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Probing the Protein Orientation on Charged Self-Assembled Monolayers on Gold Nanohole Arrays by SERS Qiuming Yu* and Greg Golden Department of Chemical Engineering, Center for Nanotechnology, UniVersity of Washington, Seattle, Washington 98195 ReceiVed March 11, 2007. In Final Form: May 28, 2007 In this work, surface-enhanced Raman scattering (SERS) was applied to probe the orientation of cytochrome c (Cyt-c) on gold nanohole arrays functionalized with self-assembled monolayers (SAMs) of alkane thiols with positively (-NH2) and negatively (-COOH) charged terminal groups. Square grid gold nanohole arrays with a nanohole diameter of 270 nm and a grating of 350 nm were fabricated by electron beam lithography (EBL) and were used as the SERS substrates. The SERS intensities of the nontotally symmetric mode (B1g mode ν11) and the totally symmetric mode (A1g mode ν4) and their ratios were used to determine the orientation of Cyt-c on surfaces. The results indicate that the heme group is close and perpendicular to the negatively charged surface but is far from and oriented at an angle to the positively charged surface. Cyt-c has a random or more flat orientation on the bare Au nanoholes surface.
Introduction Control of the orientation of proteins adsorbed on surfaces is critical to retaining and maximizing protein functionality for applications of biosensors and implanted medical devices.1 Because of the charge distribution in a protein, the orientation of proteins on a surface can be controlled with the polarity of electrostatic charges on a surface. Cytochrome c (Cyt-c) is a membrane electron-transfer protein that carries a +9 charge at neutral pH, making it a good candidate for studies on electrostatic binding interactions. The dipole of Cyt-c is due to the positive patches from lysine residues located around the heme group and the negative patches from glutamic acid residues far away from the heme group. To optimize the electron transfer, adsorbed Cyt-c should have a preferred orientation with its heme group close and perpendicular to the surface. The orientation of Cyt-c on surfaces has been studied using a variety of experimental methods, such as surface plasmon resonance (SPR) sensors,2 surface-enhanced resonance Raman spectroscopy (SERRS),3 vibrational spectroscopy,4 and polarized X-ray absorption fine structure (XAFS) spectroscopy.5 Molecular simulations were also applied to study the orientation and conformation of Cyt-c on surfaces.3,6 Both experimental and molecular simulation studies showed that Cyt-c adsorbed on a negatively charged surface with the heme group close and perpendicular to the surface.4,6 Surface-enhanced Raman spectroscopy (SERS) is a powerful technique for obtaining vibrational spectra of biomolecules adsorbed on surfaces.7,8 The significantly increased intensity of Raman scattering from the vibrational bands of a biomolecule at or near a nanostructured metal (e.g., Ag, Au, and Cu) surface * Author to whom correspondence should be addressed. E-mail:
[email protected], Phone: 206-543-5101, Fax: 206-221-2528. (1) Castner, D. G.; Ratner, B. D. Surf. Sci. 2002, 500, 28-60. (2) Chen, X. X.; Ferrigno, R.; Yang, J.; Whitesides, G. M. Langmuir 2002, 18, 7009-7015. (3) Rivas, L.; Soares, C. M.; Baptista, A. M.; Simaan, J.; Di Paolo, R. E.; Murgida, D. H.; Hildebrandt, P. Biophys. J. 2005, 88, 4188-4199. (4) Edmiston, P. L.; Lee, J. E.; Cheng, S. S.; Saavedra, S. S. J. Am. Chem. Soc. 1997, 119, 560-570. (5) Edwards, A. M.; Zhang, K.; Nordgren, C. E.; Blasie, J. K. Biophys. J. 2000, 79, 3105. (6) Zhou, J.; Zheng, J.; Jiang, S. Y. J. Phys. Chem. B 2004, 108, 1741817424. (7) Dick, L. A.; Haes, A. J.; Van Duyne, R. P. J. Phys. Chem. B 2000, 104, 11752-11762. (8) Keating, C. D.; Kovaleski, K. M.; Natan, M. J. J. Phys. Chem. B 1998, 102, 9404-9413.
is due to the extremely high local electromagnetic fields that arise from local surface plasmon resonance (LSPR). SERS has been used previously to study both the electron-transfer activity of Cyt-c on a silver electrode functionalized with alkane thiol self-assembled monolayers (SAMs) with negatively charged terminal groups and the orientation and conformation of Cyt-c in a sandwich configuration of Ag nanoparticle/Cyt-c/Au nanoparticle (Ag/Cyt-c/Au).7,8 Whereas the orientation of Cyt-c in Ag/Cyt-c/Au sandwiches can be probed with SERS, the conformation of Cyt-c also affects the SERS signal. The study of the SERS effect from Au nanoparticle and nanoring arrays has been reported.9,10 Recently, the unique optical properties of subwavelength holes in metal thin films has spurred great interest because of its potential applications in novel photonic devices and biosensors.11 However, the use of Au nanohole arrays as a SERS substrate has not yet been reported. In this work, Au nanohole arrays with well-controlled dimensions were fabricated by electron beam lithography (EBL) and were used as SERS substrates. The optimized dimensions of the nanohole arrays determined from our previous studies of SERS spectra of 4-mercaptopyridine (4-MP) adsorbed on various arrays were used in this study. The capability of SERS to probe the orientation of 4-MP on Au nanohole arrays was first demonstrated. Then, the orientation of adsorbed Cyt-c on negatively (-COOH) and positively (-NH2) charged SAMcoated surfaces and on a bare Au nanohole array surface was studied using SERS. Experimental Section Au nanohole arrays (20 µm × 20 µm) with a square grid of different diameters and spaces were fabricated by electron beam lithography (EBL), which was performed on an FEI Sirion scanning electron microscope (SEM) equipped with Nabity NPGS. A layer of 100-nm-thick poly(methyl methacrylate) (PMMA) photoresist was spin-coated on a Si substrate coated with a 20 nm Au film and exposed to a 30 kV, 16.5 pA electron beam with a linear dose in the range of 0.3-0.7 nC/cm. After development in 1:3 methyl isobutyl ketone/isopropanol (MIBK/IPA) PMMA developer for 70 s, nano(9) Fe´lidj, N.; Aubard, J.; Le´vi, G.; Krenn, J. R.; Salerno, M.; Schider, G.; Lamprecht, B.; Leitner, A.; Aussenegg, F. R. Phys. ReV. B 2002, 65, 075419. (10) Laurent, G.; Fe´lidj, N.; Grand, J.; Aubard, J.; Le´vi, G.; Hohenau, A.; Aussenegg, F. R.; Krenn, J. R. Phys. ReV. B 2006, 73, 245417. (11) Barnes, W. L.; Dereux, A.; Ebbesen, T. W. Nature 2003, 424, 824-830.
10.1021/la7007073 CCC: $37.00 © 2007 American Chemical Society Published on Web 07/13/2007
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holes were generated in the PMMA layer. Finally, the Au nanohole arrays were prepared by evaporating a 2 nm Cr, 50 nm Au film over the PMMA surface. SEM and tapping mode AFM (Veeco Dimension 3100) were used to characterize the lateral and vertical dimensions of nanostructures, respectively. 4-MP (Sigma Aldrich, St. Louis, MO) SAMs were formed by soaking a UV ozone-cleaned substrate in a 3 mM aqueous solution of 4-MP for 3 h, followed by rinsing with DI water and blowing dry in a stream of N2. 10-Mercaptoundecanoic acid (HS(CH2)10COOH) (Sigma Aldrich, St. Louis, MO) and 11-amino-1-undecanethiol (HS(CH2)11NH2) (Dojindo Molecular Technologies, Inc., Gaithersburg, MD) SAMs were formed on substrates with Au nanohole arrays using the methods developed by Wang and co-workers.12 Protein adsorption was completed by placing a 40 µL drop of 1.0 × 10-6 M horse heart cytochrome c (Sigma Aldrich, St. Louis, MO) in 4.4 mM potassium phosphate buffer (Kpi), pH 7.0, onto a -COOH or -NH2 SAM surface, covering with a cover glass, and incubating at 4 °C for 30 min before Raman spectroscopy was carried out. Raman spectroscopy was carried out on Renishaw InVia Raman spectroscope attached to a Leica DMLM upright microscope. A 50× (N.A. ) 0.8 for 4-MP) or a 100× (N.A. ) 0.75 for Cyt-c) objective was used to focus the laser on the nanohole array and to collect the scattered light from the sample surface. A 785 nm nearinfrared laser line was used to irradiate the surface.
Results and Discussion 4-MP has been widely used as a probe molecule for SERS studies because it has a large scattering cross-section and forms a SAM on metal surfaces similar to alkane thiols.13-15 The Au nanohole arrays were optimized by comparing the enhancement effects in the SERS spectra of 4-MP adsorbed on various nanohole substrates with the diameters of nanoholes varying from 40 to 425 nm and the grating of square grid nanohole arrays varying from 100 to 550 nm. It was found that when the distance between the nanoholes was maintained, the intensity of the SERS spectra increased as the diameter of the nanoholes was increased. When the diameter of the nanoholes was maintained, the intensity of the SERS spectra increased as the distance between the nanoholes was decreased.16 Figure 1a is an SEM image of the optimized nanohole array with a nanohole diameter of 270 nm and a grating of 350 nm. The SERS spectrum of 4-MP adsorbed on this array and excited with a 785 nm laser line is shown in Figure 1c. The enhancement factor calculated for 4-MP on the nanohole array on the basis of the intensity of the band at 1093 cm-1 is 1.2 × 104. It was previously found that the enhancement factor for nanohole arrays does not vary much as long as the ratio of the distance between nanoholes to the sum of the distance between the nanoholes and the radius of the nanoholes is about 0.37.16 The highest electromagnetic field enhancement arises when the irradiation wavelength is resonant with the LSPR maximum.17,18 The 514 nm laser line was also used to excite 4-MP on the nanohole arrays. However, no SERS spectrum of 4-MP was observed, indicating that the LSPR wavelength of the nanohole arrays is far from 514 nm. A recent study of using gold nanohole arrays for the application in biosensing showed that the LSPR wavelength of nanohole arrays is between 800 and 900 nm from the transmission spectrum and electrodynamics (12) Wang, H.; Chen, S. F.; Li, L. Y.; Jiang, S. Y. Langmuir 2005, 21, 26332636. (13) McLellan, J. M.; Siekkinen, A.; Chen, J. Y.; Xia, Y. N. Chem. Phys. Lett. 2006, 427, 122-126. (14) Hu, J. W.; Zhao, B.; Xu, W. Q.; Li, B. F.; Fan, Y. G. Spectrochim. Acta A 2002, 58, 2827-2834. (15) Bryant, M. A.; Joa, S. L.; Pemberton, J. E. Langmuir 1992, 8, 753-756. (16) Yu, Q. M.; Qin, D.; Golden, G. Nano Lett., 2007, submitted for publication. (17) Fe´lidj, N.; Aubard, J.; Le´vi, G. Appl. Phys. Lett. 2003, 82, 3095-3097. (18) Haynes, C. L.; Van Duyne, R. P. J. Phys. Chem. B 2003, 107, 74267433.
Figure 1. (a) SEM image of a Au nanohole array with a nanohole diameter of 270 nm and a grating of 350 nm. (b) Schematic of the side view of a Au thin film with nanoholes and 4-MP adsorbed on the surface. The molecules marked with an asterisk illustrate where the local electromagnetic field is enhanced. (c) SERS spectrum of 4-MP adsorbed on a Au nanohole array compared to the Raman spectrum of solid 4-MP.
modeling,19 which is consistent with our results. The large dimensions of the nanohole arrays make them much easier to fabricate using other methods such as soft lithography.20 The orientation of 4-MP on Au nanoholes arrays can be determined by the ratio of the intensity of the band at 1093 cm-1 to the intensity of the band at 1035 cm-1 as compared to the intensity ratio of the band at 1105 cm-1 to the intensity of the band at 1044 cm-1 of 4-MP solid (Figure 1c). The band at 1105 cm-1 of solid 4-MP arises from the ring-breathing mode coupled with the C-S stretching mode, and the band at 1044 cm-1 of solid 4-MP arises from the C-H in-plane bending mode.14,15 When 4-MP is adsorbed on the Au nanohole array via its sulfur atom (19) Stewart, M. E.; Mack, N. H.; Malyarchuk, V.; Soares, J. A. N.; Lee, T.-W.; Gray, S. K.; Nuzzo, R. G.; Rogers, J. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17143-17148. (20) Kim, E.; Xia, Y. N.; Zhao, X. M.; Whitesides, G. M. AdV. Mater. 1997, 9, 651-654.
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Figure 2. Comparison of (a) the Raman spectrum of a 1.0 × 10-3 M Cyt-c solution with the SERS spectra of 1.0 × 10-6 M Cyt-c on (b) a bare Au nanohole array, (c) a positively charged surface (-NH2), and (d) a negatively charged surface (-COOH).
with the ring perpendicular to the surface, the band at 1105 cm-1 shifts to 1093 cm-1, and the intensity is enhanced dramatically. At the same time, the band at 1044 cm-1 shifts to 1035 cm-1, and the intensity decreases. The intensity ratio of the band at 1093 cm-1 to the band at 1035 cm-1 increased from 0.08 for 4-MP solid to 3.8 for 4-MP adsorbed on the Au nanohole array surface. To study the influence of surface properties on protein orientation using SERS, three types of surfaces were prepared. SAMs with -COOH and -NH2 terminal groups on Au nanohole arrays were used as negatively and positively charged surfaces, respectively, and a bare Au nanohole array was used as a highenergy surface. To eliminate any distance effects, both -COOH and -NH2 thiols have the same alkane backbone chain length making the SAMs the same thickness. Figure 2a shows the Raman spectrum of 1.0 × 10-3 M Cyt-c in 4.4 mM Kpi, pH 7.0,
using a 785 nm laser excitation source. Previous studies of Cyt-c used resonance Raman scattering (RRS) with shorter-wavelength lasers.7,8,21 Table 1 shows that bands exhibiting in RRS21 also appear in the Raman spectrum of the Cyt-c solution using a 785 nm laser line, with the exception of the bands at 1626 and 1551 cm-1. The band at 1447 cm-1 in the Raman spectrum of the Cyt-c solution using a 785 nm laser line may come from the protein backbone bands because of the longer irradiation wavelength used. SERS spectra of 1.0 × 10-6 M Cyt-c in 4.4 mM Kpi buffer, pH 7.0, from a bare Au nanohole array surface and from SAMs with -NH2 and -COOH terminal groups are shown in Figure 2b-d, respectively. B1g mode ν11 (∼1560 cm-1), which is absent in the Raman spectrum of Cyt-c solution (Figure 2a), appears in the SERS spectra of Cyt-c on all three types of surfaces as a result of the strong electromagnetic enhancement caused by the nanohole arrays. No SERS signals were observed when the irradiation source was switched to a 514 nm laser. As discussed before, because the LSPR wavelength of the gold nanohole array is close to the irradiation wavelength of 785 nm, the SERS signals from the heme group are enhanced significantly when the heme group is close to the surface. Most of the mode assignments are the same as those from RRS. However, some of the modes appeared in the Raman spectra using a 785 nm laser line; the 1522 and 1431 cm-1 signals may come from the protein backbone bands because of the longer irradiation wavelength used. The SERS signal of Cyt-c adsorbed on the bare Au nanohole array is stronger than those from the SAM surfaces because of the strong local electromagnetic field generated by the nanohole array directly interacting with Cyt-c. The SERS intensity ratio of B1g/A1g was used on the basis of SERS selection rules to determine the orientation of Cyt-c.8 Because the orientation of the local electromagnetic field is always perpendicular to the surface, the SERS effect preferentially enhances vibrations that involve a change in polarizability along an axis perpendicular to the surface. When the heme group is standing up on the surface, the B1g modes would be enhanced because the heme group in-plane vibrations would have a large component perpendicular to the surface. The A1g modes would also be enhanced in this orientation. However, when the heme group is lying flat on the surface, only the A1g modes would be enhanced. Therefore, the SERS intensity ratio of B1g/A1g decreases as the angle of the heme group with respect to the surface normal increases. B1g mode ν11 and A1g mode ν4 are marked by short vertical lines in Figure 2. The intensities of B1g mode ν11 and A1g mode ν4 and the intensity ratios of B1g(ν11)/A1g(ν4) for Cyt-c on all three types of surfaces are listed in Table 2. For Cyt-c on the negatively charged SAM (-COOH) surface, B1g mode ν11
Table 1. Surface-Enhanced Raman Frequencies of Cyt-c on SAMs with -COOH and -NH2 Terminal Groups and on a Bare Gold Nanohole Array, Raman Frequencies of Cyt-c in Kpi Buffer, and Resonance Raman Frequencies of Cyt-c and Their Normal Mode Assignments -COOH
-NH2
bare Au
solution
RRS21
mode
symmetry
1609 1580
1626 1610 1587 1551
ν10 ν38 ν19 ν11
B1g Eu A2g B1g
ν(CR-Cm)asym ν(Cβ-Cβ) ν(CR-Cm)asym ν(Cβ-Cβ)
1512 1462 1447 1414 1382 1344
1496 1483
ν3 ν28
A1g B2g
ν(CR-Cm)sym ν(CR-Cm)sym
1407 1364 1343
ν29 ν4 ν12
B2g A1g B1g
ν(pyr quarter ring) ν(pyr half ring)sym ν(pyr half ring)sym
1309
1314
ν21
A2g
δ(Cm-H)
1621 1602 1551 1499 1468 1431 1418 1371 1341
1588 1560 1523 1493 1480 1428 1398 1372
1585 1556 1522 1508 1460 1431 1378
1359 1305
local coordinate21
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Table 2. Intensities of B1g(ν11) and A1g(ν4) and the B1g(ν11)/ A1g(ν4) Ratio for Cyt-c on Three Types of Surfaces surfaces
A1g(ν4) (cps)
B1g(ν11) (cps)
B1g(ν11)/A1g(ν4)
-COOH -NH2 bare Au
18 87 357
325 225 381
18.5 2.6 1.1
is enhanced significantly with an intensity of 325 cps whereas the intensity of A1g mode ν4 is only 18 cps. This gives an intensity ratio of B1g(ν11)/A1g(ν4) of 18.5, which indicates that the heme
group plane is perpendicular to the surface. Although the SERS signal is expected to be weaker for Cyt-c on a SAM surface because of the decay length of the local electromagnetic field, the similar intensity levels for the B1g(ν11) of Cyt-c on the negatively charged SAM surface (325 cps) and on the bare Au nanohole array (380 cps) indicate that the heme group is located close to the negatively charged surface. All of these results indicate that the heme group plane is perpendicular and close to the negatively charged surface as illustrated in Figure 3a, which is consistent with previous studies of Cyt-c adsorbed on a negatively charged surface.4,6 The moderate intensity of A1g(ν4) on the positively charged SAM (-NH2) surface (87 cps) and the lowintensity ratio of B1g(ν11)/A1g(ν4) of 2.6 indicate that the angle of the heme group with respect to the surface normal increases and the heme group is far away from the positively charged surface as illustrated in Figure 3b. Because only the A1g modes would be enhanced when the heme group is lying flat on the surface, the much stronger intensity of A1g(ν4) on the bare Au surface (357 cps) and the low-intensity ratio of B1g(ν11)/A1g(ν4) (1.1) indicate that Cyt-c has a random or more flat-lying orientation on the bare Au nanohole array surface as illustrated in Figure 3c.
Conclusions Surface-enhanced Raman scattering spectroscopy is a powerful method for studying the orientation of proteins adsorbed on nanopatterned metal surfaces. The dimensions of Au nanohole arrays can be precisely controlled by EBL, and the optimal SERS enhancement can be achieved by varying the dimensions of nanostructures. The Au nanohole arrays provide a new type of SERS substrate that has high enhancement while providing dimensions that allow for ease of fabrication with methods other than EBL. The different orientations of Cyt-c on negatively and positively charged surfaces and on the bare Au nanoholes surface can be probed by SERS.
Figure 3. Schematics of Cyt-c with the heme group (a) close and perpendicular to a negatively charged surface, (b) far from and oriented at an angle to a positively charged surface, and (c) lying flat on a bare Au surface. The arrows indicate the directions of the dipole of Cyt-c.
Acknowledgment. This work was conducted at the Nanotech User Facility at the University of Washington, a member of the National Nanotechnology Infrastructure Network (NNIN) supported by NSF. LA7007073 (21) Hu, S. Z.; Morris, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. J. Am. Chem. Soc. 1993, 115, 12446-12458.
