Langmuir 1991, 7, 479-485
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Infrared Study of N-Methylacetamide on Clean and Chemically Modified Surfaces C. Tornkvist, B. Liedberg,* and I. Lundstrom Laboratory of Applied Physics, Linkoping University, S-581 83 Linkoping, Sweden Received May 16, 1990. I n Final Form: August 22, 1990 N-Methylacetamide (NMAA)is used as a model molecule to investigate, in detail, the interaction between the peptide group and solid surfaces with different chemical properties. Monomolecular layers of NMAA are prepared on clean, oxidized, and alkyl-modified metal and semiconductor surfaces and analyzed with infrared reflection-absorption spectroscopy (IRAS) and attenuated total reflection (ATR) spectroscopy. Conclusive evidence is obtained for a coexistence between a free (non-hydrogen-bonded) and a dipolar (hydrogen-bonded)peptide group on low-energysurfaces, i.e., hydrophilic surfaces. The fraction of dipolar peptide groups varies with the properties of the surface and is generally found to be higher on oxidized surfaces, an effect that most likely is due to an increased degree of hydrogen bond interaction with surface hydroxyls. The ability to form hydrogen bonds with surface hydroxyls may also facilitate intermolecular hydrogen bonding, i.e., self-association. These two types of hydrogen bond interactions do not occur on hydrophobic (alkyl-modified) surfaces. Instead we observe a spectral signature which closely resembles that of matrix isolated trans-NMAA molecules, i.e., of completely free molecules on the surface. The lack of hydrogen bonding on the hydrophobic surface implies that the dipolar nature of the peptide group no longer exists and that the -C-N- bond has lost its double bond character. The obtained results are discussed in terms of a partial surface-induced rotational freedom around the -C-N- bond in contacting peptide groups, a phenomenon that may influence the backbone stability and thereby also the activity and function of biological macromolecules on surfaces.
Introduction Protein adsorption on solid surfaces has been the subject of extensive investigations during recent years.l Much emphasis has been focused on the structural behavior of adsorbed proteins and several attempts have been made to correlate structural changes to macroscopic parameters of the protein-surface system, such as, net charge of the protein, amino acid distribution, ionic strength, surface potential, surface energy, et^.^-^ The surface energy is perhaps the most thoroughly examined quantity, and it is often found that proteins exhibit a greater affinity to hydrophobic than to hydrophilic The structural change and the subsequent change in activity and function of the adsorbed protein molecule appear also to be more pronounced on hydrophobic surface^.^^^^^ One widespread explanation to the increased structural changes on hydrophobic surfaces is that hydrophobic regions in the interior of the protein try to get in contact with the surface during the adsorption process, in order to minimize the total energy of the system. However, there exists still no detailed microscopic understanding of how the individual amino acid side-chain groups (RIand Rz in Figure
* To whom correspondence should be addressed. (1) Brash, J. L., Horbett, T. A., Eds. In Proteins at Interfaces; ACS Symposium Series 343; American Chemical Society: Washington, DC, 1987. (2) Andrade,J. D. Ed. In Surfaceandlnterfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1985; Vol. 2 "Protein Adsorption". (3) Ivarsson, B.; Lundstrom I. In Physical Characterizationof Protein Adsorption on Metal and Metal oxide Surfaces. CRC Crit. Reu. Biocomp. 1986,2, 1. (4) Morrisey, B. W.; Stromberg, R. R. J. Colloid Interface Sci. 1974, 46, 152-164. (5) Jonsson, U.;Malmqvist, M.; Ronnberg, I. J. Colloid Interface Sci. 1985, 103, 360-372. (6) Elwing, H.; Welin, S.; Askendal, A.; Nilsson, U.; Lundstrom, I. J. Colloid Interface Sci. 1987, 119, 203-210. (7) Elwing, H.; Askendal, A.; Lundstrom, I. h o g . Colloid Polym. Sci. 1987, 74, 103-107. (8) Elwing, H.; Nilsson, B.; Svensson, K.-E.;Askendal, A.; Nilsson, U. R.; LundstrBm, I. J. Colloid Interface Sci. 1988, 125, 139-145. (9) Elwing, H.;Askendal, A.; Lundstrom, I. In Pathogenesis of Wound and Biomaterial-Associated Infections; Wadstrom, T., Eliasson, I., Holder, I., Ljungh, A., Eds.; Springer-Verlag, in press.
0743-7463/91/2407-0479$02.50/0
1)or the peptide group (shaded in Figure 1)interact with the surface. The behavior of contacting peptide groups, in particular, has only been sparsely investigated. The only work on this subject is, to our knowledge, the bound fraction measurements (number of directly attached peptide carbonyls) of albumin, prothrombin and fibrinogen on silica surfaces by Morrisey et ala4 They found that as many as 20% of the peptide carbonyls in a protein could be directly involved in the bonding to the silica surface, a value which from a purely geometrical consideration requires aquite substantial structural change of the protein molecule. Nevertheless, their results are very interesting and we have decided to initiate a survey study of the adsorption of short peptides on a variety of solid surfaces, by utilizing infrared reflection-absorption spectroscopy (1RAS)'O and attenuated total reflection (ATR)" spectroscopy. We are, of course, fully aware of the fact that specific peptide-surface interaction is only one of several phenomena that may influence the structural behavior of complex proteins on surfaces. However, it is felt that this type of model study in the long run may contribute to a deeper microscopic understanding of adsorption phenomena where proteins are involved. In this article we report on the interaction between a simple model peptide N-methylacetamide (NMAA) and solid surfaces. NMAA, Figure 1(II),was chosen as a model peptide because it is known to form a hydrogen bonding pattern in the solid and liquid state, which is closely related to that of proteins (cf. Figure 1 (I and 111)). The main objective with the study is to investigate how solid surfaces, with different chemical properties, will influence the hydrogen bonding pattern between adsorbed NMAA molecules (self-association) as well as between adsorbed NMAA and the surface. Our attention is focused on the surface energy because strongly hydrophilic (OH-saturated) and hydrophobic (CHB-saturated) surfaces are expected to contribute quite differently to the hydrogen (10) Francis, S. A.; Ellison, A. H. J. Opt. SOC.Am. 1959,49,131-138. (11) Harrick, N. J. Internal Reflection Spectroscopy; Wiley-Interscience: New York, 1967.
0 1991 American Chemical Society
480 Langmuir, Vol. 7, No. 3, 1991
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I
0
Figure 1. Schematic diagram of (I) the polypeptide backbone in a protein molecule (R1and R2 represent the amino acid sidechain groups) and (11) free (non-hydrogen-bonded) NMAA and (111) dipolar (hydrogen-bonded) NMAA. The peptide group (shaded in I and 111) is normally regarded as a stiff and planar unit in proteins and polypeptides.
bonding of contacting peptide groups. It is of particular interest to examine whether there exists any correlation between the surface energy and the presence of free (11) and dipolar (111)peptide groups on the surface. For this purpose we have chosen to compare the behavior of NMAA on a number of clean and oxidized metal and silicon surfaces as well as on alkyl-modified surfaces, which are prepared by covalently attaching long-chain alkanethiols on gold and methylsilanes on silicon/silicon dioxide (Si/ Si02). A short preliminary description of our first results was given at a recent conference.12
Experimental Section The metal substrates used for the IRAS measurements were prepared by evaporating gold and copper (a2000 A) on silicon wafers (40 X 20 X 0.3 mm), at a base pressure of lO-' Torr, and at an evaporation rate of ==5A/s. A trapezoidal silicon crystal (50 X 20 X 2 mm, 45O) was used as substrate material for the ATR measurements. Three differentcopper surfaceswere used in this investigation: (1)clean copper (oxide free); (2) copper with a 20 A thick native oxide layer (Cu/Cu20); (3) Cu/Cu20 that has been exposed to laboratory atmosphere for about 3-10 h. Clean gold surfaceswere prepared by immersingthe substrates for 5 min in a hot (80 "C) solution of 3 mL of NHB (25%) and 3 mL of H202 (30%)in 15mL of HzO (calledTL1). The cleaned surfaces were then rinsed in pure water and dried in a stream of N2. Hydrophobicgold surfaceswere prepared by immersing the TL1-cleaned substrates into a 2 mM solutionof 1-hexadecanethio1 (Fluka) in EtOH (95%)for 12 h. This treatment resulted in a close packed and vertically oriented thiol monolayer. The reflection-absorption (R-A) spectrum of this layer is in good agreement with that reported by Nuzzo et al.13 ~
~~
~~~~~~
(12) Liedberg, B.; Tiirnkvist, C.; Lundstriim,I. Proceedings of the 7th Znternational Conference on Fourier Transform Spectroscopy; Cameron, D. G., Ed.;SPIE Bellingham, WA, 1989; Vol. 1145, p 141. (13) Nuzzo, R. G.; Fuaco, F. A.; Allara, D. L. J. Am. Chem. SOC.1987, i09,2"~.
The silicon ATR-crystal was oxidized to form a 1260A thick silicon dioxide layer, as determined with a Rudolph Research, Auto El 2, ellipsometer. Cleaning of the oxidized crystal (Si/ Si02) in TL1 resulted in a OH-saturated surface, i.e., a hydrophilic surface. Hydrophobic Si/SiO2 surfaces were prepared by immersing the OH-saturated surface in a 1%solution of dimethyldichlorosilane (Merck) in trichloroethylene for 1 h. The monolayers of NMAA are prepared in the following way: A thick film (=1-5 pm) of pure NMAA (Fluka) is solution cast on the sample surface which immediately after deposition is placed in the vacuum (sample) chamber of the FT-IR spectrometer. The thick NMAA layer slowly desorbs from the surface during the pump down time of the sample chamber, and infrared spectra are continuously collectedduring the desorption process (pump down time =3-5 min), until roughly a monolayer is left on the sample surface. A program SPECPC (written by us) based on Fresnel equations has been used to calculate the thickness of the adsorbed NMAA layer. The FT-IR unit was a BRUKER IFS 113vFourier transform spectrometer equipped with a narrow band MCT detector (cuton 700 cm-'). The R-A spectra were obtained with a BRUKER GIR (grazingangleincidencereflection) accessory aligned at 85O.l' All the R-A spectra are recorded with light polarized parallel to the plane of incidence. The ATR spectra were obtained with a Wilks, Model 9, ATR accessory, aligned for 12reflectionsat 45'. The infrared spectra are taken at 8-cm-l resolution by averaging 32-128 interferograms at a mirror velocity of 0.665 cm/s. This gives a typical measurement time of about 10-40 s for each spectrum.
Results and Discussion Initial Observations. Figure 2 showsthe transmission (bulk)spectrum of pure NMAA in the liquid state and the reflection-absorption (R-A) spectrum of adsorbed NMAA on an air-exposed Cu/CueO surface. The bulk spectrum, Figure 2a, is characterized by a number of very strong and broad peaks at 3298,1657,1564 and 1300cm-', respectively. These peaks are well-known to protein spectroscopists and are normally referred to as the Amide A and Amide I, 11, I11 modes, respectively. The Amide I mode VC+, in particular, has attracted considerable interest because it has been found to be very sensitive to the local environment (hydrogen bonding) of the carbonyl group and, thereby, also to changes in the secondary structure of proteins and polypeptides.15 Much emphasis will therefore be paid to the behavior of the Amide I peak in the followingdiscussion of adsorbed NMAA. The R-A spectrum of adsorbed NMAA on air-exposed C u / C u ~ 0 ,Figure 2b, exhibits a completely different pattern of peaks as compared to that in Figure 2a. The most pronounced spectral changes occur in regions where the peptide group itself is expected to absorb. Note especially the lowering of the peak intensity and the upward shift from 3298 to 3500cm-l of the N-H stretching (Amide A) mode as well as the upward shift of the Amide I mode from 1657cm-l to approximately 1730cm-l. These observations clearly suggest that the intermolecular hydrogen bonding in NMAA is strongly perturbed by the presence of the surface and that no traces of hydrogenbonded NMAA can be identified on the surface. This latter statement is also supported by the observation that the R-A spectrum of NMAA in Figure 2b is .almost identical with that of matrix-isolated NMAA.16 A closer (14) Ihs, A,; Liedberg, B.; Uvdal, K.; Tiirnkvist, C.; Bodii, P.; Lundstriim, I. J. Colloid Interface Sci. 1990, 140, 192-206. (15) Koenig, J. L.; Tabb, D. L. In Analytical Applications of FT-IR to Molecular and Biological Systems; Durig, J. L., Ed.; Nato Advanced Study Institute Series; Riedel: Dordrecht,The Netherlands; 1980, p 229, and references therein. (16) Ataka, S.; Takeuchi, H.; Tasumi, M. J. Mol. Struct. 1984, 223, 147-160.
Infrared Study of NMAA on Surfaces
Langmuir, Vol. 7,No. 3, 1991 481
a
I
I
3298
b
2955
la3
I
'1717
1732
I
I
I
000
3000
2000
I
1000
WAVENUMBERS Icm-1) ~~
Figure 2. Infrared spectra of (a) liquid NMAA and (b) a monomolecular layer of NMAA adsorbed on an air-exposed Cu/CuzO surface. Measurement time t, = 40 s for the R-A spectrum in part b.
comparison with matrix-isolated NMAA furthermore reveals that trans-NMAA is the predominant configuration on the surface. No peaks from cis-NMAA can be observed in the spectrum, Figure 2b. Table I summarizes the observed frequencies and intensities for the strongest peaks in the spectra of liquid and matrix-isolated NMAA as well as for NMAA on the investigated surfaces. The assignments of the strongest peaks are also included in Table I. The obvious interpretation of our spectral results is that the NMAA molecules are adsorbed in such a way that they are unable to interact, hydrogen bond, with nearest neighbors as well as with the surface. Aggregation into patches (clusters) or self-association is therefore less likely to occur on this type of surface. Although this initial observation was made on a badly defined surface (airexposed CulCuzO), which presumably is saturated with organic contaminants from the laboratory atmosphere, we found the results very interesting and definitely worth a closer examination on well-defined clean and chemically modified metal and silicon surfaces. NMAA on Clean, Oxidized, and Alkyl-Modified Metals. Figures 3 and 4 show the R-A spectra of NMAA on copper and gold surfaces, respectively. The strongest peaks in the spectra are depicted in Figures 3 and 4 and are summarized in Table I. The R-A spectra of clean Cu and Cu/CuzO in Figure 3 show a slightly different spectral (17) Sugawara, Y.; Hirakawa, A. Y.; Tsuboi, M. J . Mol. Spectrosc. 1984,108,206-214. (18)Cheam, T. C.; Krimm, S. J. Chem. Phys. 1985, 82, 1631-1641. (19) Dahlgren, C.; Sunqvist, J. Immunol. Methods 1981,40, 171-179.
signature as compared to that of NMAA on air-exposed Cu/CuzO, Figure 2b. The main differences are the appearance of two peaks near 1660 and 1550 cm-1, respectively, which according to the previous discussion are characteristic of dipolar or hydrogen bonded NMAA (cf. structure I11 in Figure 1). This observation clearly shows that free and dipolar peptide groups coexist on clean Cu and Cu/CuzO but not on air-exposed (contaminated) Cu/CuzO surfaces. A thin layer of organic contaminants on the Cu/CuzO surface is apparently sufficient for disrupting the hydrogen bonding between neighboring NMAA molecules on the surface as well as between the NMAA molecule and the surface. It is also clear from Figure 3 that the amount of dipolar peptide groups increases with the presence of an oxide layer. This behavior is presumably due to a higher density of hydrogen bond donating groups (-OH) on the oxidized surface, a hypothesis that is consistent with the lowering of the contact angle eHzO for the oxidized surface, Table 11. We propose therefore that self-association and hydrogen bonding with surface hydroxyls may exist simultaneously on the Cu and Cu/CuzO surface. However, it is very difficult to distinguish between the two forms of hydrogen bonding spectroscopically and, thereby, to say something about their relative occurrence on the surface. A simple estimation of the fraction f of dipolar groups (carbonyls) in adsorbed NMAA on the investigated surfaces is also given in Table 11. It should be stressed, however, that the present calculation off is based on the assumption that the molar extinction coefficients are the same for the two peaks near 1730 and 1660 cm-', respectively, Le., €1730 = €1660 (L/(mol cm)). This implies that the listed fraction f, Table 11, should be regarded as an approximation of the degree of hydrogen bonding rather than as an exact measure of the relative number of dipolar peptide carbonyls on the investigated surface. The R-A spectra of NMAA on Au are shown in Figure 4. The results of NMAA on clean Au, Figure 4a, are very similar to those obtained on Cu, in terms of both the spectral signature, Figure 4a, and the fraction f of dipolar peptide carbonyls, Table 11. The Au and Cu surfaces exhibit also very similar values of the contact angle eHzO. Hydrogen bond interaction between the NMAA molecule and the Au surface is not expected to occur as easily as on Cu and Cu/CuzO because of the inertness of Au and the fact that no stable Au oxides can exist under the present experimental conditions. We believe therefore that the 1670-cm-l peak in the spectrum of NMAA on Au, most likely, originates from self-associated molecules. The R-A spectrum of NMAA on hydrophobic (alkyl-modified Au) shows, in analogy with that on air-exposed Cu/CuzO, Figure 2b, no traces of hydrogen bonded peptide carbonyls. Thus, the general conclusion of the results obtained so far is that the hydrophobic/hydrophilic properties of the metal surface are of great importance for the structure of adsorbed NMAA. However, one cannot really exclude the possibility that the electronic properties of the substrate material also may influence the structural changes on the surface. We have, therefore, in order to investigate how the electronic properties of the substrate will influence the behavior of contacting NMAA also included a nonconducting substrate material, Si02 on Si (Si/SiOz), in our study. NMAA on Clean and Alkyl-Modified Si/SiOz. Figure 5 shows the ATR spectra of NMAA on clean (OHsaturated) and methyl silanized (CHs-saturated) Si/SiOz. Although, the spectral range is limited to frequencies above 1500 cm-1 for Si crystals, the spectral signature for NMAA
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482 Langmuir, Vol. 7, No. 3, 1991
Table I. I n f r a r e d Frequencies (cm-') a n d Relative Intensities of N-Methylacetamide in Various Physical States a n d Tentative Assignments of the Strongest P e a k d thin layer on thin layer on thin layer on liquid
Nz matrix8 3498 w
3298 s 3108 m 2954 m
Cu
Cu/CuzO
3491 w 3329 w
3495 w 3306 m
3119 w 2947 w 1728 s 1716 s 1663 m
2958 w 1707 sh
1657 s
1624 w 1564 s
sh
1443 1416 1373 1300
1534 sh 1497 m 1440 vw 1411 vw 1377 m
1511 m 1440 vwi 1419vw 1370m
s s
1266 m 1168 vw 1161 m 1100 w 1048 w
1089vw 1037 vw
3500 w
2951 w 1732 s 1718 s 1663 s
2955 w 1732 s 1717 s
Au
Au/HS(CH&CH3
3499 vw 3314 vw 3275 vw
b
2951 vw 1732 s 1717 s 1670 m 1655 sh
b
Si/SiOZ
1530 sh 1493 m 1433 vw 1417 vw 1366 w
1497 m
1254 m 1169 w 1109 vw 1040 vw
1169 w 1097 vw 1045 vw
1250 m 1183 vw 1057 w
1539 sh 1500 m 1435 vw 1420 vw 1377 w 1300 vw 1254 m 1165 vw 1099 vw 1057 vw
Si/SiOz/ClzSi(CH3)z
assignmentsa
3503 w C
1732 s 1717 s 1663 s
1732 s 1717 s
1624 sh 1554 m
1412 w 1373 m 1301 w 1254 m
s
cu/cuzo (air expsd)
1636 sh 1558 w 1535 w 1520 w
1539 sh 1497 m 1439 vw 1416 vw 1373 w
2947 w 1730 s 1715 s 1670 vw 1655 vw 1632 vw 1528 m
I
1258 m 1192 vw 1038 vw
Based on refs 16-18. Key: Y, stretching; 6, deformation; 6,jS/,, asymmetric/symmetric/rockingdeformation. b Uncertain, diffuse peaks. No peaks are detectable above 2600 cm-* because of poor signal-to-noise. See text for further details. The Amide I band in proteins and polypeptides. e The Amide I1 band in proteins and polypeptides. f The Amide I11 band in proteins and polypeptides. 8 Based on trans-NMAA.lG Split in a t least two components. Split in two components. j Key: s, strong; m, medium; w, weak; vw, very weak; sh, shoulder. a
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1800
1
I
1600
1400
1200
800
1dOO
1717
1600
1400
1200
ld00
WAVENUMBERS (cm-1)
WAVENUMBERS Icm-ll
Figure 3. R-A spectra ( t , = 40 s) of NMAA on (a) Cu/CuzO and (b) Cu.
Figure 4. R-A spectra ( t , = 40 s) of NMAA on (a) clean Au and (b) hexadecanethiol-modified Au.
on clean Si/SiOz reveals, as in the case of hydrophilic metals, a coexistence between dipolar and free peptide carbonyls, Figure 4a. The ATR spectrum of NMAA on CH3-saturated (hydrophobic) Si/SiO:, is characterized by a single WC=O absorption near 1720 cm-1, which according to our previous discussion is due to a free peptide carbonyl. The results on the two Si/SiO:, surfaces give support to our suggestion that the hydrophobic/hydrophilic properties of the surface are of great importance for the behavior of contacting peptide groups and that the electronic properties of the substrate material appear to be of minor importance. Stability of Adsorbed NMAA and a Comparison with Previous Studies. A comparison of the spectra in Figure 4 reveals that there is a significant improvement in the signal-to-noise (S/N) ratio in the spectrum of NMAA on CHs-saturated (hydrophobic) Si/SiO:,. The reason for this improvement is the increased stability of NMAA on the CH3-saturated surface, which allows us to collect in-
terferograms over a longer period of time, and thereby obtain a better S / N ratio in the final ATR spectrum. A monolayer of NMAA on a CHB-saturated surface remains stable for a t least 30 min, whereas a monolayer on a OHsaturated surface desorbs much faster, typically within the very first minute after that monolayer has been formed on the surface. Similar differences in the stability are also observed on the investigated metal surfaces, but the effect is not as pronounced as for the two Si/SiOz surfaces. The origin of the difference in stability is a t present not fully understood. Garcia-Ramoas et aLZ0have recently investigated the adsorption of NMAA onto apatitic (Ca, Ba, and Sr) phosphate surfaces using infrared methods. They found for initially adsorbed N-deuterated NMAA a significant lowering of the Amide I frequency from about 1635 cm-l in the liquid state to about 1570 cm-l on the (20) Garcia-Ramos, J. V.; Carmona, P. Can. J. Chem. 1981,59, 222-
226.
Infrared Study of NMAA on Surfaces
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Table 11. Contact Angles with Water for the Investigated Surfaces and the Fraction f o f Dipolar Peptide Carbonyls in Adsorbed N-Methylacetamide (NMAA) surface
e(HzO) Hydrophilic
Au
cu/cuzo Si/SiO2
0
f d
25-30" 32-42' 13-17'
cu
(a)
0.27 0.28 0.46 0.47
