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J. Phys. Chem. B 2004, 108, 16508-16517
Reactions of Surface Amines with Heterobifunctional Cross-Linkers Bearing Both Succinimidyl Ester and Maleimide for Grafting Biomolecules Shou-Jun Xiao,*,†,‡ Samuel Brunner,§ and Marco Wieland†,| Laboratory for Surface Science and Technology, Department of Materials, Swiss Federal Institute of Technology, ETH-Zu¨rich, CH-8092, Zu¨rich, Switzerland, State Key Laboratory of Coordination Chemistry, College of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, China, and Swiss Federal Laboratories for Materials Testing and Research, EMPA, U ¨ berlandstrasse 129, CH-8600 Du¨bendorf, Switzerland ReceiVed: May 26, 2004; In Final Form: August 11, 2004
Surface reactions of amines with a series of heterobifunctional cross-linkers containing both maleimide and succinimidyl ester groups were investigated with infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). A specific surface derived from N-succinimidyl 6-maleimidylhexanoate (SMH) presents two linking groups, maleimide and succinimidyl ester, while surfaces from other cross-linkers present predominantly maleimidependant groups. An expected surface product by reaction of the cystamine monolayer with SMH, N,N′-bis(6-maleimidylhexanoyl)cystamine (BMHC), was synthesized independently, self-assembled, and characterized for further supporting the above conclusion. Finally, a peptide, H-Gly-Arg-Gly-Asp-Ser-Pro-Cys-OH (GRGDSPC), was immobilized on a maleimide-pendant surface as an amino-terminated structure and on a binary group (maleimide and succinimidyl ester) modified surface as a bridging structure.
Introduction
SCHEME 1
Immobilization of biomolecules onto solid surfaces has been of broad interest in biosensors, biomaterials, affinity chromatography, and biochips.1-5 It is one of the key steps for manufacturing in the above applications. The molecular structure, homogeneity, surface density, stability, and reproducibility of the functionalized surface determine the quality of the application system. Among the immobilization methods such as embodying, inclusion, coordination, and covalent immobilization, covalent immobilization is preferred because layers of immobilized biomolecules are more predictable, controllable, and reusable. A newly developed surface conjugation chemistry (Scheme 1), involving the use of heterobifunctional cross-linkers bearing both succinimidyl ester and maleimidyl groups, has been increasingly applied both in academia6-8 and industry.9 It includes three steps: (1) activation of the inert inorganic surface by self-assembling an amino-terminated species, (2) the consequent surface reaction of primary amines with heterobifunctional cross-linkers, and finally (3) covalent attachment of biomolecules.10-15 However, the details of the surface reaction, especially the side reaction of surface amine with maleimide, are not fully understood and the surface structure of immobilized molecules still remains partially unknown. An initial step in the development of this method is due to Bhatia et al.,16 who first mercaptosilanized a silica surface to react with the maleimidyl group of a cross-linker, yielding a terminal succinimidyl ester. Onto this amino-reactive surface was grafted IgG with free lysine amino groups. A radiolabeling
a A wavy line represents the substituted alkyl or aryl groups. R-SH incicates a biomolecule with an accessible thiol group. bSurface 2i represents surface products by reaction of 1 with a, b, c, d, e, and h, respectively. c3i represents the grafted biomolecule R-S- on surface 2i.
* Corresponding author: e-mail
[email protected]. † Swiss Federal Institute of Technology. ‡ Nanjing University. § Swiss Federal Laboratories for Materials Testing and Research | Present address: Institut Straumann AG, Hauptstrasse 26d, Waldenburg, CH-4437, Switzerland.
technique was used to estimate the IgG surface coverage and fluorescence labeling to image the patterned structure.17 Hong et al.10,11 were the first group to directly use the following approach: an aminosilanized surface was reacted with a heterobifunctional cross-linker, N-succinimidyl 6-maleimidylhexanoate (SMH) [it is abbreviated as EMCS from N-(-maleimidocaproyloxy)succinimide in previous literature], for grafting thiolcontaining biomolecules. They suggested that the reaction produced a maleimidyl-terminated surface (in the following, we describe a side reaction that occurs in this case). A cloned cytochrome with exposed thiols was subsequently immobilized onto the thiol-reactive silica surfaces. By use of UV-visible spectroscopy to measure the absorbance of cytochrome, the surface coverage was determined and the orientation of the grafted cytochrome was proposed. Following the same chemistry, Chrisey et al.12,13 fabricated DNA patterns on silica
10.1021/jp047726s CCC: $27.50 © 2004 American Chemical Society Published on Web 09/28/2004
Grafting Biomolecules by Use of Cross-Linkers surfaces. UV-Visible spectroscopy, fluorescence labeling, and radiolabeling techniques were used to estimate the DNA surface coverage and to characterize its patterned structure. Frey and Corn18 investigated the surface reaction between poly(l-lysine) and sulfosuccinimidyl 4-(N-maleimidylmethyl)cyclohexane-1carboxylate with infrared reflection absorption spectroscopy (IRRAS). They pointed out the possibility of a side reaction between surface amine and maleimide. We investigated this cross-linking reaction on titanium surfaces by means of IRRAS and X-ray photoelectron spectroscopy (XPS).14,15 We were puzzled while attempting to interpret the IRRAS data of the SMH-functionalized surface. The surface showed distinctive discrepancies in IRRAS compared to its analogues. Four bands were observed in the carbonyl stretching region 1700-1900 cm-1, while only one band at ∼1710 cm-1 was observed for its analogous surfaces. It is widely believed that the reaction occurs between the amino group and the succinimidyl ester group, eliminating N-hydroxysuccinimide as the leaving group and producing a maleimideterminated surface. In this case, due to the asymmetric stretching mode of the maleimidyl group, the infrared spectra should exhibit one strong band around 1710 cm-1. For the anomalous surface prepared from SMH, we guessed that either a side reaction or an orientation of maleimide was playing a role.15 In this paper, we have clarified the band assignments of the specific IRRAS spectrum produced from SMH. We used selfassembled aminothiols on gold as model surfaces instead of aminosilanes on titanium surfaces.15,19 The advantages of using aminothiol self-assembled monolayers (SAMs) on gold instead of aminosilanes are as follows: (1) They are more reproducible for surface characterization than aminosilanes. For example, due to the poor reproducibility of siloxane film formation and the variable degree of polymerization, the IRRAS spectra of aminosilanes on titanium and silicon surfaces show variations in the region 1650-1000 cm-1, corresponding to the NH2 and CH deformation and the Si-O-Si stretching vibration.14,15 These bands overlap those from the cross-linking groups, which is a situation that hinders a clear assignment and explanation of the infrared bands on the aminosilanized surfaces. (2) The expected surface product from in situ stepwise reactions on gold can be synthesized separately and therefore its surface properties can be obtained independently. By comparing their surface properties, information concerning molecular structure, orientation, and surface density can be inferred. Cystamine was first self-assembled on gold surfaces as the starting film. In a second step, the resulting surface amines were reacted with a series of analogous cross-linkers. The specific reaction with SMH and other reactions with its analogues exhibit similar IRRAS results in the region 1700-1900 cm-1 to those on aminosilanized surfaces. To clearly explain the characteristics of the surface from SMH, the expected surface product, N,N′bis(maleimidylhexanoyl)cystamine (BMHC) was synthesized separately and self-assembled on gold surfaces. In the carbonyl stretching region, it only shows one imidyl stretching band around 1710 cm-1, different from the SMH-derived surface. Therefore, two types of reactions are inferred for the stepwise reactions when linking groups are introduced: (1) Only one predominant reaction occurs between the surface amine and the succinimidyl ester group for most cross-linkers, resulting in maleimide-pendant surfaces. (2) A side reaction between amine and maleimide, accompanying the first reaction, happens for two cross-linkers, SMH and N-succinimidyl 8-maleimidyloctanoate, resulting in a heterogeneous surface covered by two linking groups, maleimide and succinimidyl ester. Finally, a cell-
J. Phys. Chem. B, Vol. 108, No. 42, 2004 16509 SCHEME 2
a
a The linkage atoms S and N (marked d) are highlighted from Cys and Gly, respectively, but the peptide sequence GRGDSPC is still kept. SMH, N-succinimidyl-6-maleimidyl hexanoate.
adhesive Arg-Gly-Asp- (RGD-) containing peptide, GRGDSPC, was grafted onto two functionalized surfaces, the self-assembled BMHC and the heterogeneous surface, respectively. The first reaction produced an amino-terminated peptide structure via a thioether linkage; the latter, a bridging peptide structure via both amide and thioether linkages. The two configurations are deduced from IRRAS measurements and additionally supported by XPS and time-of-flight secondary ion mass spectroscopy (TOF-SIMS). The different configurations of a peptide on solid surfaces might be of potential applications in biomaterials, biosensors, and biochips. The geometry and topology of attached biomolecules will affect surface charges, hydrophobic and hydrophilic properties, molecular orientations, and native conformations and thus determine both biological activity and function on surfaces. Experimental Section Chemical Reaction Route. We select analogous derivatives with different alkyl or aryl chains between the succinimidyl ester and maleimidyl groups: N-succinimidyl 3-maleimidylbenzoate (a), N-succinimidyl 4-(4-maleimidylphenyl)butyrate (b), N-succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (c), N-succinimidyl 3-maleimidylpropionate (d), N-succinimidyl 4-maleimidylbutyrate (e), SMH (f), N-succinimidyl 8-maleimidyloctanoate (g), and N-succinimidyl 11-maleimidylundecanoate (h). Their structures are shown in the Supporting Information. Scheme 1 shows the targeted surface reaction of cystamine SAMs (1) with cross-linkers to produce the maleimide-terminated surface (2i). Biomolecules bearing thiol groups (R-SH) can be grafted with a thioether linkage (3i) by the nucleophilic addition of thiol to maleimide. Scheme 2 presents a heterogeneous surface reaction resulting in both groups of maleimide and succinimidyl ester (2f) in the second step by reaction of SMH with cystamine SAMs. Besides the expected reaction of amine with succinimidyl ester, a side Michael-type addition occurs between maleimide and amine and generates a terminal succinimidyl ester. On the heterogeneous surface 2f, a peptide GRGDSPC containing both free thiol and amino groups predominantly reacts first with maleimide at pH 6.5 to produce a surface 3f* with a thioether linkage and an unreacted end amine. Followed by incubation of 3f* in a phosphate buffer (pH 9.0) within 5 h, a bridging peptide structure (3f) appears dominantly on the surface because of the nucleophilic substitution of the N-terminal amine to the excess retained succinimidyl ester. Scheme 3 illustrates the synthesis of BMHC, its self-
16510 J. Phys. Chem. B, Vol. 108, No. 42, 2004 SCHEME 3
a
a The linkage atoms S and N (marked d) are highlighted from Cys and Gly, respectively, but the peptide sequence GRGDSPC is still kept. SMH, N-succinimidyl-6-maleimidyl hexanoate; DIEA, diisopropylethylamine; BMHC, N,N′-bis(maleimidylhexanoyl)cystamine.
assembled monolayer (2m), and the immobilization of GRGDSPC with a thioether linkage (3m). The BMHC SAMs (2m) provide further evidence to demonstrate our picture of the heterogeneous surface chemistry. Materials. N-Succinimidyl 4-benzoylbenzoate (a) was purchased from Molecular Probe (Netherlands). [14C]Phenylglyoxal (27.0 mCi/mmol) from Amersham, Buckinghamshire, U.K., was stored and used as indicated. GRGDSPC (65%) was from Bachem AG, Bubendorf, Switzerland. All other chemicals were obtained from Fluka. The molecular structures and compound names of all heterobifunctional cross-linkers are shown in the Supporting Information. The substrates used for the experiments consisted of Si(100) wafers with an evaporated 200-nm-thick gold layer and a 6-nm chromium interlayer as an adhesion promoter. Preparation of Cystamine SAMs (1). The cystamine SAMs (1) were prepared by immersing gold substrates in a 2 mM solution of cystamine dihydrochloride in methanol or ethanol for 24 h and then rinsing with methanol, 1 mM NaOH aqueous solution, and water. The solubility of cystamine dihydrochloride in ethanol is very low. Therefore, some solid powder sediments can be seen even in the 2 mM solution. However, this does not interfere with the formation of cystamine SAMs. No difference was found between the SAMs prepared in either methanol or ethanol. Preparation of Linker-Modified Surfaces. The aminothiol SAMs were immersed in a 5 mM cross-linker solution in acetonitrile for 30 min, with sonication for 1 min every 10 min. After reaction, the substrates were washed with acetonitrile at least 3 times. Synthesis of N,N′-Bis(maleimidylhexanoyl)cystamine and Its SAMs (2m). BMHC is a new compound synthesized according to a method used by Doughty et al.20 A solution of SMH (62 mg, 0.2 mmol) in 1 mL of CH2Cl2 was added dropwise to a stirred suspension of cystamine dihydrochloride (23.0 mg, 0.1 mmol) and diisopropylethylamine (72 µL, 0.42 mmol) in CH2Cl2 (2 mL) under nitrogen. After the reaction mixture was allowed to stir for 16 h, the precipitated solid was removed by filtration, and the solvent of the filtrate was removed under reduced pressure to give a viscous oil. The crude residue was purified by flash chromatography (silica gel, 2.5 × 30 cm, EtOH:ethyl acetate ) 1:9) to afford a white solid (25 mg). NMR (CDCl3): δ 6.70 (s, 4 H, dCH), 6.42 (t, 2 H, CONH), 3.52 (m, 8 H, CH2NH, CH2N), 2.82 (t, 4 H, CH2S), 2.21 (t, 4 H, CH2CdO), 1.63 (m, 8 H, CH2), 1.32 (m, 4 H, CH2). Anal. calcd (%) for C24H34N4O6S2: C, 53.51; H, 6.36; N, 10.40; O, 17.82; S, 11.91. Found: C, 53.74; H, 6.57; N, 10.13. The BMHC
Xiao et al. SAMs (2m) were prepared by immersing gold substrates in a 2 mM solution (BMHC in methanol) for 12 h and then rinsing with methanol. Preparation of Peptide-Modified Surfaces 3m, 3f*, and 3f. Surfaces 2m and 2f were incubated at 20 °C for 1 h in a 2.0 mM peptide solution (pH 6.5) to produce surfaces 3m and 3f*, respectively. Then the samples were taken out, washed with water, dried and stored. Surface 3f was prepared by immersing 3f* in a phosphate buffer (pH 9.0) for 5 h, after which it was washed thoroughly with water, dried, and stored. X-ray Photoelectron Spectroscopy Measurements. XPS spectra were recorded on a Specs SAGE 100 system with unmonochromatized Mg KR radiation at 300 W (12 kV). Measurements were carried out with a takeoff angle of 90° with respect to the sample surface. The analyzed area was typically 9 × 9 mm2. Survey scans over a binding energy range of 0-1150 eV were taken for each sample with a constant detector pass energy range of 50 eV, followed by a high-resolution XPS measurement (pass energy 14 eV) for the quantitative determination of binding energies and atomic concentrations. Background subtraction, peak integration, and fitting were carried out by using the SpecsLab software. Electron binding energies were calibrated to Au 4f7/2 at 84.0 eV on the pure gold surface. To convert peak areas to surface concentration, sensitivity factors published by Evans et al.21 were applied. Infrared Reflection Absorption Spectroscopy Measurements. The IRRAS measurements were performed on a Bruker IFS 66V spectrometer operating at ca. 100 Pa. A mercurycadmium-telluride (MCT) detector was used to collect spectra with a resolution of 2 cm-1. The angle of incidence was 80° from the surface normal. A freshly evaporated gold film was used as the reference. For both sample and reference, 500 scans were collected. Time-of-Flight Secondary Ion Mass Spectroscopy. TOFSIMS measurements were performed on a commercial model, PHI 7200 (Physical Electronics). Three to five areas were usually analyzed per sample. Most mass spectra obtained from the same sample type were nearly identical and only one spectrum is discussed. Calibration of the mass spectra in the positive mass range was based on hydrogen and saturated molecular hydrocarbon fragments (amu) CH3+ (15.025), C2H5+ (29.04), C3H7+ (43.05), and C4H9+ (57.07). In the negative mass range, the calibration was based on CH- (13.01), C2H- (25.01), C3H- (37.01), and C4H- (49.01). Near-Edge X-ray Absorption Fine Structure Spectroscopy Measurements. NEXAFS experiments were carried out at the National Synchrotron Light Source (NSLS), Brookhaven National Lab (New York), on Exxon beamline U1A in the partial electron yield mode for changeable irradiation angles between normal and grazing incidence. Data analysis was carried out by means of difference spectra according to the analysis procedure described previously.22 NEXAFS spectroscopy probes transitions of core electrons into unoccupied molecular orbitals. Since these excitations are governed by dipole selection rules, the polarization dependence of their signal intensities can be used to extract information on the orientation of molecular adsorbates. Radiolabeling Procedure. The radiolabeling procedure was described in our previous publication.15 The GRGDSPC-grafted surface was held in a self-fabricated plate with two wells. To each well was injected 50 µL of sodium phosphate (25 mM) buffer (pH 7.4) containing 5 mM [14C]phenylglyoxal. The sample plate was incubated for 24 h at 20 °C and then washed thoroughly with water, dried with N2, and counted.
Grafting Biomolecules by Use of Cross-Linkers
J. Phys. Chem. B, Vol. 108, No. 42, 2004 16511
Figure 1. IR spectrum of SMH in KBr pellets.
Results Monitoring Surface Reactions with IRRAS: Cystamine Self-Assembled Monolayers (1). Kudelski and Hill23 studied the surface-enhanced Raman scattering (SERS) spectra of cysteamine and cystamine SAMs on Ag, and Wirde and Gelius24 reported the XPS and voltammetry results of the two aminothiol SAMs on gold surfaces. Both groups suggested that the two aminothiols form densely packed SAMs with surface coverage larger than 80% of that for an octadecanethiol monolayer. Our measurement of the cystamine SAMs with IRRAS does not show any informative signals in the frequency range 2700700 cm-1, in accordance with Kudelski’s SERS investigations. This enables the convenient monitoring of surface reactions with the IRRAS technique. IR Spectra of Cross-Linkers. Before discussion of the IRRAS results of the functionalized gold surfaces, IR spectra of the analogous cross-linkers in the solid state are introduced. These cross-linkers have maleimidyl and succinimidyl ester groups on both ends and an alkyl or aryl connection between them. Obviously, one’s attention is drawn to the two major groups: maleimide and succinimidyl ester. Maleimide has a five-membered, cyclic imide ring. Most authors apply the C2V symmetry on maleimide or succinimide derivatives, while others use Cs instead of C2V for the N-substituted moieties.25-28 The imidyl group (OdC-N-CdO) in maleimide and succinimide generally shows an intense peak between 1700 and 1750 cm-1, due to the asymmetric stretching mode, and one or several medium/weak peaks between 1740 and 1850 cm-1, due to the symmetric stretching modes. The symmetric peaks are much more complex because their appearance relates to many parameters such as the imide N-substituted groups, solvents, solid and liquid states, etc. An example IR spectrum of a cross-linker, SMH, is shown in Figure 1. Its band assignment is listed in Table 2. Four peaks are observed in the carbonyl stretching region: 1710, 1740, 1780, and 1820 cm-1. According to the literature,27-30 we can easily assign the 1710 cm-1 peak to the asymmetric stretching mode of the maleimidyl group. The other three peaks are due to the succinimidyl ester group. Both Frey and Corn18 and Lahiri et al.31 made the same assignment of the three peaks: 1740 and 1780 cm-1 to the imide asymmetric and symmetric stretching modes, respectively, and 1820 cm-1 to the ester stretching mode. However, Dordi et al.32 suggested that both weak bands 1780 and 1820 cm-1 be attributed to the ester splitting. Frey observed a strong peak at 1820 cm-1 and a weak peak around 1780 cm-1 for the sulfo-modified succinimidyl ester, while Lahiri, Dordi, and we observed two weak peaks at 1780 and 1820 cm-1 for succinimidyl ester. The weak band is in contrast with the usual strong ester stretching band. Therefore
Figure 2. IRRAS spectra of linker-functionalized surfaces 2a-2h.
we agree with Frey’s and Lahiri’s assignments. The weak band 1820 is supposed to be attenuated by a coupling between succinimide and the ester carbonyl (CdO) group. Three medium peaks are shown at 1445, 1413, and 1372 cm-1, respectively, due to the hydrocarbon and C-N-C group. The symmetric C-N-C stretching band is suggested between 1350 and 1440 cm-1 because very strong Raman lines appear for the imide derivatives around the same region, where no phenyl and carbonyl modes are expected.33 We assign the peak 1445 to the CH2 bending mode, 1413 to the C-N-C symmetric vibration mode, and 1372 to the C-N stretching mode. The main difference between succinimide and maleimide is that succinimide has a strong peak around 1250 cm-1, corresponding to the asymmetric vibration mode of C-N-C, while maleimide has a weak peak here. Therefore, the peak 1210 cm-1 is assigned to the C-N-C asymmetric vibration mode of succinimide. The peak 1060 cm-1 can be assigned to either -C(dO)-O-N- stretching or CH2 skeletal bending mode. Since N-alkyl-substituted succinimide does not have such a strong peak, we assign this peak to the ester -C(dO)-O-N- skeletal stretching mode. The alkenyl C-H stretching band of maleimide appears at 3092 cm-1 and its out-of-plane bending band at 833 cm-1. IRRAS Spectra of Linker-Functionalized Surfaces (2a-2h). It is well-known that IRRAS yields information not only on the chemical structure of a molecule but also on its orientation with respect to an external reference system. In the case of the grazing incidence infrared measurement configuration, the introduced light interacts with a thin organic film adsorbed on a metallic support. Consequently, only the electric field normal to the support interacts with the IR transition dipole moments of the adsorbed molecules. Therefore, the transition dipole moment perpendicular to the surface is IR-active, while that parallel to the surface is IR-inactive. This is the so-called “surface selection rule” for IRRAS. Surface reactions of amines with cross-linkers generate maleimide-terminated surfaces shown in Scheme 1 and heterogeneous surfaces with both maleimide and succinimidyl ester groups in Scheme 2. In the IRRAS spectra (Figure 2), maleimide-covered surfaces show one imide asymmetric stretching band around 1710 cm-1 and no symmetric stretching bands. For example, surfaces 2a-2e and 2h exhibit such properties. The wavenumbers of surface maleimide bands are listed in Table 1. All bands show a blue shift compared to those in the bulk around 1700 cm-1. We believe it is caused by a surface effect, especially the molecular oscillator model with one-end-fixed, in which the vibration frequency increases. The relatively larger
16512 J. Phys. Chem. B, Vol. 108, No. 42, 2004
Xiao et al.
TABLE 1: IRRAS Region 1700-1900 cm-1 of Linker-Functionalized Surfaces surface
2a
2b
2c
2d
2e
2f
2g
2h
frequency (cm-1)
1723
1721
1715
1717
1712
1707, 1745, 1784, 1822
1705, 1743, 1783, 1820
1710
TABLE 2: IR Frequencies and Assignments of SMH in KBr Pellets and of Surface 2fa frequency (cm-1) SMH 3504 w 3092 m 2946 s 2871 m 1817 m 1784 m 1741 s 1701 s 1445 w 1413 m 1372 m 1205 s 1065 s 863 m 833 m 695 m 650 m
2f
assignment
1821 w 1784 m 1745 s 1707 s 1540 w 1442 w 1410 m 1370 w 1210 m 1071 w
2 × 1741 mal CH stretching asym CH2 stretching sym CH2 stretching ester CdO stretching sym suc imide stretching asym suc imide stretching asym mal imide stretching amide II CH2 deformation sym mal C-N-C stretching mal C-N stretching asym suc C-N-C stretching ester C-O-N stretching suc CH2 rocking mal CH bending mal ring deformation suc ring deformation
Figure 3. IR spectrum of BMHC in KBr pellets.
a Abbreviations: mal, maleimide; suc, succinimidyl ester; sym, symmetric; asym, asymmetric; w, weak; m, medium; s, strong; b, broad; vw, very weak; vs, very strong.
blue shift on 2a, 2b, and 2d might relate to the stiff benzene, cyclohexane, and short alkyl chains, which strain the connection between surface and maleimide and thus increase the imide stretching band. The newly formed linkage-amide group does not show any strong amide I and/or II bands, except in the case of 2f. Two possibilities can explain this phenomenon: either the amide plane is parallel to the surface or the concentration of amide groups is too low to be detected. The same factors can account for the missing bands of the benzene ring stretching modes at ∼1590 and ∼1488 cm-1 on surfaces 2a and 2b. Surfaces 2f and 2g are examples of Scheme 2. They show additional multiple bands in the range 1700-1900 cm-1. On surface 2f, four groups contribute to the IRRAS spectrum: succinimidyl ester, maleimide, succinimide, and amide. By comparison of the IRRAS spectrum of 2f (Figure 2, 2f) with the IR spectrum of its precursor SMH in KBr pellets (Figure 1), it is obvious that nearly every peak on 2f has its corresponding one in SMH (KBr), except the amide II at 1550 cm-1. This makes the band assignments relatively easy and they are listed in Table 2. An additional band, amide II existing only on 2f, indicates the high concentration of amide groups and thus a high reaction yield between surface amine and succinimidyl ester. Amide II also supports the covalent linkage of linkers to surfaces. Self-Assembled Monolayers of BMHC (2m). We have suggested a mixture of maleimide and succinimidyl ester groups on surface 2f due to the side reaction of amine with maleimide. However, other factors such as molecular orientation can also influence the IRRAS spectrum. To prove our suggestion, the expected surface product, N,N′-bis(maleimidylhexanoyl)cystamine (BMHC), was synthesized separately and purified. When BMHC is self-assembled on Au (2m), it is considered to be the expected model of the surface product between the selfassembled monolayer of cystamine and SMH. Studies of the IR spectrum of BMHC in KBr pellets (Figure 3) and the IRRAS spectrum of its SAMs (Figure 4) give the detail of peak assignments and some information about the orientation of
Figure 4. IRRAS spectrum of the self-assembled monolayer of BMHC (2m).
TABLE 3: Frequencies and Assignments of IR Bands for BMHC in KBr Pellets and of IRRAS Bands for 2ma frequency (cm-1) BMHC 3450 b 3310 vs 3093 m 2940 s 2866 m 1767 vw 1700 vs 1646 s 1546 s 1443 w 1418 m 1375 w 1337 w 1307 w 1254 w 1210 w 1186 w 1129 w 838 m 698 m a
2m
1774 vw 1712 s 1543 m 1443 w 1412 m 1377 w
1217 vw 830 w
assignment 1700 + 1745 amide NH stretching mal CH stretching asym CH2 stretching sym CH2 stretching sym mal imide CdO stretching asym mal imide CdO stretching amide I amide II CH2 bending sym C-N-C stretching C-N stretching CH2 bending CH2 bending amide III asym C-N-C stretching mal CH bending mal ring deformation
Abbreviations are defined in Table 2.
functional groups. Comparison of the IRRAS spectra between 2f and 2m provides more evidence for the mixed species on 2f. The spectra of BMHC in the regions 3500-2700 and 1500500 cm-1 are assigned in Table 3 without a detailed discussion. In the region 1800-1500 cm-1, the imide group [-C(dO)N(O))C-] of BMHC in the bulk exhibits a strong asymmetric stretching band at 1700 cm-1 and a weak symmetric stretching shoulder around 1770 cm-1. The amide carbonyl (CdO) functionality usually exists in a trans configuration with the
Grafting Biomolecules by Use of Cross-Linkers N-H group, with the CdO stretching band (amide I) appearing at 1646 cm-1 and amide II at 1546 cm-1 very strongly in the bulk. On surface, this region is the most informative for deciphering the surface structure of HMBC. The strong asymmetric stretching band of the imide group shifts from 1700 in the bulk to 1712 cm-1 on the surface, suggests that the interchain interactions, e.g., the van der Waals interaction, are stronger on the surface than in the bulk. Amide II appears on the surface at 1543 cm-1 as a strong peak, similar to that in the bulk; however, amide I disappears on the surface. From the surface selection rule, a conclusion can be deduced that the amide I (CdO) stretching mode within the monolayers is parallel to and the amide II (C-N-H) stretching mode is perpendicular to the surface plane. Comparison of the IRRAS Spectra between 2f and 2m. The SAMs of 2m were independently characterized to support our deduction of the surface side reaction between cystamine SAMs and SMH. The obvious IRRAS difference from 2f is the disappearance of the succinimidyl ester frequencies 1745, 1782, and 1821 cm-1. The other subtle differences in the IRRAS spectra are as follows: (1) The amide II (1543 cm-1) is a sharp and strong peak on 2m but a weak and broad peak on 2f, due to the orientation of amide groups on the surface. (2) The asymmetric stretching vibration peak of maleimide on 2m is at 1712 cm-1; however, it is at 1707 cm-1 on 2f. The most possible reason for 1707 cm-1 on 2f is the overlap of maleimide and succinimide. (3) The out-of-plane bending peak of the maleimide C-H at 830 cm-1 appears on 2m but not on 2f due to the random orientation of maleimidyl groups. (4) Two frequencies, 1210 and 1071 cm-1, related to the succinimidyl ester C-N-C and C(dO)-O-N vibrations, appear on 2f but not on 2m. In summary, all peaks on 2m, except 830 cm-1, can be found on 2f. On the other hand, the peaks from the succinimidyl ester group on 2f do not appear on 2m. This strongly supports the presence of a mixture of maleimide and succinimidyl ester groups on 2f. IRRAS Spectra of Peptide-Modified Surfaces. After we know the detail of the second step reaction, it is straightforward to deduce the structure of biomolecules grafted on surfaces. It is well-known that maleimide reacts easily with biomolecules bearing accessible thiols and that succinimidyl ester is a specific reactive group for coupling biomolecules bearing amino groups. Without a doubt, the existence of both linking groups will affect the coupling of biomolecules carrying both amino and thiol groups. Here we show an example for coupling a cell-adhesive, RGD-containing peptide, GRGDSPC, on surfaces 2f and 2m. The peptide contains both reactive groups: the N-terminal amine on glycine and the thiol on cysteine. The IRRAS spectra of their corresponding surface products 3m, 3f*, and 3f are shown in Figure 5. Surface 3m is produced by the reaction of 2m with GRGDSPC at pH 6.5 for 1 h. The obvious changes are the appearance of amide I at 1665 cm-1 and the disappearance of the maleimidyl alkenyl dC-H bending at 830 cm-1, indicative of the covalent attachment of the peptide and the complete conversion of maleimide to succinimide, respectively. The asymmetric stretching band of imidyl OdC-N-CdO at 1712 cm-1 on 2m shifts slightly to 1710 cm-1 on 3m because of the change of maleimide to succinimide. The band amide II around 1543 cm-1 can be deconvoluted into two parts: a sharp, narrow band due to the orientated amide linkage on 2m and a broad band due to the peptide bonds. Three possible products can be anticipated by reaction of 2f with GRGDSPC: amino-terminated, thiol-terminated, and bridg-
J. Phys. Chem. B, Vol. 108, No. 42, 2004 16513
Figure 5. IRRAS spectra of GRGDSPC-grafted surfaces 3f*, 3f, and 3m.
ing peptides. After reaction of 2f with GRGDSPC at pH 6.5 for 1 h by mimicking the reaction condition of 3m, an intermediate surface 3f* is obtained. Its IRRAS spectrum (Figure 5) shows a new amide I at 1665 cm-1 and an intensity decrease of the bands from succinimidyl ester. The most possible structure for 3f* is a mixture of amino-terminated and bridging peptides. The thiol-terminated structure is difficult to obtain because the thiol group can react with maleimide much faster. The bridging structure is proposed according to the decrease of succinimidyl ester bands, indicating a partial reaction of succinimidyl esters with glycine amines. Furthermore, 3f* was incubated in a phosphate buffer at pH 9.0 for 5 h. The resulting product 3f exhibits nearly the same spectrum as that of 3m. The only difference is that amide II of 3f is shorter than that of 3m because 3f does not have an orientated amide group in which the carbonyl CdO is parallel to the gold surface. This process strongly supports the bridging peptide because the available amino groups can only come from the thioether-linked peptide. To exclude the hydrolysis of succinimidyl ester under the above condition, a control sample of 2f was placed in the same buffer for 5 h and virtually no hydrolysis of succinimidyl ester was observed, in accordance with a previous report.34 The higher amide I intensity of 3f compared to 3f* is attributed to the major portion of amide CdO bonds of the bridging peptide being perpendicular to the substrate surface plane. Monitoring Surface Reactions with XPS: Atomic Concentrations. In our studies, XPS was used to monitor the stepwise reactions. Five elements, C, N, O, S, and Au, were detected on all functionalized surfaces. Gold is easily detected, indicating very thin organic films (
