pubs.acs.org/Langmuir © 2009 American Chemical Society
Semiquantitative Study of the EDC/NHS Activation of Acid Terminal Groups at Modified Porous Silicon Surfaces S. Sam,†,‡,§ L. Touahir,† J. Salvador Andresa,† P. Allongue,† J.-N. Chazalviel,† A. C. Gouget-Laemmel,† C. Henry de Villeneuve,† A. Moraillon,† F. Ozanam,*,† N. Gabouze,‡ and S. Djebbar§ †
Physique de la Mati ere Condens ee, Ecole Polytechnique, CNRS, 91128 Palaiseau, France, ‡UDTS, 2 bd Frantz Fanon, BP 399, Alger-Gare, Algiers, Algeria, and §Laboratoire d’hydrom etallurgie et chimie inorganique mol eculaire, Facult e de Chimie, USTHB, BP 32 El Alia, 16111 Bab-Ezzouar, Algiers, Algeria Received June 19, 2009
Infrared spectroscopy is used to investigate the transformation of carboxyl-terminated alkyl chains immobilized on a surface into succinimidyl ester-terminated chains by reaction with an aqueous solution of N-ethyl-N0 -(3(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The acid chains are covalently grafted at the surface of hydrogenated porous silicon whose large specific surface area allows for assessing the activation yield in a semiquantitative way by infrared (IR) spectroscopy and detecting trace amounts of surface products and/or reaction products of small IR cross section. In this way, we rationalize the different reaction paths and optimize the reaction conditions to obtain as pure as possible succinimidyl ester-terminated surfaces. A diagram mapping the surface composition after activation was constructed by systematically varying the solution composition. Results are accounted for by NHS surface adsorption and a kinetic competition between the various EDC-induced surface reactions.
Introduction Biological sensors are built on different kinds of solid supports (metals, oxides, carbon, semiconductors) and make use of various detection schemes (fluorescence, surface plasmon resonance (SPR), electrochemical or electric-field-induced detection).1 In many cases, the reliability of the sensor strongly depends on the control of the immobilization of biological probes on the solid surface.2 From this viewpoint, covalent immobilization of the probes has been recognized as an attractive strategy, since in this case the sensor can withstand assay protocols more easily without probe loss. Many aspects of covalent probe immobilization procedures are common to all of the above-mentioned biological sensors. One standard procedure to immobilize biological probes (DNA or proteins) on molecular layers consists in preparing a succinimidyl ester (-COOSuc)-terminated surface layer and reacting it with an amino linker appended to the probe oligonucleotide3 or with the lysine groups of the outer envelope of the probe protein4 to form strong covalent peptide bonds. In most cases, the -COOSuc surface is obtained by reacting a surface bearing carboxyl end groups with N-hydroxysuccinimide (NHS), in the presence of a water-soluble carbodiimide such as N-ethylN0 -(3-(dimethylamino)propyl)carbodiimide (EDC) or other similar coupling agents.5,6 This reaction is often referred to as surface “activation”. In a few cases, the -COOSuc surface was directly *Corresponding author. E-mail:
[email protected]. (1) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev. 2008, 108, 109. (2) Borisov, S. M.; Wolfbeis, O. S. Chem. Rev. 2008, 108, 423. (3) Luderer, F.; Walschus, U. Top. Curr. Chem. 2005, 260, 37. (4) Rusmini, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2007, 8, 1775. (5) Staros, J. V.; Wright, R. W.; Swingle, D. M. Anal. Biochem. 1986, 156, 220. (6) Hermanson, G. T. Bioconjugate Techniques, 2nd ed.; Academic Press: San Diego, 2008. (7) Dordi, B.; Sch€onherr, H.; Vansco, G. J. Langmuir 2003, 19, 5780. (8) Taratula, O.; Galoppini, E.; Mendelsohn, R.; Reyes, P. I.; Zhang, Z.; Duan, Z.; Zhong, J.; Lu, Y. Langmuir 2009, 25, 2107. (9) Wojtyk, J. T. C.; Morin, K. A.; Boukherroub, R.; Wayner, D. D. M. Langmuir 2002, 18, 6081.
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obtained by using the relevant molecular precursor bearing the -COOSuc end group.7-10 This attractive procedure requires specific organic synthesis. This is probably why surface activation remains widely used, e.g., on self-assembled molecular layers at Au surfaces,11 even though the direct obtention of succinimidyl ester-functionalized surfaces from succinimidyl ester disulfide has been described more than one decade ago.12 In our group, we are more specifically interested in the immobilization of biological probes on molecular layers grafted onto oxide-free silicon surfaces. Since the pioneering work of Chidsey,13 many grafting procedures have been described on such surfaces,14 so that many routes have been tested on various hydrogenated silicon surfaces (porous, rough or atomically flat crystalline silicon,14 amorphous silicon,15 silicon nanowires16). These new possibilities have soon been recognized as attractive for biosensors,17 which has motivated the development of multistep (10) (a) B€ocking, T.; James, M.; Coster, H. G. L.; Chilcott, T. C.; Barrow, K. D. Langmuir 2004, 20, 9235. (b) Yang, M.; Teeuwen, R. L. M.; Giesbers, M.; Baggerman, J.; Arafat, A.; de Wolf, F. A.; van Hest, J. C. M.; Zuilhof, H. Langmuir 2008, 24, 7931. (11) (a) Patel, N.; Davies, M. C.; Hartshorne, M.; Heaton, R. J.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1997, 13, 6485. (b) Wolf, L. K.; Fullenkamp, D. E.; Georgiadis, R. M. J. Am. Chem. Soc. 2005, 127, 17453. (c) Lee, J. W.; Sim, S. J.; Cho, S. M.; Lee, J. Biosens. Bioelectron. 2005, 20, 1422. (d) Jang, L.-S.; Keng, H.-K. Biomed. Microdevices 2008, 10, 203. (12) (a) Nakano, K.; Taira, H.; Takagi, M. Anal. Sci. 1993, 9, 133. (b) Wagner, P.; Kernen, P.; Hegner, M.; Ungewickell, E.; Semenza, G. FEBS Lett. 1994, 356, 267. (13) Linford, M. R.; Fenter, P.; Eisenberger, P. M.; Chidsey, C. E. D. J. Am. Chem. Soc. 1995, 117, 3145. (14) (a) Buriak, J. M. Chem. Rev. 2002, 102, 1271. (b) Wayner, D. D. M.; Wolkow, R. A. J. Chem. Soc., Perkin Trans. 2 2002, 2, 23. (c) Boukherroub, R. Curr. Opin. Solid State Mater. Sci. 2005, 9, 66. (d) Shirahata, N.; Hozumi, A.; Yonezawa, T. Chem. Rec. 2005, 5, 145. (15) Lehner, A.; Steinhoff, G.; Brandt, M. S.; Eickhoff, M.; Stutzmann, M. J. Appl. Phys. 2003, 94, 2289. (16) (a) Bunimovich, Y. L.; Ge, G.; Beverly, K. C.; Ries, R. S.; Hood, L.; Heath, J. R. Langmuir 2004, 20, 10630. (b) Streifer, J. A.; Kim, H.; Nichols, B. M.; Hamers, R. J. Nanotechnology 2005, 16, 1868. (c) Scheibal, Z. R.; Xu, W.; Audiffred, J. F.; Flake, J. C. Electrochem. Solid-State Lett. 2008, 11, K81. (17) Wagner, P.; Nock, S.; Spudick, J. A.; Volkmuth, W. D.; Chu, S.; Cicero, R. L.; Wade, C. P.; Linford, M. R.; Chidsey, C. E. D. J. Struct. Biol. 1997, 119, 189.
Published on Web 09/02/2009
DOI: 10.1021/la902220a
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processes for the probe immobilization.18,19 In parallel, grafting methods in milder conditions have also been developed in order to reduce the number of processing steps,20 but approaching the maximum packing density at the surface21 by using such roomtemperature conditions remains a difficult target.22 In this framework, we have been led to make use of the standard EDC/NHS activation reaction,23 even though, here again, the direct attachment of succinimidyl ester is achievable.9,10 A literature survey about the activation step (-COOH + NHS/ EDC f -COOSuc) of molecular layers on Au or Si substrates shows that very different conditions have been used: namely, the concentrations in EDC and NHS span a wide range (from a few 0.1 M down to the mM range),19,24,25 the relative concentrations of EDC and NHS strongly vary from one study to another one,11,19,24c,25 and the temperature and pH conditions seem quite important.11d,26 Moreover, the infrared spectra of the molecular layers obtained on various substrates present some differences depending upon the conditions used for the activation treatment19,24 as well as when compared to spectra of layers grafted in one step.27,10b These considerations suggest that the NHS/EDC activation of acid surfaces remains empirical, although the reaction has been carefully optimized in solution28 and is well understood in this context at the molecular scale.5 Because, as others, we encountered some difficulties in controlling the biological probe immobilization after this reaction at a surface, this paper presents the results of a systematic study of the reaction at a surface. To the best of our knowledge there is no such work available. We study the activation reaction at porous silicon layers grafted with carboxyl-terminated alkyl chains, on which activation by EDC/NHS treatment has already been demonstrated,29 using infrared (IR) spectroscopy. This approach allows for assessing the activation yield in a semiquantitative way and detecting trace amounts of surface products and/or products of small IR absorption cross section. A diagram mapping the surface composition after activation is constructed as a function of the solution composition and discussed on the basis of the elementary reactions involved in the activation process.
Experimental Details Materials. N-Ethyl-N0 -(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) (∼98%) and N-hydroxysuccinimide (18) (a) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205. (b) Pike, A. R.; Lie, L. H.; Eagling, R. A.; Ryder, L. C.; Patole, S. N.; Connoly, B. A.; Horrocks, B. R.; Houlton, A. Angew. Chem., Int. Ed. 2002, 41, 615. (c) Guo, D.-J.; Xiao, S.-J.; Xia, B.; Wei, S.; Pei, J.; Pan, Y.; You, X.-Z.; Gu, Z.-Z.; Lu, Z. J. Phys. Chem. B 2005, 109, 20620. (d) B€ocking, T.; Kilian, K. A.; Hanley, T.; Ilyas, S.; Gaus, K.; Gal, M.; Gooding, J. J. Langmuir 2005, 21, 10522. (19) Voicu, R.; Boukherroub, R.; Bartzoka, V.; Ward, T.; Wojtyk, J. T. C.; Wayner, D. D. M. Langmuir 2004, 20, 11713. (20) (a) Stewart, M. P.; Buriak, J. M. J. Am. Chem. Soc. 2001, 123, 7821. (b) Sun, Q.-Y.; de Smet, L. C. P. M.; van Lagen, B.; Wright, A.; Zuilhof, H.; Sudh€olter, E. J. R. Angew. Chem., Int. Ed. 2004, 43, 1352. (c) Liu, Y.; Yamazaki, S.; Yamabe, S.; Nakato, Y. J. Mater. Chem. 2005, 15, 4906. (21) (a) Sieval, A. B.; van den Hout, B.; Zuilhof, H.; Sudh€olter, E. R. J. Langmuir 2001, 17, 2172. (b) Zhang, L.; Wesley, K.; Jiang, S. Langmuir 2001, 17, 6275. (22) Cicero, R. L.; Linford, M. R.; Chidsey, C. E. D. Langmuir 2000, 16, 5688. (23) Moraillon, A.; Gouget-Laemmel, A. C.; Ozanam, F.; Chazalviel, J.-N. J. Phys. Chem. C 2008, 112, 7158. (24) (a) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777. (b) Chi, Y. S.; Jung, Y. H.; Choi, I. S.; Kim, Y.-G. Langmuir 2005, 21, 4669. (c) Briand, E.; Salmain, M.; Compere, C.; Pradier, C.-M. Colloids Surf., B 2006, 53, 215. (25) Wei, F.; Sun, B.; Guo, Y.; Zhao, X. S. Biosens. Bioelectron. 2003, 18, 1157. (26) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosens. Bioelectron. 1997, 12, 977. (27) Grubor, N. M.; Shinar, R.; Jankowiak, R.; Porter, M. D.; Small, G. J. Biosens. Bioelectron. 2004, 19, 547. (28) Seghal, D.; Vijay, I. K. Anal. Biochem. 1994, 218, 87. (29) Boukherroub, R.; Wojtyk, J. T. C.; Wayner, D. D. M.; Lockwood, D. J. J. Electrochem. Soc. 2002, 149, H59.
810 DOI: 10.1021/la902220a
(NHS) (98%) were purchased from Sigma-Aldrich and undecylenic acid (99%) and chromosulfuric acid from Acros Organics. The chemicals used for porous silicon preparation were of VLSI grade: acetic acid glacial and absolute ethanol were supplied by Carlo Erba and hydrofluoric acid (HF) (50%) by SDS. Ultrapure water (Milli-Q, 18 MΩ cm) was used for the preparation of the solutions and for all rinses. Porous Silicon Preparation. The 500 μm thick silicon wafers used in these experiments were CZ-grown, double-side polished, (100)-oriented, boron-doped (p-type). Their resistivity was either 0.08-0.12 or 1 Ω cm. The porous silicon layers were prepared by anodic dissolution at a constant current density. The silicon anode was horizontal and was mounted at the bottom of a polytrifluorochloroethylene cell. An ohmic contact was provided by a gold grid coating deposited by evaporation on the silicon wafer backside. A platinum grid was used as the counter electrode. The cell was designed in order to ensure a good homogeneity of the current density over the 0.8 cm2 silicon surface exposed to the electrolyte and to obtain uniform porous layers. The electrolyte was a 1:1 mixture (by volume) of 50% aqueous HF and absolute ethanol for low-resistivity wafers or a similar 2:1 mixture for the more resistive ones. The etching time was 30 s, and the anodization current density was 80 and 150 mA cm-2 for the low-resistivity and high-resistivity wafers, respectively. These conditions were chosen to yield mesoporous silicon formation for ensuring a uniform chemical modification throughout the whole porous silicon layer. Prior to anodization, the samples were cleaned in chromosulfuric acid for 5 min, rinsed with a copious amount of water, and then immersed in HF for 1 min to remove the native oxide. After etching, the samples were copiously rinsed with water and thoroughly dried under a nitrogen stream. Thermal Hydrosilylation. First, 10 mL of neat undecylenic acid was outgassed in a Schlenk tube by bubbling argon for 15 min. The freshly prepared porous silicon samples were then transferred into the Schlenk tube under continuous argon bubbling for 15 min and allowed to react at 150 °C for 16 h. The functionalized surface was rinsed twice for 30 min in an outgassed Schlenk tube containing acetic acid at 75 °C.30 The sample was removed from hot acetic acid and blown dry under a nitrogen stream. Activation. The EDC and NHS solutions were prepared with cold water. The appropriate concentration mixture of EDC and NHS was deoxygenated with argon for 15 min in a Schlenk tube placed in a water bath at 15 °C. The freshly prepared acidterminated alkyl surface was placed in the Schlenk tube and allowed to react under continuous argon bubbling for 90 min at 15 °C. The resulting succinimidyl ester-terminated surface was copiously rinsed with water and dried under a stream of nitrogen. Infrared Measurements. The Fourier transform infrared (FT-IR) spectra were recorded using a Bruker (Equinox 55) spectrometer equipped with a deuterated triglycine sulfate (DTGS) detector. The samples were mounted in a purged sample chamber in transmission geometry at normal incidence. All FTIR spectra were collected with 200 scans in the 400-4000 cm-1 spectral region at 4 cm-1 resolution. Background spectra were obtained by using an untreated deoxidized flat silicon wafer mounted in the same geometry.
Results Infrared Characterization of Activated Layers. Figure 1 shows the spectrum of a porous silicon layer after its fabrication by anodic dissolution of silicon in HF (a) and after grafting in undecylenic acid (b). Sharp vibrational peaks appear superimposed on a sine-wave-modulated baseline due to optical interferences in the low-optical-index porous layer. The peak (30) Faucheux, A.; Gouget-Laemmel, A. C.; Henry de Villeneuve, C.; Boukherroub, R.; Ozanam, F.; Allongue, P.; Chazalviel, J.-N. Langmuir 2006, 22, 153.
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Article Table 1. Peak Assignments in the IR Spectra position [cm-1]
mode
description
Silicon Related Bands 2250 2140 2115 2085 1000-1200
ν(O3SiH) ν(SiH3) ν(SiH2) ν(SiH) νas(SiO)
910 880 680 610
δ(SiH2) δ(O-SiH2) δ(SiH) ω(SiH2)
oxidized SiH stretchd silicon trihydride stretchb silicon dihydride stretchb silicon monohydride stretchb silicon oxide antisymmetric Si-O-Si stretch (several modes)c SiH2 scissor deformationb oxidized SiH2 scissor deformationd SiH bendingb SiH2 waggingb
Saturated-Chain Related Bands
Figure 1. Transmission IR spectra of porous silicon layers: hydrogenated surface, after electrochemical fabrication (a); acid surface, after thermal grafting of undecylenic acid (b); after immersion of grafted acid surface in a 0.1 mol/L aqueous EDC solution for 1 h (c). Spectra have been vertically shifted for clarity. Spectra (a) and (b) have been recorded at different processing steps of the same sample.
assignments are listed in Table 1. In the spectrum of the freshly prepared porous silicon layer, one observes peaks characteristic of the SiH bonds present at the surface.31 The absence of any sizable contribution in the 1000-1200 cm-1 range demonstrates that the porous silicon surface is oxide-free.32 After reaction with undecylenic acid, the signature of acid-terminated acid chains grafted at the surface30 appears clearly. At this stage, the porous silicon surface remains essentially oxide-free, and the integrated intensity of the SiH stretching bands has decreased by a factor of ∼1.5 due to the partial substitution of the surface SiH species by the grafted chains. In order to obtain specific information about the interaction of the surface acid groups with the EDC and NHS reactants, we first exposed the surface to EDC and NHS separately. Therefore, we left a porous silicon layer bearing a grafted acid-terminated layer in contact with either a 0.1 M NHS solution or a 0.1 M EDC solution during 1 h and then rinsed the sample. The IR spectrum recorded after exposure to NHS does not reveal any surface modification. In contrast, besides indications of limited surface oxidation,32,33 the spectrum recorded after exposure to EDC (Figure 1c) evidenced the presence of new bands in the spectral range characteristic of carbonyl groups at 1750 and 1820 cm-1. Such a high-frequency doublet is characteristic of anhydride groups.34 A new peak (labeled A in Figure 1c) also appears at 1700 cm-1. Though tempting, the assignment of this band to the O-acylurea adduct formed by the reaction of EDC with the surface carboxyl group35,36 remains uncertain.37 Figure 2 shows infrared spectra recorded after activation of a grafted acid-terminated layer in EDC and NHS mixtures of various concentrations. In all of these experiments, the (31) Ogata, Y.; Niki, H.; Sakka, T.; Iwasaki, M. J. Electrochem. Soc. 1995, 142, 195. (32) da Fonseca, C.; Ozanam, F.; Chazalviel, J.-N. Surf. Sci. 1995, 365, 1. (33) (a) Lucovsky, G. Solid State Commun. 1979, 29, 571. (b) Ubara, H.; Imura, T.; Hiraki, A. Solid State Commun. 1984, 50, 673. (c) Chazalviel, J.-N.; Ozanam, F. In EMIS Datarev., Ser. 18 (Properties of Porous Silicon); Canham, L., Ed.; The Institution of Electrical Engineers: London, 1997; pp 59-65. (34) (a) Yan, L.; Marzolin, C.; Terfort, A.; Whitesides, G. M. Langmuir 1997, 13, 6704. (b) Faucheux, A.; Gouget-Laemmel, A. C.; Allongue, P.; Henry de Villeneuve, C.; Ozanam, F.; Chazalviel, J.-N. Langmuir 2007, 23, 1326. (35) Khorana, H. G. Chem. Rev. 1953, 53, 145. (36) Hoare, D. G.; Koshland, D. E., Jr. J. Biol. Chem. 1967, 242, 2447. (37) Iwasawa, T.; Wash, P.; Gibson, C.; Rebek, J., Jr. Tetrahedron 2007, 63, 6506.
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2925 2855 1465
νas(CH2) νs(CH2) δ(CH2)
methylene antisymmetric stretcha methylene symmetric stretcha methylene scissor deformationa
Functional-Group Related Bands 1820 1820 1785 1750 1745 1715 1650 1550 1415 1370 1285 1205 1065
ν(CdO) νs(CdO) νs(CdO) νas(CdO) νas(CdO) ν(CdO) ν(CdO) ν(N-H) δ(C-O-H) νs(C-N-C) ν(C-OH) νas(C-N-C) ν(N-C-O)
suc-ester carbonyl stretchf anhydride symmetric CdO stretche suc-cycle CdO symmetric stretchf anhydride antisymmetric CdO stretche suc-cycle CdO antisymmetric stretchf carboxylic acid CdO stretcha peptide CdO stretch (amide I)g mostly peptide N-H bend (amide II)g acid C-O-H in-plane deformationa suc-cycle sym CNC stretchf acid C-OH stretcha suc-cycle antisym CNC stretchf suc N-C-O stretchf
From ref 30. b From ref 31. c From ref 32. d From ref 33. e From ref 34. f From ref 38. g From ref 39. a
attachment of a succinimidyl ester termination to the grafted chains is evidenced by the prominent triplet in the spectral range of the CdO stretching vibrations.38 The activation yield does not always appear to be total, as manifested in some spectra by a shoulder or an asymmetry of the 1745 cm-1 peak ascribed to the contribution of the CdO stretching mode of unreacted acid groups. Furthermore, two small and broad additional bands, which cannot be ascribed to the succinimidyl ester termination or to acid groups, are observed at 1650 and 1550 cm-1. Though other assignments might be considered, these bands are strongly evocative of amide bonds.39 Finally, one can notice that the shape of the NHS triplet is not identical in all the spectra of Figure 2. In particular, one may notice that the relative intensity of the two high-wavenumber peaks is changing from spectrum to spectrum. This can be straightforwardly ascribed to the contribution of two distinct species (anhydride and succinimidyl ester) to the 1820 cm-1 peak. In the infrared spectrum of pure succinimidyl ester-terminated layers obtained by reacting N-succinimidyl undecylenate with porous silicon9 or flat Si(111) surfaces,10b the 1785 cm-1 peak of the carbonyl triplet is larger than the 1820 cm-1 one. Therefore, one may conclude that, depending upon EDC and NHS concentrations, the activated layers may suffer from several defects: (i) incomplete activation reaction, characterized by a remaining acid νCdO vibration at 1715 cm-1; (ii) the presence of urea derivative byproduct (as discussed below), evidenced by amide I and II bands at 1650 and 1550 cm-1; (iii) the presence of anhydride byproduct, evidenced by a peak at 1820 cm-1 larger than that at 1785 cm-1. Influence of NHS and EDC Concentrations during Activation. We have used the above criteria in order to rationalize the presence of these defects as a function of NHS and EDC DOI: 10.1021/la902220a
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Figure 2. Transmission IR spectra of porous silicon layers grafted with undecylenic acid after an activation treatment of 90 min in an aqueous solution of EDC and NHS. EDC and NHS concentrations are indicated on the right-hand side of each spectrum. Spectrum (e) refers to a sample formed on a substrate of lower resistivity, absorbing at low wavenumbers.
concentrations. Moreover, an absolute determination of the surface concentration of activated chains would be desirable, since it avoids implicit assumptions concerning the surface in the reference state.40 However, it would require the determination of the specific surface area of porous silicon, a difficult task indeed. Therefore, we have used a relative criterion, implicitly considering the νasCH2 band intensity as a measurement of the number of acid chains initially present at the porous silicon surface. Specifically, in order to have a semiquantitative criterion for assessing the overall yield of activation, we have used the ratio η of the height of the main succinimidyl ester band at 1745 cm-1 to that of the νasCH2 at 2920 cm-1. The measured η value should be compared to the reference value ηmax ≈ 2.5, i.e., the value measured in FTIR spectra of N-succinimidyl ester monolayers prepared in one step on Si(111)10b or on porous silicon.9 When comparing the spectra shown in Figure 2a-c, it appears that when the NHS concentration is varied while keeping the EDC concentration at a fixed value (5 mM), the activation yield varies nonmonotonously. When [NHS] > [EDC] (Figure 2a, [NHS] = 50 mM), the activation reaction is clearly incomplete (η = 1.1 < ηmax). Unreacted acid groups remain at the surface, and no byproduct is detected on the surface. Conversely, when [EDC] > [NHS] (Figure 2b, [NHS] = 0.5 mM), no real contribution of the νCdO acid vibration is detectable although the activation is again incomplete (η = 1.5). The carbonyl line shape clearly reveals the presence of anhydride, and the contribution of urea derivatives (amide bands) is also discernible. An activation close to maximum is observed when [EDC] = [NHS] = 5 mM since η = 2.5 with no extra IR amide bands (Figure 2c). When choosing an identical value for EDC and NHS concentrations, the resulting spectra depend on this value (Figure 2c-e). For [EDC] = [NHS] = 0.5 mM (Figure 2e), the activation yield is quite low (η = 1), acid groups remain on the surface, and the presence of anhydride is detected. For [EDC] = [NHS] = 50 mM (Figure 2d), the activation yield is larger (η = 1.8); no unreacted acid groups are detected at the surface, but some urea derivatives (38) (a) Frey, B. L.; Corn, R. M. Anal. Chem. 1996, 68, 3187. (b) Xiao, S.-J.; Brunner, S.; Wieland, M. J. Phys. Chem. B 2004, 108, 16508. (39) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: San Diego, 1991. (40) Aureau, D.; Ozanam, F.; Allongue, P.; Chazalviel, J.-N. Langmuir 2008, 24, 9440.
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Figure 3. Diagram showing the composition of the porous silicon surface after an activation of 90 min in EDC-NHS solutions of various compositions given by the coordinates of the points in the [EDC]-[NHS] plane. The boundaries of the zones are approximately drawn from the shape of the experimental infrared spectra and do not result from a quantitative reaction model. Each of the first three zones (which partly overlap) corresponds to the presence of the signature of a given product, as indicated on the legend at the right-hand side of the diagram. Optimum conditions correspond to zone 4 at the center of the graph. Solid circles correspond to conditions for which the activation yield (as defined in the text) is larger than 2.3, i.e., close to complete activation.
are found instead. The optimum result is then obtained with [EDC] = [NHS] = 5 mM (Figure 2c). In this case, the measured activation yield η = 2.5 ≈ ηmax suggests that the activation is essentially complete. The diagram in Figure 3 was constructed for summarizing the influence of solution composition on the chemical composition of the surface after activation. It should be emphasized that the boundaries between the different domains are only indicative. They have been drawn across the experimental points to define regions where specific reaction byproducts are detected on the surface at the end of the activation treatment: unreacted acid groups (zone 1), urea (zone 2), and anhydride (zone 3). They are by no means the result of a quantitative reaction model. Overlaps between the various zones correspond to conditions where several byproducts are simultaneously found at the surface. In zone 1, η is between 1 and 1.6 and drops to values lower than 1 for low EDC and NHS concentrations. In zone 2, η is generally smaller than 1.7, except for large concentrations of EDC and NHS where η may reach 2.3, close to the value corresponding to complete activation. In zone 3, η does not exceed 1.6. The optimum conditions are realized for 5 mM < [EDC] ∼ [NHS] < 10 mM (zone 4). Under these conditions, η values in excess of 2.3 are found (highlighted points on the diagram).
Discussion Reaction Mechanism. Figure 4 presents the complete reaction scheme thought to come into play for the activation of an acid-terminated surface. It is drawn according to reactions derived from the established reaction paths in homogeneous phase.5,41 For the sake of simplicity, EDC has been represented in neutral form, though it is in cationic form in the reaction conditions. The first step (reaction 1 in Figure 4) is the addition of the OH group of the carboxylic acid across one of the double bonds of the carbodiimide reactant, forming an O-acylurea adduct. Then, the surface O-acylurea can be transformed (41) Nakajima, N.; Ikada, Y. Bioconjugate Chem. 1995, 6, 123.
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Figure 4. Scheme of the elementary activation reactions. At the first step (reaction 1), the interaction between surface acid groups and EDC results in the formation of O-acylurea. Subsequently, various paths (reactions 2-5) account for the experimentally detected products (succinimidyl ester, anhydride, urea). The kinetic competition between the various paths determines the final surface composition.
according to three competitive paths: (i) a nucleophilic attack by NHS can yield the succinimidyl ester product with the release of the urea corresponding to the initial carbodiimide reactant (reaction 2); (ii) a reaction with a neighboring carboxyl group can yield an anhydride product grafted at the surface, with a similar urea release (reaction 3); (iii) a rearrangement via an intramolecular acyl transfer can yield an N-acylurea (reaction 4). NHS is precisely used to efficiently compete with the latter reaction in order to provide a more stable intermediate aimed at reacting with a primary amine to form an amide bond (in principle, the O-acylurea adduct can directly react with a primary amine and yield the wanted peptide coupling, but the reaction rate is low).5,6,28 Reaction 3, documented in the literature,41 straightforwardly accounts for the observation of anhydride species at the surface. These anhydride species can undergo further reaction with NHS, yielding the desired succinimidyl ester and regeneration of an acid group (reaction 5). Therefore, anhydride formation appears as a secondary pathway for succinimidyl ester formation, available at high acid concentration but which could hardly yield full surface activation. On the contrary, the N-acylurea product formed at the surface is unreactive. It is likely to be responsible for the observation of the amide signature in the infrared spectra rather than the urea released during the formation of the succinimidyl ester (reactions 2 and 5), which is expected to be removable from the surface during the final rinses. Byproducts Formation. The presence of byproducts at the surface (Figure 3) results from a kinetic competition between the different reaction paths depicted in Figure 4. Examination of some limiting cases helps in rationalizing the experimental findings. • First, let us consider the case where the EDC and NHS concentrations are both large (top right part of the graph in Figure 3) and focus on the formation of the Langmuir 2010, 26(2), 809–814
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urea byproduct, identified to N-acylurea. The N-acylurea generation rate is proportional to the surface concentration in O-acylurea, which results from the detailed balance between generation (reaction 1) and consumption (reactions 2-4, likely dominated by reaction 2 at large NHS concentration). At large EDC concentration, the surface concentration in O-acylurea can reach a significant level, yielding the generation of a detectable amount of N-acylurea at the surface. When EDC concentration remains large, but NHS concentration is low (bottom right part of the graph in Figure 3), the situation is similar except that slow kinetics are expected for reactions 2 and 5, which should favor byproduct formation through reactions 3 and 4. Anhydride and N-acylurea are therefore formed at the surface.42 When NHS and EDC concentrations are both low (bottom left part of the graph in Figure 3), anhydride formation through reaction 3 appears as the most likely consumption pathway of O-acylurea, at least in the first stage of the electrode modification when surface acid concentration remains sizable. Under these conditions, slow kinetics are expected for reactions 1, 2, and 5, which accounts for the incomplete modification of the electrode. Anhydride formation
(42) In previous experiments [see ref 23 and (a) Douarche, C.; Cortes, R.; Henry de Villeneuve, C.; Roser, S. J.; Braslau, A. J. Chem. Phys. 2008, 128, 225108. (b) Douarche, C. PhD Thesis, Universite de Lille 1 (USTL), 2007], surface activation was performed by exposing the acid surface to EDC alone (30 min) and then adding NHS (60 min). Applying this procedure here with [EDC] = [NHS] = 5 mM yields an incomplete reaction (η = 1.6), consistent with irreversible formation of N-acylurea during the first step of the procedure.
DOI: 10.1021/la902220a
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(i.e., reaction 4 dominant) and absence of N-acylurea (negligible O-acylurea surface concentration during reaction) are then expected, in agreement with experimental observations. • When EDC concentration is low but NHS concentration is large (top left part of the graph in Figure 3), fast kinetics of reactions 2 and 5 suggest that a slow succinimidyl ester formation should take place, with no other byproduct formation. The same situation should prevail as long as the NHS concentration remains much larger than that of EDC. Therefore, a complete reaction would be expected within the experiment duration if EDC concentration is raised at a value making reaction 1 fast enough. However, incomplete reaction is found instead. Regeneration of acid species via hydrolysis reactions (not taken into account in the simple scheme of Figure 4) can contribute to the presence of surface acid species, but it should become negligible at high NHS concentrations. The incomplete reaction observed under these conditions is more satisfactorily explained if NHS adsorption takes place at the acid surface and impedes reaction 1. Such an adsorption might appear unlikely, since no change in the infrared spectrum is observed after exposure of porous silicon grafted with an acidterminated layer to a concentrated (0.1 mol/L) NHS solution. However, in situ infrared experiments give clear indication of such an adsorption,43 which supports the conjecture of the impeding role of adsorbed NHS on reaction 1 when NHS is in excess in the activation solution. (43) Touahir, L.; Moraillon, A.; Sam, S.; Gouget-Laemmel, A. C.; Allongue, P.; Chazalviel, J.-N.; Henry de Villeneuve, C.; Ozanam, F. ECS Trans. 2009, 19, 283.
814 DOI: 10.1021/la902220a
Sam et al.
Since no quantitative measurement of the concentration of all the species involved in this reaction scheme is available at the various stages of the surface modification, a detailed simulation of the kinetics of the reactions could hardly make sense. Nonetheless, it can be concluded that the general trends can be accounted for by the set of reactions 1-5, plus an adsorption reaction of NHS, assuming that sites where NHS adsorbs at the surface become inactive.
Conclusion The transformation of the acid termination of a molecular layer grafted at the surface of porous silicon into succinimidyl (“activated”) ester appears practicable with a high yield by using an EDC/NHS treatment. However, the respective concentrations of EDC and NHS must be carefully chosen to yield complete modification and prevent formation of unwanted surface byproducts. Equimolar mixing of EDC and NHS in a rather restricted concentration range around 5 mM appears to achieve the wanted compromise. In fact, the EDC concentration must be high enough in order to obtain a complete reaction, but small enough to prevent N-acyl urea formation. Similarly, NHS concentration must be high enough to favor succinimidyl ester formation, but small enough to obtain a complete reaction. The experimental results can be accounted for by a set of five reactions, some of which being in kinetic competition, and the further assumption that sites where NHS adsorbs at the surface become inactive. The applicability of these findings to flat hydrogenated silicon surfaces remains to be demonstrated. Besides specific applications, working on flat surfaces also offers the opportunity to have a deeper insight into reaction kinetics, by measuring the kinetic constants associated with the reactions of interest using in situ infrared spectroscopy. These issues will be addressed in a subsequent work.
Langmuir 2010, 26(2), 809–814
