carboxy groups of the 10-carboxy1-decanethiol SAM on gold are observed in the VSFG spectra, the transition frequency of the CdO stretch indicates monomeric H-bonds rather than H-bonded dimers, where two carboxy groups are stacked “head-to-tail” forming two H-bonds between the carbonyl oxygen and the hydroxyl hydrogen.49 This can be attributed to the steric factor imposed onto the arrangement of the carboxy groups by the flat substrate surface. The carbonyl stretch frequency is observed in the VSFG spectra of carboxyterminated SAMs at 1767 cm-1 (Tables 4 and 5). FTIR spectra of 10-carboxy-1-decanethiol recorded before preparation of the SAMs (data not presented) show this mode at ∼1725 cm-1,
Molecular Organization in SAMs
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Table 4. VSFG Experimental Results for CdO Stretch Region of 1:1 Mix-Terminated and Carboxy-Terminated SAMs on Gold Type A with PPP Polarization terminal group ω (cm-1) carboxy 1:1 mix
1766.8 1767.0 1706.0 1653.9 1839.0
b
gamma (Γ)
-0.684 -1.089 -0.172 -0.789 -0.419
22.694 14.117 5.736 13.067 6.85
|b/Γ|2
aNR
phi (φ)
0.00091 0.823 -2.20 0.006 1.04 -1.97 0.0009 0.004 0.004
Table 5. VSFG Experimental Results for CdO Stretch Region of 1:1mix Terminated, and Carboxy Terminated SAMs on Gold Type B with PPP Polarization terminal group carboxy 1:1 mix
ω (cm-1)
b
gamma (Γ)
|b/Γ|2
aNR
phi (φ)
1766.5 1762.8 1694.2 1654.1 1842.0
-1.342 -1.063 -2.995 -0.643 -0.216
21.117 14.953 43.939 5.883 9.498
0.004 0.005 0.005 0.012 0.0005
0.82 0.92
-1.56 -1.33
which is typical of the CdO vibrational frequency for hydrogenbonded carboxylic acids dimers.49 Therefore, before SAM deposition, the carboxylic acid molecules in solvent were in the form of hydrogen-bonded dimers. However, after deposition on gold and formation of SAM, the frequency of 1767 cm-1 found in the VSFG spectra of mixed and carboxy-terminated SAMs indicates that carboxylic acid molecules are present in the monomeric H-bond form. The CO transition for COOH (1765 cm-1) and for COOgroups (1654 cm-1) present in mixed SAMs has equal intensities, see Figure 7A,B. This indicates that by mixing SAMs with aminoand carboxy-terminal groups we obtain a two-dimensional homogeneous mixture of these molecules in the SAMs deposited on gold, i.e. no domain formation occurs. Our reasoning is as follows. In the case of domain formation for COOH groups, the intensity of the COOH transition would be proportional to the area covered by nondissociated molecules, whereas for the COOgroup, the intensity of CO transition would be proportional to the boundary length between domains of purely amino- and purely carboxy-terminated SAM. As the intensities of the CO transition for both groups are equal, we have to conclude that the number of molecules in such domains is approximately equal to the number of molecules at the boundaries of the domains. This is possible only when the size of the domain is of the order of magnitude of the intermolecular distance. Therefore, the formation of separate domains with carboxy- and amino-terminated molecules does not occur under current conditions. The ionic content of the substrate and the environment of molecular groups responsible for cell adhesion determine the efficiency of adhesion and network growth. The human body has a homeostatic mechanism that maintains a constant pH of 7.4 including numerous buffering systems to prevent pH changes; these include proteins and inorganic buffers. We maintain our system in a buffer solution which mimics the pH conditions in the human body. The presence of a buffer usually prevents major changes in pH even if a strong acid or base is added to the system, but local conditions can affect pH. Different kinds of cells can distinguish these minor changes. At pH 7.4, the amino groups of the amino-terminated SAMs become protonated and therefore positively charged. Positive electrical charges attract negatively charged buffer counterions, which form a double electric layer. When the neuron comes in direct contact with the substrate covered by amino-terminated SAMs, the labile nature of the negative counterions in the double electric layer of the
Figure 8. (a) The well ordered and dense amino-terminated SAM under pH 7.4 becomes partially protonated. The neuron penetrates through the double layer thus gaining access to the positively charged surface, resulting in efficient adhesion. (b) Mixed SAM of the carboxy- and amino-terminated molecules presents a catanionic structure on which no cell adhesion was observed.
buffer allows efficient cell binding to the amino groups. This is usually considered to be the main reason for successful cell attachment (Figure 8a). In the case of carboxy-terminated SAMs, we did not observe any embryonic neuronal cell attachment. In aqueous solution of neutral pH, carboxylic acids partially dissociate. Therefore, negatively charged surface with positively charged screening layer will be formed in the case of pure carboxy-terminated SAMs. In mixed SAMs, when the static negative charges are present in the form of carboxylate SAM molecules next to amino groups at the surface, no adhesion takes place even though the total number of the amino groups is only reduced by a factor of 2 relative to the pure amino-terminated SAMs. As we showed above, carboxy-terminated molecules of mixed SAMs can partially dissociate due to interaction with water and neighboring amino groups. This will cause formation of the catanionic structure of SAM, where neighboring molecules become oppositely charged. But in the case of catanionic surface, when neuronal cell contacts the surface of mixed SAM, it meets closely spaced negatively and positively charged ions which cannot be neutralized. This seems to be most probably the mechanism prohibiting neuron attachment in spite of the high concentration and ordered structure of surface amino groups (Figure 8b). Also this could indicate that the binding sites on the neuronal cell surface could require simultaneous interactions with more than one amino group. Our results indicate that, in addition to displaying the amino groups at the surface, an overall net positive charge of the surface is essential in determining the neuronal adhesion properties. Therefore, amino groups are very effective adhesion promoters despite being hydrogen bonded and screened by counterions as long as there is no static, oppositely charged site in nearest neighborhood. Surface carboxyl groups, which are partially dissociated and thus negatively charged, show no ability to bind neurons. We hope that the approach presented here can help to find the optimal conditions necessary to grow cells and control the quality of supporting substrate, namely, content and structure of SAM and metal film parameters.
5. Conclusions Successful neuron adhesion and the neuronal network development were achieved when depositing neurons onto aminoterminated alkanethiol SAM without a protein adhesive layer. Equal adhesion efficiency for the ordered amine groups of SAM and disordered amine groups of PDL allows us to conclude that the steric factors and ordering of amine groups is not critical for
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adhesion of neurons and the development of neuron networks, as long as the amine groups are accessible to the binding sites of the cell. Therefore, relatively high rates of gold deposition, which result in higher surface roughness but are less expensive in manufacturing of adhesive substrates, can be used. Aminoterminated alkanethiol SAMs may be good implant coating material due to their efficient cell adhesion, inability to be metabolized by surrounding cells, and capacity to create patterns of neurons and to control axon and neural process growth without any additional protein layer coating. Differences in surface morphology of the deposited metal, corresponding to an rms roughness range of 0.7-2 nm, do not have considerable influence on cell attachment or on conformation of alkane chains with carboxy-and/or amino-terminal groups. However, the ordering of the carboxy-terminal groups is better for smaller values of surface roughness. Amino-terminated SAMs have better ordered hydrocarbon chains than carboxy-terminated SAMs, presumably due to the lateral hydrogen bonding of the terminal groups disrupting the alkane chain ordering in the latter case. Alkane chains of all the SAMs under investigation are in liquidlike phase, unlike in the case on pure alkanethiol SAMs, which are usually found in a near crystalline state. Interactions between amino and carboxy groups in mixed SAMs provide additional alignment of
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alkane chains and terminal groups. Mixed SAMs form homogeneous mixtures without segregation or domain formation. By using model substrates with well-controlled surface properties, we demonstrate that surface chemical functionality and electrostatics alter the neuronal cell binding properties. This approach may offer a pathway to analyze and regulate the composition of artificial structural and signaling proteins localized to focal adhesion complexes and manipulate the binding of adhesive proteins, representing a versatile approach to elicit specific cellular responses in biomaterial applications. Acknowledgment. This work was supported by the Michigan Life Sciences Corridor Grant GR-358. The authors would like to acknowledge Wayne State University Nano Initiative Program and Smart Sensors and Integrated Microsystems for financial and technical support. The surface spectroscopy component of this research (A.N.B., A.K.Y., and A.V.B.) is supported by NSF CAREER Grant No. CHE-0449720. We would like to thank the group of Dr. G. Mao for AFM measurements (Department of Chemical Engineering and Materials Science, Wayne State University). LA7032675
